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Klf5 maintains the balance of primitive endoderm to epiblast specification during mouse embryonic development by suppression of Fgf4Takuya Azami1,2, Tsuyoshi Waku3, Ken Matsumoto2, Hyojung Jeon1, Masafumi Muratani4, Akihiro Kawashima1, Jun Yanagisawa5,6, Ichiro Manabe7, Ryozo Nagai8, Tilo Kunath9, Tomonori Nakamura10,11, Kazuki Kurimoto10,11, Mitinori Saitou10,11,12,13, Satoru Takahashi1,6,14, and Masatsugu Ema2,151Department of Anatomy and Embryology, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba, Ibaraki 305-8575, Japan2Department of Stem Cells and Human Disease Models, Research Center for Animal Life Science, Shiga University of Medical Science, Seta, Tsukinowa-cho, Otsu, Shiga 520-2192, Japan3 Graduate School of Pharmaceutical Sciences, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan4Department of Genome Biology, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan5Graduate School of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8577, Japan6Center for Tsukuba Advanced Research Alliance, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8577, Japan7Department of Cardiovascular Medicine, The University of Tokyo Graduate School of Medicine, Bunkyo, Tokyo 113-8655, Japan8Jichi Medical University, 3311-1 Yakushiji, Shimotsuke, Tochigi 329-0498, Japan9MRC Centre for Regenerative Medicine, School of Biological Sciences, University of Edinburgh, 5 Little France Drive, Edinburgh EH16 4UU, UK10Department of Anatomy and Cell Biology, Graduate School of Medicine, Kyoto University, Yoshida-Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan11JST, ERATO, Yoshida-Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan12Department of Reprogramming Science, Center for iPS Cell Research and Application, Kyoto University, 53 Kawahara-cho, Shogoin Yoshida, Sakyo-ku, Kyoto 606-8507, Japan13Institute for Integrated Cell-Material Sciences, Kyoto University, Yoshida-Ushinomiya-cho, Sakyo-ku, Kyoto 606-8501, Japan14International Institute for Integrative Sleep Medicine, Life Science Center, and Laboratory Animal Resource Center, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8577, Japan15PRESTO, Japan Science and Technology Agency, 4-1-8 Honcho Kawaguchi, Saitama 332-0012, JapanCorrespondence to: Masatsugu Ema, E-mail: mema@belle.shiga-med.ac.jpKey words: inner cell mass, primitive endoderm, epiblast, Fgf4, Klf5Summary StatementKlf5 represses Fgf4–Fgfr–ERK signalling to suppress its activation of the PrE-specification programme, thus ensuring the emergence of Nanog-positive na?ve pluripotent cells.AbstractThe inner cell mass of the mouse blastocyst gives rise to the pluripotent epiblast (EPI), which forms the embryo proper, and the primitive endoderm (PrE), which forms extra-embryonic yolk sac tissues. All inner cells co-express lineage markers such as Nanog and Gata6 at embryonic day (E) 3.25, and the EPI and PrE precursor cells eventually segregate to exclusively express Nanog and Gata6, respectively. Fibroblast growth factor (FGF)/extracellular signal-regulated kinase (ERK) signalling is involved in segregation of the EPI and PrE lineages; however, the mechanism involved in Fgf4-regulation is poorly understood. Here, we identified Klf5 as an upstream repressor of Fgf4. While Fgf4 was markedly upregulated in Klf5 knockout (KO) embryos at E3.0, it was downregulated in embryos overexpressing Klf5. Furthermore, Klf5 KO and overexpressing blastocysts showed skewed lineage specification phenotypes, similar to FGF4-treated preimplantation embryos and Fgf4 KO embryos, respectively. Inhibitors of the FGF receptor and ERK pathways reversed the skewed lineage specification of Klf5 KO blastocysts. These data demonstrate that Klf5 suppresses Fgf4-Fgfr-ERK signalling, thus preventing precocious activation of the PrE specification programme.IntroductionMammalian preimplantation embryo development segregates into three fundamental cell lineages. The first lineage segregation event separates, an epithelial cell layer called the trophectoderm (TE) on the surface of the embryo, which gives rise to trophoblast tissues of the placenta, and the inner cell mass (ICM), which gives rise to the embryo proper and extra-embryonic mesoderm. The second lineage segregation event further differentiates the ICM into epiblast (EPI) cells and the primitive endoderm (PrE). EPI cells generate most of the embryo proper and are a source for pluripotent embryonic stem (ES) cells, whereas the PrE generates visceral and parietal endoderm tissues, and these become the visceral and parietal yolk sacs ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1242/dev.017178", "ISSN" : "0950-1991", "PMID" : "19201946", "abstract" : "The investigation into lineage allocation and early asymmetries in the pre- and peri-implantation mouse embryo is gaining momentum. As we review here, new insights have been gained into the cellular and molecular events that lead to the establishment of the three lineages of the blastocyst, to the determination of the origin and the fates of the visceral endoderm in the peri-implantation mouse embryo, and to the generation of cellular and molecular activities that accompany the emergence of asymmetries in the pre-gastrulation embryo. We also discuss the continuing debate that surrounds the relative impacts of early lineage bias versus the stochastic allocation of cells with respect to the events that pattern the blastocyst and initiate its later asymmetries.", "author" : [ { "dropping-particle" : "", "family" : "Rossant", "given" : "Janet", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Tam", "given" : "Patrick P L", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Development (Cambridge, England)", "id" : "ITEM-1", "issue" : "5", "issued" : { "date-parts" : [ [ "2009", "3" ] ] }, "page" : "701-13", "title" : "Blastocyst lineage formation, early embryonic asymmetries and axis patterning in the mouse.", "type" : "article-journal", "volume" : "136" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(Rossant and Tam, 2009)", "plainTextFormattedCitation" : "(Rossant and Tam, 2009)", "previouslyFormattedCitation" : "(Rossant and Tam, 2009)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(Rossant and Tam, 2009). After compaction, inner cells generated in the first wave of cell division (8- to 16-cell stage) mainly contribute to EPI cells, while inner cells generated in the second wave (16- to 32-cell stage) are biased towards PrE cells ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1073/pnas.0915063107", "ISBN" : "0915063107", "ISSN" : "1091-6490", "PMID" : "20308546", "abstract" : "A crucial question in mammalian development is how cells of the early embryo differentiate into distinct cell types. The first decision is taken when cells undertake waves of asymmetric division that generate one daughter on the inside and one on the outside of the embryo. After this division, some cells on the inside remain pluripotent and give rise to the epiblast, and hence the future body, whereas others develop into the primitive endoderm, an extraembryonic tissue. How the fate of these inside cells is decided is unknown: Is the process random, or is it related to their developmental origins? To address this question, we traced all cells by live-cell imaging in intact, unmanipulated embryos until the epiblast and primitive endoderm became distinct. This analysis revealed that inner cell mass (ICM) cells have unrestricted developmental potential. However, cells internalized by the first wave of asymmetric divisions are biased toward forming pluripotent epiblast, whereas cells internalized in the next two waves of divisions are strongly biased toward forming primitive endoderm. Moreover, we show that cells internalized by the second wave up-regulate expression of Gata6 and Sox17, and changing the expression of these genes determines whether the cells become primitive endoderm. Finally, with our ability to determine the origin of cells, we find that inside cells that are mispositioned when they are born can sort into the correct layer. In conclusion, we propose a model in which the timing of cell internalization, cell position, and cell sorting combine to determine distinct lineages of the preimplantation mouse embryo.", "author" : [ { "dropping-particle" : "", "family" : "Morris", "given" : "Samantha a", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Teo", "given" : "Roy T Y", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Li", "given" : "Huiliang", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Robson", "given" : "Paul", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Glover", "given" : "David M", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Zernicka-Goetz", "given" : "Magdalena", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Proceedings of the National Academy of Sciences of the United States of America", "id" : "ITEM-1", "issue" : "14", "issued" : { "date-parts" : [ [ "2010", "4", "6" ] ] }, "page" : "6364-9", "title" : "Origin and formation of the first two distinct cell types of the inner cell mass in the mouse embryo.", "type" : "article-journal", "volume" : "107" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(Morris et al., 2010)", "plainTextFormattedCitation" : "(Morris et al., 2010)", "previouslyFormattedCitation" : "(Morris et al., 2010)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(Morris et al., 2010). However, a previous study did not observe such predetermination in EPI and PrE specification ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1242/dev.043471", "ISSN" : "1477-9129", "PMID" : "20147376", "abstract" : "Primitive endoderm (PE) and epiblast (EPI) are two lineages derived from the inner cell mass (ICM) of the E3.5 blastocyst. Recent studies showed that EPI and PE progenitors expressing the lineage-specific transcriptional factors Nanog and Gata6, respectively, arise progressively as the ICM develops. Subsequent sorting of the two progenitors during blastocyst maturation results in the ormation of morphologically distinct EPI and PE layers at E4.5. It is, however, unknown how the initial differences between the two populations become established in the E3.5 blastocyst. Because the ICM cells are derived from two distinct rounds of polarized cell divisions during cleavage, a possible role for cell lineage history in promoting EPI versus PE fate has been proposed. We followed cell lineage from the eight-cell stage by live cell tracing and could find no clear linkage between developmental history of individual ICM cells and later cell fate. However, modulating FGF signaling levels by inhibition of the receptor/MAP kinase pathway or by addition of exogenous FGF shifted the fate of ICM cells to become either EPI or PE, respectively. Nanog- or Gata6-expressing progenitors could still be shifted towards the alternative fate by modulating FGF signaling during blastocyst maturation, suggesting that the ICM progenitors are not fully committed to their final fate at the time that initial segregation of gene expression occurs. In conclusion, we propose a model in which stochastic and progressive specification of EPI and PE lineages occurs during maturation of the blastocyst in an FGF/MAP kinase signal-dependent manner.", "author" : [ { "dropping-particle" : "", "family" : "Yamanaka", "given" : "Yojiro", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Lanner", "given" : "Fredrik", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Rossant", "given" : "Janet", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Development (Cambridge, England)", "id" : "ITEM-1", "issue" : "5", "issued" : { "date-parts" : [ [ "2010", "3" ] ] }, "page" : "715-24", "title" : "FGF signal-dependent segregation of primitive endoderm and epiblast in the mouse blastocyst.", "type" : "article-journal", "volume" : "137" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(Yamanaka et al., 2010)", "plainTextFormattedCitation" : "(Yamanaka et al., 2010)", "previouslyFormattedCitation" : "(Yamanaka et al., 2010)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(Yamanaka et al., 2010). Single-cell analysis revealed that inner cells at embryonic day (E) 3.25 randomly co-express EPI and PrE markers and eventually acquire either an EPI or a PrE identity ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1016/j.devcel.2010.02.012", "ISSN" : "1878-1551", "PMID" : "20412781", "abstract" : "Three distinct cell types are present within the 64-cell stage mouse blastocyst. We have investigated cellular development up to this stage using single-cell expression analysis of more than 500 cells. The 48 genes analyzed were selected in part based on a whole-embryo analysis of more than 800 transcription factors. We show that in the morula, blastomeres coexpress transcription factors specific to different lineages, but by the 64-cell stage three cell types can be clearly distinguished according to their quantitative expression profiles. We identify Id2 and Sox2 as the earliest markers of outer and inner cells, respectively. This is followed by an inverse correlation in expression for the receptor-ligand pair Fgfr2/Fgf4 in the early inner cell mass. Position and signaling events appear to precede the maturation of the transcriptional program. These results illustrate the power of single-cell expression analysis to provide insight into developmental mechanisms. The technique should be widely applicable to other biological systems.", "author" : [ { "dropping-particle" : "", "family" : "Guo", "given" : "Guoji", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Huss", "given" : "Mikael", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Tong", "given" : "Guo Qing", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Wang", "given" : "Chaoyang", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Li Sun", "given" : "Li", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Clarke", "given" : "Neil D", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Robson", "given" : "Paul", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Developmental cell", "id" : "ITEM-1", "issue" : "4", "issued" : { "date-parts" : [ [ "2010", "4", "20" ] ] }, "page" : "675-85", "publisher" : "Elsevier Ltd", "title" : "Resolution of cell fate decisions revealed by single-cell gene expression analysis from zygote to blastocyst.", "type" : "article-journal", "volume" : "18" }, "uris" : [ "" ] }, { "id" : "ITEM-2", "itemData" : { "DOI" : "10.1093/nar/gkl050", "ISSN" : "1362-4962", "PMID" : "16547197", "abstract" : "A systems-level understanding of a small but essential population of cells in development or adulthood (e.g. somatic stem cells) requires accurate quantitative monitoring of genome-wide gene expression, ideally from single cells. We report here a strategy to globally amplify mRNAs from single cells for highly quantitative high-density oligonucleotide microarray analysis that combines a small number of directional PCR cycles with subsequent linear amplification. Using this strategy, both the representation of gene expression profiles and reproducibility between individual experiments are unambiguously improved from the original method, along with high coverage and accuracy. The immediate application of this method to single cells in the undifferentiated inner cell masses of mouse blastocysts at embryonic day (E) 3.5 revealed the presence of two populations of cells, one with primitive endoderm (PE) expression and the other with pluripotent epiblast-like gene expression. The genes expressed differentially between these two populations were well preserved in morphologically differentiated PE and epiblast in the embryos one day later (E4.5), demonstrating that the method successfully detects subtle but essential differences in gene expression at the single-cell level among seemingly homogeneous cell populations. This study provides a strategy to analyze biophysical events in medicine as well as in neural, stem cell and developmental biology, where small numbers of distinctive or diseased cells play critical roles.", "author" : [ { "dropping-particle" : "", "family" : "Kurimoto", "given" : "Kazuki", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Yabuta", "given" : "Yukihiro", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Ohinata", "given" : "Yasuhide", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Ono", "given" : "Yukiko", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Uno", "given" : "Kenichiro D", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Yamada", "given" : "Rikuhiro G", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Ueda", "given" : "Hiroki R", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Saitou", "given" : "Mitinori", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Nucleic acids research", "id" : "ITEM-2", "issue" : "5", "issued" : { "date-parts" : [ [ "2006", "1" ] ] }, "page" : "e42", "title" : "An improved single-cell cDNA amplification method for efficient high-density oligonucleotide microarray analysis.", "type" : "article-journal", "volume" : "34" }, "uris" : [ "" ] }, { "id" : "ITEM-3", "itemData" : { "DOI" : "10.1038/ncb2881", "ISSN" : "1476-4679", "PMID" : "24292013", "abstract" : "It is now recognized that extensive expression heterogeneities among cells precede the emergence of lineages in the early mammalian embryo. To establish a map of pluripotent epiblast (EPI) versus primitive endoderm (PrE) lineage segregation within the inner cell mass (ICM) of the mouse blastocyst, we characterized the gene expression profiles of individual ICM cells. Clustering analysis of the transcriptomes of 66 cells demonstrated that initially they are non-distinguishable. Early in the segregation, lineage-specific marker expression exhibited no apparent correlation, and a hierarchical relationship was established only in the late blastocyst. Fgf4 exhibited a bimodal expression at the earliest stage analysed, and in its absence, the differentiation of PrE and EPI was halted, indicating that Fgf4 drives, and is required for, ICM lineage segregation. These data lead us to propose a model where stochastic cell-to-cell expression heterogeneity followed by signal reinforcement underlies ICM lineage segregation by antagonistically separating equivalent cells.", "author" : [ { "dropping-particle" : "", "family" : "Ohnishi", "given" : "Yusuke", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Huber", "given" : "Wolfgang", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Tsumura", "given" : "Akiko", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Kang", "given" : "Minjung", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Xenopoulos", "given" : "Panagiotis", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Kurimoto", "given" : "Kazuki", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Ole\u015b", "given" : "Andrzej K.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Ara\u00fazo-Bravo", "given" : "Marcos J.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Saitou", "given" : "Mitinori", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Hadjantonakis", "given" : "Anna-Katerina", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Hiiragi", "given" : "Takashi", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Nature cell biology", "id" : "ITEM-3", "issue" : "1", "issued" : { "date-parts" : [ [ "2014", "1" ] ] }, "page" : "27-37", "publisher" : "Nature Publishing Group", "title" : "Cell-to-cell expression variability followed by signal reinforcement progressively segregates early mouse lineages.", "type" : "article-journal", "volume" : "16" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(Guo et al., 2010; Kurimoto et al., 2006; Ohnishi et al., 2014)", "plainTextFormattedCitation" : "(Guo et al., 2010; Kurimoto et al., 2006; Ohnishi et al., 2014)", "previouslyFormattedCitation" : "(Guo et al., 2010; Kurimoto et al., 2006; Ohnishi et al., 2014)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(Guo et al., 2010; Kurimoto et al., 2006; Ohnishi et al., 2014). The mechanism involved in the emergence and specification of EPI and PrE precursor cells is not fully understood ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1002/bies.201400147", "ISSN" : "1521-1878", "PMID" : "25640415", "abstract" : "Shortly after implantation the embryonic lineage transforms from a coherent ball of cells into polarized cup shaped epithelium. Recently we elucidated a previously unknown apoptosis-independent morphogenic event that reorganizes the pluripotent lineage. Polarization cues from the surrounding basement membrane rearrange the epiblast into a polarized rosette-like structure, where subsequently a central lumen is established. Thus, we provided a new model revising the current concept of apoptosis-dependent epiblast morphogenesis. Cell death however has to be tightly regulated during embryogenesis to ensure developmental success. Here, we follow the stages of early mouse development and take a glimpse at the critical signaling and morphogenic events that determine cells destiny and reshape the embryonic lineage.", "author" : [ { "dropping-particle" : "", "family" : "Bedzhov", "given" : "Ivan", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Zernicka-Goetz", "given" : "Magdalena", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "BioEssays : news and reviews in molecular, cellular and developmental biology", "id" : "ITEM-1", "issue" : "4", "issued" : { "date-parts" : [ [ "2015", "4" ] ] }, "page" : "372-8", "title" : "Cell death and morphogenesis during early mouse development: are they interconnected?", "type" : "article-journal", "volume" : "37" }, "uris" : [ "" ] }, { "id" : "ITEM-2", "itemData" : { "DOI" : "10.1098/rstb.2013.0537", "ISSN" : "1471-2970", "PMID" : "25349446", "abstract" : "In amniotes, primitive endoderm (PrE) plays important roles not only for nutrient support but also as an inductive tissue required for embryo patterning. PrE is an epithelial monolayer that is visible shortly before embryo implantation and is one of the first three cell lineages produced by the embryo. We review here the molecular mechanisms that have been uncovered during the past 10 years on PrE and epiblast cell lineage specification within the inner cell mass of the blastocyst and on their subsequent steps of differentiation.", "author" : [ { "dropping-particle" : "", "family" : "Hermitte", "given" : "St\u00e9phanie", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Chazaud", "given" : "Claire", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Philosophical transactions of the Royal Society of London. Series B, Biological sciences", "id" : "ITEM-2", "issue" : "1657", "issued" : { "date-parts" : [ [ "2014", "12", "5" ] ] }, "title" : "Primitive endoderm differentiation: from specification to epithelium formation.", "type" : "article-journal", "volume" : "369" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(Bedzhov and Zernicka-Goetz, 2015; Hermitte and Chazaud, 2014)", "plainTextFormattedCitation" : "(Bedzhov and Zernicka-Goetz, 2015; Hermitte and Chazaud, 2014)", "previouslyFormattedCitation" : "(Bedzhov and Zernicka-Goetz, 2015; Hermitte and Chazaud, 2014)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(Bedzhov and Zernicka-Goetz, 2015; Hermitte and Chazaud, 2014). The cell fate of EPI and PrE precursors is still plastic at E3.75 and is fully committed to EPI and PrE cells around E4.0–4.25 ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1242/dev.067702", "ISBN" : "1477-9129 (Electronic)\\r0950-1991 (Linking)", "ISSN" : "1477-9129", "PMID" : "22096072", "abstract" : "Cell differentiation during pre-implantation mammalian development involves the formation of two extra-embryonic lineages: trophoblast and primitive endoderm (PrE). A subset of cells within the inner cell mass (ICM) of the blastocyst does not respond to differentiation signals and forms the pluripotent epiblast, which gives rise to all of the tissues in the adult body. How this group of cells is set aside remains unknown. Recent studies documented distinct sequential phases of marker expression during the segregation of epiblast and PrE within the ICM. However, the connection between marker expression and lineage commitment remains unclear. Using a fluorescent reporter for PrE, we investigated the plasticity of epiblast and PrE precursors. Our observations reveal that loss of plasticity does not coincide directly with lineage restriction of epiblast and PrE markers, but rather with exclusion of the pluripotency marker Oct4 from the PrE. We note that individual ICM cells can contribute to all three lineages of the blastocyst until peri-implantation. However, epiblast precursors exhibit less plasticity than precursors of PrE, probably owing to differences in responsiveness to extracellular signalling. We therefore propose that the early embryo environment restricts the fate choice of epiblast but not PrE precursors, thus ensuring the formation and preservation of the pluripotent foetal lineage.", "author" : [ { "dropping-particle" : "", "family" : "Grabarek", "given" : "Joanna B", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Zyzy\u0144ska", "given" : "Krystyna", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Saiz", "given" : "N\u00e9stor", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Piliszek", "given" : "Anna", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Frankenberg", "given" : "Stephen", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Nichols", "given" : "Jennifer", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Hadjantonakis", "given" : "Anna-Katerina", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Plusa", "given" : "Berenika", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Development (Cambridge, England)", "id" : "ITEM-1", "issue" : "1", "issued" : { "date-parts" : [ [ "2012", "1" ] ] }, "page" : "129-39", "publisher" : "Oxford University Press for The Company of Biologists Limited", "title" : "Differential plasticity of epiblast and primitive endoderm precursors within the ICM of the early mouse embryo.", "type" : "article-journal", "volume" : "139" }, "uris" : [ "" ] }, { "id" : "ITEM-2", "itemData" : { "DOI" : "10.1242/dev.038893", "ISSN" : "1477-9129", "PMID" : "19710168", "abstract" : "Embryonic stem (ES) cells can be derived and propagated from multiple strains of mouse and rat through application of small-molecule inhibitors of the fibroblast growth factor (FGF)/Erk pathway and of glycogen synthase kinase 3. These conditions shield pluripotent cells from differentiation-inducing stimuli. We investigate the effect of these inhibitors on the development of pluripotent epiblast in intact pre-implantation embryos. We find that blockade of Erk signalling from the 8-cell stage does not impede blastocyst formation but suppresses development of the hypoblast. The size of the inner cell mass (ICM) compartment is not reduced, however. Throughout the ICM, the epiblast-specific marker Nanog is expressed, and in XX embryos epigenetic silencing of the paternal X chromosome is erased. Epiblast identity and pluripotency were confirmed by contribution to chimaeras with germline transmission. These observations indicate that segregation of hypoblast from the bipotent ICM is dependent on FGF/Erk signalling and that in the absence of this signal, the entire ICM can acquire pluripotency. Furthermore, the epiblast does not require paracrine support from the hypoblast. Thus, na\u00efve epiblast and ES cells are in a similar ground state, with an autonomous capacity for survival and replication, and high vulnerability to Erk signalling. We probed directly the relationship between na\u00efve epiblast and ES cells. Dissociated ICM cells from freshly harvested late blastocysts gave rise to up to 12 ES cell clones per embryo when plated in the presence of inhibitors. We propose that ES cells are not a tissue culture creation, but are essentially identical to pre-implantation epiblast cells.", "author" : [ { "dropping-particle" : "", "family" : "Nichols", "given" : "Jennifer", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Silva", "given" : "Jose", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Roode", "given" : "Mila", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Smith", "given" : "Austin", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Development (Cambridge, England)", "id" : "ITEM-2", "issue" : "19", "issued" : { "date-parts" : [ [ "2009", "10" ] ] }, "page" : "3215-22", "title" : "Suppression of Erk signalling promotes ground state pluripotency in the mouse embryo.", "type" : "article-journal", "volume" : "136" }, "uris" : [ "" ] }, { "id" : "ITEM-3", "itemData" : { "DOI" : "10.1242/dev.043471", "ISSN" : "1477-9129", "PMID" : "20147376", "abstract" : "Primitive endoderm (PE) and epiblast (EPI) are two lineages derived from the inner cell mass (ICM) of the E3.5 blastocyst. Recent studies showed that EPI and PE progenitors expressing the lineage-specific transcriptional factors Nanog and Gata6, respectively, arise progressively as the ICM develops. Subsequent sorting of the two progenitors during blastocyst maturation results in the ormation of morphologically distinct EPI and PE layers at E4.5. It is, however, unknown how the initial differences between the two populations become established in the E3.5 blastocyst. Because the ICM cells are derived from two distinct rounds of polarized cell divisions during cleavage, a possible role for cell lineage history in promoting EPI versus PE fate has been proposed. We followed cell lineage from the eight-cell stage by live cell tracing and could find no clear linkage between developmental history of individual ICM cells and later cell fate. However, modulating FGF signaling levels by inhibition of the receptor/MAP kinase pathway or by addition of exogenous FGF shifted the fate of ICM cells to become either EPI or PE, respectively. Nanog- or Gata6-expressing progenitors could still be shifted towards the alternative fate by modulating FGF signaling during blastocyst maturation, suggesting that the ICM progenitors are not fully committed to their final fate at the time that initial segregation of gene expression occurs. In conclusion, we propose a model in which stochastic and progressive specification of EPI and PE lineages occurs during maturation of the blastocyst in an FGF/MAP kinase signal-dependent manner.", "author" : [ { "dropping-particle" : "", "family" : "Yamanaka", "given" : "Yojiro", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Lanner", "given" : "Fredrik", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Rossant", "given" : "Janet", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Development (Cambridge, England)", "id" : "ITEM-3", "issue" : "5", "issued" : { "date-parts" : [ [ "2010", "3" ] ] }, "page" : "715-24", "title" : "FGF signal-dependent segregation of primitive endoderm and epiblast in the mouse blastocyst.", "type" : "article-journal", "volume" : "137" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(Grabarek et al., 2012; Nichols et al., 2009; Yamanaka et al., 2010)", "plainTextFormattedCitation" : "(Grabarek et al., 2012; Nichols et al., 2009; Yamanaka et al., 2010)", "previouslyFormattedCitation" : "(Grabarek et al., 2012; Nichols et al., 2009; Yamanaka et al., 2010)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(Grabarek et al., 2012; Nichols et al., 2009; Yamanaka et al., 2010).Nanog and Gata6 mRNAs are detectable as early as the 2-cell stage ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1016/j.devcel.2010.02.012", "ISSN" : "1878-1551", "PMID" : "20412781", "abstract" : "Three distinct cell types are present within the 64-cell stage mouse blastocyst. We have investigated cellular development up to this stage using single-cell expression analysis of more than 500 cells. The 48 genes analyzed were selected in part based on a whole-embryo analysis of more than 800 transcription factors. We show that in the morula, blastomeres coexpress transcription factors specific to different lineages, but by the 64-cell stage three cell types can be clearly distinguished according to their quantitative expression profiles. We identify Id2 and Sox2 as the earliest markers of outer and inner cells, respectively. This is followed by an inverse correlation in expression for the receptor-ligand pair Fgfr2/Fgf4 in the early inner cell mass. Position and signaling events appear to precede the maturation of the transcriptional program. These results illustrate the power of single-cell expression analysis to provide insight into developmental mechanisms. The technique should be widely applicable to other biological systems.", "author" : [ { "dropping-particle" : "", "family" : "Guo", "given" : "Guoji", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Huss", "given" : "Mikael", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Tong", "given" : "Guo Qing", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Wang", "given" : "Chaoyang", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Li Sun", "given" : "Li", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Clarke", "given" : "Neil D", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Robson", "given" : "Paul", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Developmental cell", "id" : "ITEM-1", "issue" : "4", "issued" : { "date-parts" : [ [ "2010", "4", "20" ] ] }, "page" : "675-85", "publisher" : "Elsevier Ltd", "title" : "Resolution of cell fate decisions revealed by single-cell gene expression analysis from zygote to blastocyst.", "type" : "article-journal", "volume" : "18" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(Guo et al., 2010)", "plainTextFormattedCitation" : "(Guo et al., 2010)", "previouslyFormattedCitation" : "(Guo et al., 2010)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(Guo et al., 2010), and both Nanog and Gata6 proteins are co-expressed in all inner cells at E3.25 (approximately 32 cells) ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1242/dev.003798", "ISBN" : "0950-1991 (Print)\\r0950-1991 (Linking)", "ISSN" : "0950-1991", "PMID" : "17978007", "abstract" : "Mouse pre-implantation development gives rise to the blastocyst, which is made up of at least three distinct cell types: the trophectoderm (TE) that surrounds a cavity, and an inner cell mass (ICM) comprising the primitive endoderm (PE) and epiblast (EPI). However, the underlying mechanisms involved in patterning the cleavage-stage embryo are still unresolved. By analyzing the distribution of the transcription factors Oct4 (Pou5f1), Cdx2 and Nanog at precisely defined stages in pre-implantation development, we were able to identify critical events leading to the divergence of TE, EPI and PE lineages. We found that Oct4 is present in all cells until late blastocyst, gradually disappearing from the TE thereafter. The expression patterns of both Cdx2 and Nanog exhibit two specific phases, culminating in their restriction to TE and EPI, respectively. In the first phase, starting after compaction, blastomeres show highly variable Cdx2 and Nanog protein levels. Importantly, the variability in Nanog levels is independent of position within the morula, whereas Cdx2 variability may originate from asymmetric cell divisions at the 8-cell stage in a non-stereotypic way. Furthermore, there is initially no reciprocal relationship between Cdx2 and Oct4 or between Cdx2 and Nanog protein levels. In the second phase, a definite pattern is established, possibly by a sorting process that accommodates intrinsic and extrinsic cues. Based on these results, we propose a model in which early embryonic mouse patterning includes stochastic processes, consistent with the highly regulative capacity of the embryo. This may represent a feature unique to early mammalian development.", "author" : [ { "dropping-particle" : "", "family" : "Dietrich", "given" : "Jens-Erik", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Hiiragi", "given" : "Takashi", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Development (Cambridge, England)", "id" : "ITEM-1", "issue" : "23", "issued" : { "date-parts" : [ [ "2007", "12" ] ] }, "page" : "4219-31", "title" : "Stochastic patterning in the mouse pre-implantation embryo.", "type" : "article-journal", "volume" : "134" }, "uris" : [ "" ] }, { "id" : "ITEM-2", "itemData" : { "DOI" : "10.1242/dev.021519", "ISSN" : "0950-1991", "PMID" : "18725515", "abstract" : "The first two lineages to differentiate from a pluripotent cell population during mammalian development are the extraembryonic trophectoderm (TE) and the primitive endoderm (PrE). Whereas the mechanisms of TE specification have been extensively studied, segregation of PrE and the pluripotent epiblast (EPI) has received comparatively little attention. A current model of PrE specification suggests PrE precursors exhibit an apparently random distribution within the inner cell mass of the early blastocyst and then segregate to their final position lining the cavity by the late blastocyst. We have identified platelet-derived growth factor receptor alpha (Pdgfralpha) as an early-expressed protein that is also a marker of the later PrE lineage. By combining live imaging of embryos expressing a histone H2B-GFP fusion protein reporter under the control of Pdgfra regulatory elements with the analysis of lineage-specific markers, we investigated the events leading to PrE and EPI lineage segregation in the mouse, and correlated our findings using an embryo staging system based on total cell number. Before blastocyst formation, lineage-specific factors are expressed in an overlapping manner. Subsequently, a gradual progression towards a mutually exclusive expression of PrE- and EPI-specific markers occurs. Finally, cell sorting is achieved by a variety of cell behaviours and by selective apoptosis.", "author" : [ { "dropping-particle" : "", "family" : "Plusa", "given" : "Berenika", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Piliszek", "given" : "Anna", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Frankenberg", "given" : "Stephen", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Artus", "given" : "J\u00e9r\u00f4me", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Hadjantonakis", "given" : "Anna-Katerina", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Development (Cambridge, England)", "id" : "ITEM-2", "issue" : "18", "issued" : { "date-parts" : [ [ "2008", "9" ] ] }, "page" : "3081-91", "title" : "Distinct sequential cell behaviours direct primitive endoderm formation in the mouse blastocyst.", "type" : "article-journal", "volume" : "135" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(Dietrich and Hiiragi, 2007; Plusa et al., 2008)", "plainTextFormattedCitation" : "(Dietrich and Hiiragi, 2007; Plusa et al., 2008)", "previouslyFormattedCitation" : "(Dietrich and Hiiragi, 2007; Plusa et al., 2008)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(Dietrich and Hiiragi, 2007; Plusa et al., 2008). Variability in the initial Nanog expression level shows no correlation with that of Gata6 in individual inner cells at E3.25 ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1038/ncb2881", "ISSN" : "1476-4679", "PMID" : "24292013", "abstract" : "It is now recognized that extensive expression heterogeneities among cells precede the emergence of lineages in the early mammalian embryo. To establish a map of pluripotent epiblast (EPI) versus primitive endoderm (PrE) lineage segregation within the inner cell mass (ICM) of the mouse blastocyst, we characterized the gene expression profiles of individual ICM cells. Clustering analysis of the transcriptomes of 66 cells demonstrated that initially they are non-distinguishable. Early in the segregation, lineage-specific marker expression exhibited no apparent correlation, and a hierarchical relationship was established only in the late blastocyst. Fgf4 exhibited a bimodal expression at the earliest stage analysed, and in its absence, the differentiation of PrE and EPI was halted, indicating that Fgf4 drives, and is required for, ICM lineage segregation. These data lead us to propose a model where stochastic cell-to-cell expression heterogeneity followed by signal reinforcement underlies ICM lineage segregation by antagonistically separating equivalent cells.", "author" : [ { "dropping-particle" : "", "family" : "Ohnishi", "given" : "Yusuke", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Huber", "given" : "Wolfgang", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Tsumura", "given" : "Akiko", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Kang", "given" : "Minjung", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Xenopoulos", "given" : "Panagiotis", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Kurimoto", "given" : "Kazuki", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Ole\u015b", "given" : "Andrzej K.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Ara\u00fazo-Bravo", "given" : "Marcos J.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Saitou", "given" : "Mitinori", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Hadjantonakis", "given" : "Anna-Katerina", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Hiiragi", "given" : "Takashi", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Nature cell biology", "id" : "ITEM-1", "issue" : "1", "issued" : { "date-parts" : [ [ "2014", "1" ] ] }, "page" : "27-37", "publisher" : "Nature Publishing Group", "title" : "Cell-to-cell expression variability followed by signal reinforcement progressively segregates early mouse lineages.", "type" : "article-journal", "volume" : "16" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(Ohnishi et al., 2014)", "plainTextFormattedCitation" : "(Ohnishi et al., 2014)", "previouslyFormattedCitation" : "(Ohnishi et al., 2014)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(Ohnishi et al., 2014). As embryos develop, the salt-and-pepper distributions of Nanog and Gata6 are evident in ICM cells until E3.5–4.0 ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1016/j.devcel.2006.02.020", "ISBN" : "1534-5807 (Print)\\r1534-5807 (Linking)", "ISSN" : "1534-5807", "PMID" : "16678776", "abstract" : "It has been thought that early inner cell mass (ICM) is a homogeneous population and that cell position in the ICM leads to the formation of two lineages, epiblast (EPI) and primitive endoderm (PE), by E4.5. Here, however, we show that the ICM at E3.5 is already heterogeneous. The EPI- and PE-specific transcription factors, Nanog and Gata6, were expressed in the ICM in a random \"salt and pepper\" pattern, as early as E3.5, in a mutually exclusive manner. Lineage tracing showed predominant lineage restriction of single ICM cells at E3.5 to either lineage. In embryos lacking Grb2 where no PE forms, Gata6 expression was lost and all ICM cells were Nanog positive. We propose a model in which the ICM develops as a mosaic of EPI and PE progenitors at E3.5, dependent on Grb2-Ras-MAP kinase signaling, followed by later segregation of the progenitors into the appropriate cell layers.", "author" : [ { "dropping-particle" : "", "family" : "Chazaud", "given" : "Claire", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Yamanaka", "given" : "Yojiro", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Pawson", "given" : "Tony", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Rossant", "given" : "Janet", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Developmental cell", "id" : "ITEM-1", "issue" : "5", "issued" : { "date-parts" : [ [ "2006", "5" ] ] }, "page" : "615-24", "title" : "Early lineage segregation between epiblast and primitive endoderm in mouse blastocysts through the Grb2-MAPK pathway.", "type" : "article-journal", "volume" : "10" }, "uris" : [ "" ] }, { "id" : "ITEM-2", "itemData" : { "DOI" : "10.1242/dev.021519", "ISSN" : "0950-1991", "PMID" : "18725515", "abstract" : "The first two lineages to differentiate from a pluripotent cell population during mammalian development are the extraembryonic trophectoderm (TE) and the primitive endoderm (PrE). 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The establishment of this salt-and-pepper distribution of Nanog and Gata6 is poorly understood; however, fibroblast growth factor (FGF)-extracellular signal-regulated kinase (ERK) signalling is postulated to play key roles ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1016/j.devcel.2006.02.020", "ISBN" : "1534-5807 (Print)\\r1534-5807 (Linking)", "ISSN" : "1534-5807", "PMID" : "16678776", "abstract" : "It has been thought that early inner cell mass (ICM) is a homogeneous population and that cell position in the ICM leads to the formation of two lineages, epiblast (EPI) and primitive endoderm (PE), by E4.5. Here, however, we show that the ICM at E3.5 is already heterogeneous. The EPI- and PE-specific transcription factors, Nanog and Gata6, were expressed in the ICM in a random \"salt and pepper\" pattern, as early as E3.5, in a mutually exclusive manner. Lineage tracing showed predominant lineage restriction of single ICM cells at E3.5 to either lineage. In embryos lacking Grb2 where no PE forms, Gata6 expression was lost and all ICM cells were Nanog positive. We propose a model in which the ICM develops as a mosaic of EPI and PE progenitors at E3.5, dependent on Grb2-Ras-MAP kinase signaling, followed by later segregation of the progenitors into the appropriate cell layers.", "author" : [ { "dropping-particle" : "", "family" : "Chazaud", "given" : "Claire", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Yamanaka", "given" : "Yojiro", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Pawson", "given" : "Tony", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Rossant", "given" : "Janet", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Developmental cell", "id" : "ITEM-1", "issue" : "5", "issued" : { "date-parts" : [ [ "2006", "5" ] ] }, "page" : "615-24", "title" : "Early lineage segregation between epiblast and primitive endoderm in mouse blastocysts through the Grb2-MAPK pathway.", "type" : "article-journal", "volume" : "10" }, "uris" : [ "" ] }, { "id" : "ITEM-2", "itemData" : { "DOI" : "10.1242/dev.084996", "ISSN" : "1477-9129", "PMID" : "23193166", "abstract" : "The emergence of pluripotent epiblast (EPI) and primitive endoderm (PrE) lineages within the inner cell mass (ICM) of the mouse blastocyst involves initial co-expression of lineage-associated markers followed by mutual exclusion and salt-and-pepper distribution of lineage-biased cells. Precisely how EPI and PrE cell fate commitment occurs is not entirely clear; however, previous studies in mice have implicated FGF/ERK signaling in this process. Here, we investigated the phenotype resulting from zygotic and maternal/zygotic inactivation of Fgf4. Fgf4 heterozygous blastocysts exhibited increased numbers of NANOG-positive EPI cells and reduced numbers of GATA6-positive PrE cells, suggesting that FGF signaling is tightly regulated to ensure specification of the appropriate numbers of cells for each lineage. Although the size of the ICM was unaffected in Fgf4 null mutant embryos, it entirely lacked a PrE layer and exclusively comprised NANOG-expressing cells at the time of implantation. An initial period of widespread EPI and PrE marker co-expression was however established even in the absence of FGF4. Thus, Fgf4 mutant embryos initiated the PrE program but exhibited defects in its restriction phase, when lineage bias is acquired. Consistent with this, XEN cells could be derived from Fgf4 mutant embryos in which PrE had been restored and these cells appeared indistinguishable from wild-type cells. Sustained exogenous FGF failed to rescue the mutant phenotype. Instead, depending on concentration, we noted no effect or conversion of all ICM cells to GATA6-positive PrE. We propose that heterogeneities in the availability of FGF produce the salt-and-pepper distribution of lineage-biased cells.", "author" : [ { "dropping-particle" : "", "family" : "Kang", "given" : "Minjung", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Piliszek", "given" : "Anna", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Artus", "given" : "J\u00e9r\u00f4me", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Hadjantonakis", "given" : "Anna-Katerina", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Development (Cambridge, England)", "id" : "ITEM-2", "issue" : "2", "issued" : { "date-parts" : [ [ "2013", "1", "15" ] ] }, "page" : "267-79", "title" : "FGF4 is required for lineage restriction and salt-and-pepper distribution of primitive endoderm factors but not their initial expression in the mouse.", "type" : "article-journal", "volume" : "140" }, "uris" : [ "" ] }, { "id" : "ITEM-3", "itemData" : { "DOI" : "10.1016/j.ydbio.2013.09.023", "ISSN" : "1095-564X", "PMID" : "24063807", "abstract" : "The primitive endoderm (PE) and epiblast (EPI) are two lineages derived from the inner cell mass (ICM) of the E3.5 blastocyst. Although it has been shown that FGF signaling is necessary and sufficient for PE specification in the ICM, it is unknown what mechanisms control the PE/EPI proportion in the embryo. Because modulation of FGF signaling alone is sufficient to convert all ICM cells to either PE or EPI, a model has been proposed in which the amount of FGF in the embryo controls the PE/EPI proportion. To test this model, we reduced the amount of FGF4, the major FGF in the preimplantation embryo, using various genotypes of Fgf4 mutants. We observed a maternal contribution of Fgf4 in PE specification, but it was dispensable for development. In addition, upon treatment of Fgf4 mutant embryos with exogenous FGF4, we observed a progressive increase of PE proportions in an FGF4 dose dependent manner, regardless of embryo genotype. We conclude that the amount of FGF4 is limited and regulates PE/EPI proportions in the mouse embryo.", "author" : [ { "dropping-particle" : "", "family" : "Krawchuk", "given" : "Dayana", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Honma-Yamanaka", "given" : "Nobuko", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Anani", "given" : "Shihadeh", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Yamanaka", "given" : "Yojiro", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Developmental biology", "id" : "ITEM-3", "issue" : "1", "issued" : { "date-parts" : [ [ "2013", "12", "1" ] ] }, "page" : "65-71", "publisher" : "Elsevier", "title" : "FGF4 is a limiting factor controlling the proportions of primitive endoderm and epiblast in the ICM of the mouse blastocyst.", "type" : "article-journal", "volume" : "384" }, "uris" : [ "" ] }, { "id" : "ITEM-4", "itemData" : { "DOI" : "10.1242/dev.038893", "ISSN" : "1477-9129", "PMID" : "19710168", "abstract" : "Embryonic stem (ES) cells can be derived and propagated from multiple strains of mouse and rat through application of small-molecule inhibitors of the fibroblast growth factor (FGF)/Erk pathway and of glycogen synthase kinase 3. These conditions shield pluripotent cells from differentiation-inducing stimuli. We investigate the effect of these inhibitors on the development of pluripotent epiblast in intact pre-implantation embryos. We find that blockade of Erk signalling from the 8-cell stage does not impede blastocyst formation but suppresses development of the hypoblast. The size of the inner cell mass (ICM) compartment is not reduced, however. Throughout the ICM, the epiblast-specific marker Nanog is expressed, and in XX embryos epigenetic silencing of the paternal X chromosome is erased. Epiblast identity and pluripotency were confirmed by contribution to chimaeras with germline transmission. These observations indicate that segregation of hypoblast from the bipotent ICM is dependent on FGF/Erk signalling and that in the absence of this signal, the entire ICM can acquire pluripotency. Furthermore, the epiblast does not require paracrine support from the hypoblast. Thus, na\u00efve epiblast and ES cells are in a similar ground state, with an autonomous capacity for survival and replication, and high vulnerability to Erk signalling. We probed directly the relationship between na\u00efve epiblast and ES cells. Dissociated ICM cells from freshly harvested late blastocysts gave rise to up to 12 ES cell clones per embryo when plated in the presence of inhibitors. We propose that ES cells are not a tissue culture creation, but are essentially identical to pre-implantation epiblast cells.", "author" : [ { "dropping-particle" : "", "family" : "Nichols", "given" : "Jennifer", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Silva", "given" : "Jose", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Roode", "given" : "Mila", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Smith", "given" : "Austin", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Development (Cambridge, England)", "id" : "ITEM-4", "issue" : "19", "issued" : { "date-parts" : [ [ "2009", "10" ] ] }, "page" : "3215-22", "title" : "Suppression of Erk signalling promotes ground state pluripotency in the mouse embryo.", "type" : "article-journal", "volume" : "136" }, "uris" : [ "" ] }, { "id" : "ITEM-5", "itemData" : { "DOI" : "10.1038/ncb2881", "ISSN" : "1476-4679", "PMID" : "24292013", "abstract" : "It is now recognized that extensive expression heterogeneities among cells precede the emergence of lineages in the early mammalian embryo. To establish a map of pluripotent epiblast (EPI) versus primitive endoderm (PrE) lineage segregation within the inner cell mass (ICM) of the mouse blastocyst, we characterized the gene expression profiles of individual ICM cells. Clustering analysis of the transcriptomes of 66 cells demonstrated that initially they are non-distinguishable. Early in the segregation, lineage-specific marker expression exhibited no apparent correlation, and a hierarchical relationship was established only in the late blastocyst. Fgf4 exhibited a bimodal expression at the earliest stage analysed, and in its absence, the differentiation of PrE and EPI was halted, indicating that Fgf4 drives, and is required for, ICM lineage segregation. These data lead us to propose a model where stochastic cell-to-cell expression heterogeneity followed by signal reinforcement underlies ICM lineage segregation by antagonistically separating equivalent cells.", "author" : [ { "dropping-particle" : "", "family" : "Ohnishi", "given" : "Yusuke", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Huber", "given" : "Wolfgang", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Tsumura", "given" : "Akiko", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Kang", "given" : "Minjung", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Xenopoulos", "given" : "Panagiotis", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Kurimoto", "given" : "Kazuki", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Ole\u015b", "given" : "Andrzej K.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Ara\u00fazo-Bravo", "given" : "Marcos J.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Saitou", "given" : "Mitinori", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Hadjantonakis", "given" : "Anna-Katerina", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Hiiragi", "given" : "Takashi", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Nature cell biology", "id" : "ITEM-5", "issue" : "1", "issued" : { "date-parts" : [ [ "2014", "1" ] ] }, "page" : "27-37", "publisher" : "Nature Publishing Group", "title" : "Cell-to-cell expression variability followed by signal reinforcement progressively segregates early mouse lineages.", "type" : "article-journal", "volume" : "16" }, "uris" : [ "" ] }, { "id" : "ITEM-6", "itemData" : { "DOI" : "10.1242/dev.043471", "ISSN" : "1477-9129", "PMID" : "20147376", "abstract" : "Primitive endoderm (PE) and epiblast (EPI) are two lineages derived from the inner cell mass (ICM) of the E3.5 blastocyst. Recent studies showed that EPI and PE progenitors expressing the lineage-specific transcriptional factors Nanog and Gata6, respectively, arise progressively as the ICM develops. Subsequent sorting of the two progenitors during blastocyst maturation results in the ormation of morphologically distinct EPI and PE layers at E4.5. It is, however, unknown how the initial differences between the two populations become established in the E3.5 blastocyst. Because the ICM cells are derived from two distinct rounds of polarized cell divisions during cleavage, a possible role for cell lineage history in promoting EPI versus PE fate has been proposed. We followed cell lineage from the eight-cell stage by live cell tracing and could find no clear linkage between developmental history of individual ICM cells and later cell fate. However, modulating FGF signaling levels by inhibition of the receptor/MAP kinase pathway or by addition of exogenous FGF shifted the fate of ICM cells to become either EPI or PE, respectively. Nanog- or Gata6-expressing progenitors could still be shifted towards the alternative fate by modulating FGF signaling during blastocyst maturation, suggesting that the ICM progenitors are not fully committed to their final fate at the time that initial segregation of gene expression occurs. In conclusion, we propose a model in which stochastic and progressive specification of EPI and PE lineages occurs during maturation of the blastocyst in an FGF/MAP kinase signal-dependent manner.", "author" : [ { "dropping-particle" : "", "family" : "Yamanaka", "given" : "Yojiro", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Lanner", "given" : "Fredrik", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Rossant", "given" : "Janet", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Development (Cambridge, England)", "id" : "ITEM-6", "issue" : "5", "issued" : { "date-parts" : [ [ "2010", "3" ] ] }, "page" : "715-24", "title" : "FGF signal-dependent segregation of primitive endoderm and epiblast in the mouse blastocyst.", "type" : "article-journal", "volume" : "137" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(Chazaud et al., 2006; Kang et al., 2013; Krawchuk et al., 2013; Nichols et al., 2009; Ohnishi et al., 2014; Yamanaka et al., 2010)", "plainTextFormattedCitation" : "(Chazaud et al., 2006; Kang et al., 2013; Krawchuk et al., 2013; Nichols et al., 2009; Ohnishi et al., 2014; Yamanaka et al., 2010)", "previouslyFormattedCitation" : "(Chazaud et al., 2006; Kang et al., 2013; Krawchuk et al., 2013; Nichols et al., 2009; Ohnishi et al., 2014; Yamanaka et al., 2010)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(Chazaud et al., 2006; Kang et al., 2013; Krawchuk et al., 2013; Nichols et al., 2009; Ohnishi et al., 2014; Yamanaka et al., 2010). Fgf4-Fgfr-ERK signalling determines the balance of the EPI and PrE cell lineages: over-activation of Fgf signalling caused by exogenous FGF4 converts all ICM cells to PrE cells ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1242/dev.043471", "ISSN" : "1477-9129", "PMID" : "20147376", "abstract" : "Primitive endoderm (PE) and epiblast (EPI) are two lineages derived from the inner cell mass (ICM) of the E3.5 blastocyst. Recent studies showed that EPI and PE progenitors expressing the lineage-specific transcriptional factors Nanog and Gata6, respectively, arise progressively as the ICM develops. Subsequent sorting of the two progenitors during blastocyst maturation results in the ormation of morphologically distinct EPI and PE layers at E4.5. It is, however, unknown how the initial differences between the two populations become established in the E3.5 blastocyst. Because the ICM cells are derived from two distinct rounds of polarized cell divisions during cleavage, a possible role for cell lineage history in promoting EPI versus PE fate has been proposed. We followed cell lineage from the eight-cell stage by live cell tracing and could find no clear linkage between developmental history of individual ICM cells and later cell fate. However, modulating FGF signaling levels by inhibition of the receptor/MAP kinase pathway or by addition of exogenous FGF shifted the fate of ICM cells to become either EPI or PE, respectively. Nanog- or Gata6-expressing progenitors could still be shifted towards the alternative fate by modulating FGF signaling during blastocyst maturation, suggesting that the ICM progenitors are not fully committed to their final fate at the time that initial segregation of gene expression occurs. In conclusion, we propose a model in which stochastic and progressive specification of EPI and PE lineages occurs during maturation of the blastocyst in an FGF/MAP kinase signal-dependent manner.", "author" : [ { "dropping-particle" : "", "family" : "Yamanaka", "given" : "Yojiro", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Lanner", "given" : "Fredrik", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Rossant", "given" : "Janet", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Development (Cambridge, England)", "id" : "ITEM-1", "issue" : "5", "issued" : { "date-parts" : [ [ "2010", "3" ] ] }, "page" : "715-24", "title" : "FGF signal-dependent segregation of primitive endoderm and epiblast in the mouse blastocyst.", "type" : "article-journal", "volume" : "137" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(Yamanaka et al., 2010)", "plainTextFormattedCitation" : "(Yamanaka et al., 2010)", "previouslyFormattedCitation" : "(Yamanaka et al., 2010)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(Yamanaka et al., 2010), while all ICM cells acquire EPI identity when Fgf signalling is blocked by small chemical inhibitors for Fgfr and ERK ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1242/dev.038893", "ISSN" : "1477-9129", "PMID" : "19710168", "abstract" : "Embryonic stem (ES) cells can be derived and propagated from multiple strains of mouse and rat through application of small-molecule inhibitors of the fibroblast growth factor (FGF)/Erk pathway and of glycogen synthase kinase 3. These conditions shield pluripotent cells from differentiation-inducing stimuli. We investigate the effect of these inhibitors on the development of pluripotent epiblast in intact pre-implantation embryos. We find that blockade of Erk signalling from the 8-cell stage does not impede blastocyst formation but suppresses development of the hypoblast. The size of the inner cell mass (ICM) compartment is not reduced, however. Throughout the ICM, the epiblast-specific marker Nanog is expressed, and in XX embryos epigenetic silencing of the paternal X chromosome is erased. Epiblast identity and pluripotency were confirmed by contribution to chimaeras with germline transmission. These observations indicate that segregation of hypoblast from the bipotent ICM is dependent on FGF/Erk signalling and that in the absence of this signal, the entire ICM can acquire pluripotency. Furthermore, the epiblast does not require paracrine support from the hypoblast. Thus, na\u00efve epiblast and ES cells are in a similar ground state, with an autonomous capacity for survival and replication, and high vulnerability to Erk signalling. We probed directly the relationship between na\u00efve epiblast and ES cells. Dissociated ICM cells from freshly harvested late blastocysts gave rise to up to 12 ES cell clones per embryo when plated in the presence of inhibitors. We propose that ES cells are not a tissue culture creation, but are essentially identical to pre-implantation epiblast cells.", "author" : [ { "dropping-particle" : "", "family" : "Nichols", "given" : "Jennifer", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Silva", "given" : "Jose", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Roode", "given" : "Mila", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Smith", "given" : "Austin", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Development (Cambridge, England)", "id" : "ITEM-1", "issue" : "19", "issued" : { "date-parts" : [ [ "2009", "10" ] ] }, "page" : "3215-22", "title" : "Suppression of Erk signalling promotes ground state pluripotency in the mouse embryo.", "type" : "article-journal", "volume" : "136" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(Nichols et al., 2009)", "plainTextFormattedCitation" : "(Nichols et al., 2009)", "previouslyFormattedCitation" : "(Nichols et al., 2009)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(Nichols et al., 2009), by gene knockout (KO) of Fgf4 ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1242/dev.084996", "ISSN" : "1477-9129", "PMID" : "23193166", "abstract" : "The emergence of pluripotent epiblast (EPI) and primitive endoderm (PrE) lineages within the inner cell mass (ICM) of the mouse blastocyst involves initial co-expression of lineage-associated markers followed by mutual exclusion and salt-and-pepper distribution of lineage-biased cells. Precisely how EPI and PrE cell fate commitment occurs is not entirely clear; however, previous studies in mice have implicated FGF/ERK signaling in this process. Here, we investigated the phenotype resulting from zygotic and maternal/zygotic inactivation of Fgf4. Fgf4 heterozygous blastocysts exhibited increased numbers of NANOG-positive EPI cells and reduced numbers of GATA6-positive PrE cells, suggesting that FGF signaling is tightly regulated to ensure specification of the appropriate numbers of cells for each lineage. Although the size of the ICM was unaffected in Fgf4 null mutant embryos, it entirely lacked a PrE layer and exclusively comprised NANOG-expressing cells at the time of implantation. An initial period of widespread EPI and PrE marker co-expression was however established even in the absence of FGF4. Thus, Fgf4 mutant embryos initiated the PrE program but exhibited defects in its restriction phase, when lineage bias is acquired. Consistent with this, XEN cells could be derived from Fgf4 mutant embryos in which PrE had been restored and these cells appeared indistinguishable from wild-type cells. Sustained exogenous FGF failed to rescue the mutant phenotype. Instead, depending on concentration, we noted no effect or conversion of all ICM cells to GATA6-positive PrE. We propose that heterogeneities in the availability of FGF produce the salt-and-pepper distribution of lineage-biased cells.", "author" : [ { "dropping-particle" : "", "family" : "Kang", "given" : "Minjung", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Piliszek", "given" : "Anna", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Artus", "given" : "J\u00e9r\u00f4me", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Hadjantonakis", "given" : "Anna-Katerina", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Development (Cambridge, England)", "id" : "ITEM-1", "issue" : "2", "issued" : { "date-parts" : [ [ "2013", "1", "15" ] ] }, "page" : "267-79", "title" : "FGF4 is required for lineage restriction and salt-and-pepper distribution of primitive endoderm factors but not their initial expression in the mouse.", "type" : "article-journal", "volume" : "140" }, "uris" : [ "" ] }, { "id" : "ITEM-2", "itemData" : { "DOI" : "10.1016/j.ydbio.2013.09.023", "ISSN" : "1095-564X", "PMID" : "24063807", "abstract" : "The primitive endoderm (PE) and epiblast (EPI) are two lineages derived from the inner cell mass (ICM) of the E3.5 blastocyst. Although it has been shown that FGF signaling is necessary and sufficient for PE specification in the ICM, it is unknown what mechanisms control the PE/EPI proportion in the embryo. Because modulation of FGF signaling alone is sufficient to convert all ICM cells to either PE or EPI, a model has been proposed in which the amount of FGF in the embryo controls the PE/EPI proportion. To test this model, we reduced the amount of FGF4, the major FGF in the preimplantation embryo, using various genotypes of Fgf4 mutants. We observed a maternal contribution of Fgf4 in PE specification, but it was dispensable for development. In addition, upon treatment of Fgf4 mutant embryos with exogenous FGF4, we observed a progressive increase of PE proportions in an FGF4 dose dependent manner, regardless of embryo genotype. We conclude that the amount of FGF4 is limited and regulates PE/EPI proportions in the mouse embryo.", "author" : [ { "dropping-particle" : "", "family" : "Krawchuk", "given" : "Dayana", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Honma-Yamanaka", "given" : "Nobuko", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Anani", "given" : "Shihadeh", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Yamanaka", "given" : "Yojiro", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Developmental biology", "id" : "ITEM-2", "issue" : "1", "issued" : { "date-parts" : [ [ "2013", "12", "1" ] ] }, "page" : "65-71", "publisher" : "Elsevier", "title" : "FGF4 is a limiting factor controlling the proportions of primitive endoderm and epiblast in the ICM of the mouse blastocyst.", "type" : "article-journal", "volume" : "384" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(Kang et al., 2013; Krawchuk et al., 2013)", "plainTextFormattedCitation" : "(Kang et al., 2013; Krawchuk et al., 2013)", "previouslyFormattedCitation" : "(Kang et al., 2013; Krawchuk et al., 2013)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(Kang et al., 2013; Krawchuk et al., 2013), and by KO of the expression of the adapter molecule Grb2 ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1016/j.devcel.2006.02.020", "ISBN" : "1534-5807 (Print)\\r1534-5807 (Linking)", "ISSN" : "1534-5807", "PMID" : "16678776", "abstract" : "It has been thought that early inner cell mass (ICM) is a homogeneous population and that cell position in the ICM leads to the formation of two lineages, epiblast (EPI) and primitive endoderm (PE), by E4.5. Here, however, we show that the ICM at E3.5 is already heterogeneous. The EPI- and PE-specific transcription factors, Nanog and Gata6, were expressed in the ICM in a random \"salt and pepper\" pattern, as early as E3.5, in a mutually exclusive manner. Lineage tracing showed predominant lineage restriction of single ICM cells at E3.5 to either lineage. In embryos lacking Grb2 where no PE forms, Gata6 expression was lost and all ICM cells were Nanog positive. We propose a model in which the ICM develops as a mosaic of EPI and PE progenitors at E3.5, dependent on Grb2-Ras-MAP kinase signaling, followed by later segregation of the progenitors into the appropriate cell layers.", "author" : [ { "dropping-particle" : "", "family" : "Chazaud", "given" : "Claire", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Yamanaka", "given" : "Yojiro", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Pawson", "given" : "Tony", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Rossant", "given" : "Janet", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Developmental cell", "id" : "ITEM-1", "issue" : "5", "issued" : { "date-parts" : [ [ "2006", "5" ] ] }, "page" : "615-24", "title" : "Early lineage segregation between epiblast and primitive endoderm in mouse blastocysts through the Grb2-MAPK pathway.", "type" : "article-journal", "volume" : "10" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(Chazaud et al., 2006)", "plainTextFormattedCitation" : "(Chazaud et al., 2006)", "previouslyFormattedCitation" : "(Chazaud et al., 2006)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(Chazaud et al., 2006). Fgf4 KO embryos show normal Nanog and Gata6 expression levels at E3.25; thus, the initial co-expression of Nanog and Gata6 itself is independent of Fgf4 ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1242/dev.084996", "ISSN" : "1477-9129", "PMID" : "23193166", "abstract" : "The emergence of pluripotent epiblast (EPI) and primitive endoderm (PrE) lineages within the inner cell mass (ICM) of the mouse blastocyst involves initial co-expression of lineage-associated markers followed by mutual exclusion and salt-and-pepper distribution of lineage-biased cells. Precisely how EPI and PrE cell fate commitment occurs is not entirely clear; however, previous studies in mice have implicated FGF/ERK signaling in this process. Here, we investigated the phenotype resulting from zygotic and maternal/zygotic inactivation of Fgf4. Fgf4 heterozygous blastocysts exhibited increased numbers of NANOG-positive EPI cells and reduced numbers of GATA6-positive PrE cells, suggesting that FGF signaling is tightly regulated to ensure specification of the appropriate numbers of cells for each lineage. Although the size of the ICM was unaffected in Fgf4 null mutant embryos, it entirely lacked a PrE layer and exclusively comprised NANOG-expressing cells at the time of implantation. An initial period of widespread EPI and PrE marker co-expression was however established even in the absence of FGF4. Thus, Fgf4 mutant embryos initiated the PrE program but exhibited defects in its restriction phase, when lineage bias is acquired. Consistent with this, XEN cells could be derived from Fgf4 mutant embryos in which PrE had been restored and these cells appeared indistinguishable from wild-type cells. Sustained exogenous FGF failed to rescue the mutant phenotype. Instead, depending on concentration, we noted no effect or conversion of all ICM cells to GATA6-positive PrE. We propose that heterogeneities in the availability of FGF produce the salt-and-pepper distribution of lineage-biased cells.", "author" : [ { "dropping-particle" : "", "family" : "Kang", "given" : "Minjung", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Piliszek", "given" : "Anna", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Artus", "given" : "J\u00e9r\u00f4me", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Hadjantonakis", "given" : "Anna-Katerina", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Development (Cambridge, England)", "id" : "ITEM-1", "issue" : "2", "issued" : { "date-parts" : [ [ "2013", "1", "15" ] ] }, "page" : "267-79", "title" : "FGF4 is required for lineage restriction and salt-and-pepper distribution of primitive endoderm factors but not their initial expression in the mouse.", "type" : "article-journal", "volume" : "140" }, "uris" : [ "" ] }, { "id" : "ITEM-2", "itemData" : { "DOI" : "10.1016/j.ydbio.2013.09.023", "ISSN" : "1095-564X", "PMID" : "24063807", "abstract" : "The primitive endoderm (PE) and epiblast (EPI) are two lineages derived from the inner cell mass (ICM) of the E3.5 blastocyst. Although it has been shown that FGF signaling is necessary and sufficient for PE specification in the ICM, it is unknown what mechanisms control the PE/EPI proportion in the embryo. Because modulation of FGF signaling alone is sufficient to convert all ICM cells to either PE or EPI, a model has been proposed in which the amount of FGF in the embryo controls the PE/EPI proportion. To test this model, we reduced the amount of FGF4, the major FGF in the preimplantation embryo, using various genotypes of Fgf4 mutants. We observed a maternal contribution of Fgf4 in PE specification, but it was dispensable for development. In addition, upon treatment of Fgf4 mutant embryos with exogenous FGF4, we observed a progressive increase of PE proportions in an FGF4 dose dependent manner, regardless of embryo genotype. We conclude that the amount of FGF4 is limited and regulates PE/EPI proportions in the mouse embryo.", "author" : [ { "dropping-particle" : "", "family" : "Krawchuk", "given" : "Dayana", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Honma-Yamanaka", "given" : "Nobuko", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Anani", "given" : "Shihadeh", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Yamanaka", "given" : "Yojiro", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Developmental biology", "id" : "ITEM-2", "issue" : "1", "issued" : { "date-parts" : [ [ "2013", "12", "1" ] ] }, "page" : "65-71", "publisher" : "Elsevier", "title" : "FGF4 is a limiting factor controlling the proportions of primitive endoderm and epiblast in the ICM of the mouse blastocyst.", "type" : "article-journal", "volume" : "384" }, "uris" : [ "" ] }, { "id" : "ITEM-3", "itemData" : { "DOI" : "10.1038/ncb2881", "ISSN" : "1476-4679", "PMID" : "24292013", "abstract" : "It is now recognized that extensive expression heterogeneities among cells precede the emergence of lineages in the early mammalian embryo. To establish a map of pluripotent epiblast (EPI) versus primitive endoderm (PrE) lineage segregation within the inner cell mass (ICM) of the mouse blastocyst, we characterized the gene expression profiles of individual ICM cells. Clustering analysis of the transcriptomes of 66 cells demonstrated that initially they are non-distinguishable. Early in the segregation, lineage-specific marker expression exhibited no apparent correlation, and a hierarchical relationship was established only in the late blastocyst. Fgf4 exhibited a bimodal expression at the earliest stage analysed, and in its absence, the differentiation of PrE and EPI was halted, indicating that Fgf4 drives, and is required for, ICM lineage segregation. These data lead us to propose a model where stochastic cell-to-cell expression heterogeneity followed by signal reinforcement underlies ICM lineage segregation by antagonistically separating equivalent cells.", "author" : [ { "dropping-particle" : "", "family" : "Ohnishi", "given" : "Yusuke", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Huber", "given" : "Wolfgang", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Tsumura", "given" : "Akiko", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Kang", "given" : "Minjung", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Xenopoulos", "given" : "Panagiotis", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Kurimoto", "given" : "Kazuki", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Ole\u015b", "given" : "Andrzej K.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Ara\u00fazo-Bravo", "given" : "Marcos J.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Saitou", "given" : "Mitinori", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Hadjantonakis", "given" : "Anna-Katerina", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Hiiragi", "given" : "Takashi", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Nature cell biology", "id" : "ITEM-3", "issue" : "1", "issued" : { "date-parts" : [ [ "2014", "1" ] ] }, "page" : "27-37", "publisher" : "Nature Publishing Group", "title" : "Cell-to-cell expression variability followed by signal reinforcement progressively segregates early mouse lineages.", "type" : "article-journal", "volume" : "16" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(Kang et al., 2013; Krawchuk et al., 2013; Ohnishi et al., 2014)", "plainTextFormattedCitation" : "(Kang et al., 2013; Krawchuk et al., 2013; Ohnishi et al., 2014)", "previouslyFormattedCitation" : "(Kang et al., 2013; Krawchuk et al., 2013; Ohnishi et al., 2014)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(Kang et al., 2013; Krawchuk et al., 2013; Ohnishi et al., 2014). Subsequently, the segregation of EPI and PrE precursor cells is believed to be mediated by reciprocal repression between Nanog and Gata6 ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1634/stemcells.2007-0126", "ISSN" : "1549-4918", "PMID" : "17615266", "abstract" : "Nanog is a critical homeodomain factor responsible for maintaining embryonic stem (ES) cell self-renewal and pluripotency. Of interest, Nanog expression is not homogeneous in the conventional culture of murine ES cells. A Nanog-high population expresses markers for pluripotent ES cells, whereas a Nanog-low population expresses markers for primitive endoderm, such as Gata6. Since the inner cell mass of early blastocysts has recently been reported to be heterogeneous in terms of Nanog and Gata6 expression, ES cells appear to closely resemble the developing stage from which they originate. We further demonstrate that Nanog can directly repress Gata6 expression through its binding to the proximal promoter region of the Gata6 gene and that overexpression of Nanog reduces heterogeneity during ES cell maintenance. Interestingly, Nanog heterogeneity does not correlate with the heterogeneous expression of stage-specific embryonic antigen-1, suggesting that multiple but overlapping levels of heterogeneity may exist in ES cells. These findings provide insight into the factors that control ES cell self-renewal and the earliest lineage commitment to primitive endoderm while also suggesting methods to promote homogeneity during ES cell maintenance. Disclosure of potential conflicts of interest is found at the end of this article.", "author" : [ { "dropping-particle" : "", "family" : "Singh", "given" : "Amar M.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Hamazaki", "given" : "Takashi", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Hankowski", "given" : "Katherine E.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Terada", "given" : "Naohiro", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Stem cells (Dayton, Ohio)", "id" : "ITEM-1", "issue" : "10", "issued" : { "date-parts" : [ [ "2007", "10" ] ] }, "page" : "2534-42", "title" : "A heterogeneous expression pattern for Nanog in embryonic stem cells.", "type" : "article-journal", "volume" : "25" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(Singh et al., 2007)", "plainTextFormattedCitation" : "(Singh et al., 2007)", "previouslyFormattedCitation" : "(Singh et al., 2007)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(Singh et al., 2007). In agreement with this model, the loss of Gata6 results in a complete shift into the EPI lineage, while there is no effect on the initial Nanog expression level at E3.0–3.25; thus, the initial expression of Nanog expression is independent of Gata6 at E3.0 – 3.25. ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1242/dev.109678", "ISSN" : "1477-9129", "PMID" : "25209243", "abstract" : "During blastocyst formation, inner cell mass (ICM) cells differentiate into either epiblast (Epi) or primitive endoderm (PrE) cells, labeled by Nanog and Gata6, respectively, and organized in a salt-and-pepper pattern. Previous work in the mouse has shown that, in absence of Nanog, all ICM cells adopt a PrE identity. Moreover, the activation or the blockade of the Fgf/RTK pathway biases cell fate specification towards either PrE or Epi, respectively. We show that, in absence of Gata6, all ICM cells adopt an Epi identity. Furthermore, the analysis of Gata6(+/-) embryos reveals a dose-sensitive phenotype, with fewer PrE-specified cells. These results and previous findings have enabled the development of a mathematical model for the dynamics of the regulatory network that controls ICM differentiation into Epi or PrE cells. The model describes the temporal dynamics of Erk signaling and of the concentrations of Nanog, Gata6, secreted Fgf4 and Fgf receptor 2. The model is able to recapitulate most of the cell behaviors observed in different experimental conditions and provides a unifying mechanism for the dynamics of these developmental transitions. The mechanism relies on the co-existence between three stable steady states (tristability), which correspond to ICM, Epi and PrE cells, respectively. Altogether, modeling and experimental results uncover novel features of ICM cell fate specification such as the role of the initial induction of a subset of cells into Epi in the initiation of the salt-and-pepper pattern, or the precocious Epi specification in Gata6(+/-) embryos.", "author" : [ { "dropping-particle" : "", "family" : "Bessonnard", "given" : "Sylvain", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Mot", "given" : "Laurane", "non-dropping-particle" : "De", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Gonze", "given" : "Didier", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Barriol", "given" : "Manon", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Dennis", "given" : "Cynthia", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Goldbeter", "given" : "Albert", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Dupont", "given" : "Genevi\u00e8ve", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Chazaud", "given" : "Claire", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Development (Cambridge, England)", "id" : "ITEM-1", "issue" : "19", "issued" : { "date-parts" : [ [ "2014", "10" ] ] }, "page" : "3637-48", "title" : "Gata6, Nanog and Erk signaling control cell fate in the inner cell mass through a tristable regulatory network.", "type" : "article-journal", "volume" : "141" }, "uris" : [ "" ] }, { "id" : "ITEM-2", "itemData" : { "DOI" : "10.1016/j.devcel.2014.04.011", "ISSN" : "1878-1551", "PMID" : "24835466", "abstract" : "Cells of the inner cell mass (ICM) of the mouse blastocyst differentiate into the pluripotent epiblast or the primitive endoderm (PrE), marked by the transcription factors NANOG and GATA6, respectively. To investigate the mechanistic regulation of this process, we applied an unbiased, quantitative, single-cell-resolution image analysis pipeline to analyze embryos lacking or exhibiting reduced levels of GATA6. We find that Gata6 mutants exhibit a complete absence of PrE and demonstrate that GATA6 levels regulate the timing and speed of lineage commitment within the ICM. Furthermore, we show that GATA6 is necessary for PrE specification by FGF signaling and propose a model where interactions between NANOG, GATA6, and the FGF/ERK pathway determine ICM cell fate. This study provides a framework for quantitative analyses of mammalian embryos and establishes GATA6 as a nodal point in the gene regulatory network driving ICM lineage specification.", "author" : [ { "dropping-particle" : "", "family" : "Schrode", "given" : "Nadine", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Saiz", "given" : "N\u00e9stor", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Talia", "given" : "Stefano", "non-dropping-particle" : "Di", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Hadjantonakis", "given" : "Anna-Katerina", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Developmental cell", "id" : "ITEM-2", "issue" : "4", "issued" : { "date-parts" : [ [ "2014", "5", "27" ] ] }, "page" : "454-67", "title" : "GATA6 levels modulate primitive endoderm cell fate choice and timing in the mouse blastocyst.", "type" : "article-journal", "volume" : "29" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(Bessonnard et al., 2014; Schrode et al., 2014)", "plainTextFormattedCitation" : "(Bessonnard et al., 2014; Schrode et al., 2014)", "previouslyFormattedCitation" : "(Bessonnard et al., 2014; Schrode et al., 2014)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(Bessonnard et al., 2014; Schrode et al., 2014). Importantly, single-cell analysis showed that bimodal Fgf4 expression precedes asymmetric Nanog and Gata6 expression and is the first sign of the segregation of the EPI and PrE lineages ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1016/j.devcel.2010.02.012", "ISSN" : "1878-1551", "PMID" : "20412781", "abstract" : "Three distinct cell types are present within the 64-cell stage mouse blastocyst. We have investigated cellular development up to this stage using single-cell expression analysis of more than 500 cells. The 48 genes analyzed were selected in part based on a whole-embryo analysis of more than 800 transcription factors. We show that in the morula, blastomeres coexpress transcription factors specific to different lineages, but by the 64-cell stage three cell types can be clearly distinguished according to their quantitative expression profiles. We identify Id2 and Sox2 as the earliest markers of outer and inner cells, respectively. This is followed by an inverse correlation in expression for the receptor-ligand pair Fgfr2/Fgf4 in the early inner cell mass. Position and signaling events appear to precede the maturation of the transcriptional program. These results illustrate the power of single-cell expression analysis to provide insight into developmental mechanisms. 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To establish a map of pluripotent epiblast (EPI) versus primitive endoderm (PrE) lineage segregation within the inner cell mass (ICM) of the mouse blastocyst, we characterized the gene expression profiles of individual ICM cells. Clustering analysis of the transcriptomes of 66 cells demonstrated that initially they are non-distinguishable. Early in the segregation, lineage-specific marker expression exhibited no apparent correlation, and a hierarchical relationship was established only in the late blastocyst. Fgf4 exhibited a bimodal expression at the earliest stage analysed, and in its absence, the differentiation of PrE and EPI was halted, indicating that Fgf4 drives, and is required for, ICM lineage segregation. These data lead us to propose a model where stochastic cell-to-cell expression heterogeneity followed by signal reinforcement underlies ICM lineage segregation by antagonistically separating equivalent cells.", "author" : [ { "dropping-particle" : "", "family" : "Ohnishi", "given" : "Yusuke", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Huber", "given" : "Wolfgang", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Tsumura", "given" : "Akiko", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Kang", "given" : "Minjung", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Xenopoulos", "given" : "Panagiotis", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Kurimoto", "given" : "Kazuki", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Ole\u015b", "given" : "Andrzej K.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Ara\u00fazo-Bravo", "given" : "Marcos J.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Saitou", "given" : "Mitinori", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Hadjantonakis", "given" : "Anna-Katerina", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Hiiragi", "given" : "Takashi", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Nature cell biology", "id" : "ITEM-2", "issue" : "1", "issued" : { "date-parts" : [ [ "2014", "1" ] ] }, "page" : "27-37", "publisher" : "Nature Publishing Group", "title" : "Cell-to-cell expression variability followed by signal reinforcement progressively segregates early mouse lineages.", "type" : "article-journal", "volume" : "16" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(Guo et al., 2010; Ohnishi et al., 2014)", "plainTextFormattedCitation" : "(Guo et al., 2010; Ohnishi et al., 2014)", "previouslyFormattedCitation" : "(Guo et al., 2010; Ohnishi et al., 2014)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(Guo et al., 2010; Ohnishi et al., 2014). Currently, what regulates Fgf4 at this developmental stage is unknown ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1007/s00018-014-1630-3", "ISBN" : "1420-9071 (Electronic)\\r1420-682X (Linking)", "ISSN" : "1420-9071", "PMID" : "24794628", "abstract" : "During early development, the mammalian embryo undergoes a series of profound changes that lead to the formation of two extraembryonic tissues--the trophectoderm and the primitive endoderm. These tissues encapsulate the pluripotent epiblast at the time of implantation. The current model proposes that the formation of these lineages results from two consecutive binary cell fate decisions. The first controls the formation of the trophectoderm and the inner cell mass, and the second controls the formation of the primitive endoderm and the epiblast within the inner cell mass. While early mammalian embryos develop with extensive plasticity, the embryonic pattern prior to implantation is remarkably reproducible. Here, we review the molecular mechanisms driving the cell fate decision between primitive endoderm and epiblast in the mouse embryo and integrate data from recent studies into the current model of the molecular network regulating the segregation between these lineages and their subsequent differentiation.", "author" : [ { "dropping-particle" : "", "family" : "Artus", "given" : "J\u00e9r\u00f4me", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Chazaud", "given" : "Claire", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Cellular and molecular life sciences : CMLS", "id" : "ITEM-1", "issue" : "17", "issued" : { "date-parts" : [ [ "2014", "9" ] ] }, "page" : "3327-38", "title" : "A close look at the mammalian blastocyst: epiblast and primitive endoderm formation.", "type" : "article-journal", "volume" : "71" }, "uris" : [ "" ] }, { "id" : "ITEM-2", "itemData" : { "DOI" : "10.1242/dev.128314", "ISSN" : "1477-9129", "PMID" : "27048685", "abstract" : "During mouse preimplantation embryo development, totipotent blastomeres generate the first three cell lineages of the embryo: trophectoderm, epiblast and primitive endoderm. In recent years, studies have shown that this process appears to be regulated by differences in cell-cell interactions, gene expression and the microenvironment of individual cells, rather than the active partitioning of maternal determinants. Precisely how these differences first emerge and how they dictate subsequent molecular and cellular behaviours are key questions in the field. As we review here, recent advances in live imaging, computational modelling and single-cell transcriptome analyses are providing new insights into these questions.", "author" : [ { "dropping-particle" : "", "family" : "Chazaud", "given" : "Claire", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Yamanaka", "given" : "Yojiro", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Development (Cambridge, England)", "id" : "ITEM-2", "issue" : "7", "issued" : { "date-parts" : [ [ "2016", "4", "1" ] ] }, "page" : "1063-74", "publisher" : "Oxford University Press for The Company of Biologists Limited", "title" : "Lineage specification in the mouse preimplantation embryo.", "type" : "article-journal", "volume" : "143" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(Artus and Chazaud, 2014; Chazaud and Yamanaka, 2016)", "plainTextFormattedCitation" : "(Artus and Chazaud, 2014; Chazaud and Yamanaka, 2016)", "previouslyFormattedCitation" : "(Artus and Chazaud, 2014; Chazaud and Yamanaka, 2016)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(Artus and Chazaud, 2014; Chazaud and Yamanaka, 2016). Klf5, a member of the Krüppel-like factor (Klf) family of transcription factors, functions in the maintenance of pluripotency and in somatic cell reprogramming ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1016/j.cell.2006.07.024", "ISSN" : "0092-8674", "PMID" : "16904174", "abstract" : "Differentiated cells can be reprogrammed to an embryonic-like state by transfer of nuclear contents into oocytes or by fusion with embryonic stem (ES) cells. Little is known about factors that induce this reprogramming. Here, we demonstrate induction of pluripotent stem cells from mouse embryonic or adult fibroblasts by introducing four factors, Oct3/4, Sox2, c-Myc, and Klf4, under ES cell culture conditions. Unexpectedly, Nanog was dispensable. These cells, which we designated iPS (induced pluripotent stem) cells, exhibit the morphology and growth properties of ES cells and express ES cell marker genes. Subcutaneous transplantation of iPS cells into nude mice resulted in tumors containing a variety of tissues from all three germ layers. Following injection into blastocysts, iPS cells contributed to mouse embryonic development. These data demonstrate that pluripotent stem cells can be directly generated from fibroblast cultures by the addition of only a few defined factors.", "author" : [ { "dropping-particle" : "", "family" : "Takahashi", "given" : "Kazutoshi", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Yamanaka", "given" : "Shinya", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Cell", "id" : "ITEM-1", "issue" : "4", "issued" : { "date-parts" : [ [ "2006", "8", "25" ] ] }, "page" : "663-76", "publisher" : "Elsevier Inc.", "title" : "Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.", "type" : "article-journal", "volume" : "126" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(Takahashi and Yamanaka, 2006)", "plainTextFormattedCitation" : "(Takahashi and Yamanaka, 2006)", "previouslyFormattedCitation" : "(Takahashi and Yamanaka, 2006)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(Takahashi and Yamanaka, 2006). Klf5 marks a na?ve state of human pluripotent stem cells ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1016/j.stem.2013.11.015", "ISBN" : "1875-9777 (Electronic)", "ISSN" : "1875-9777", "PMID" : "24315441", "abstract" : "Human embryonic stem cells (hESCs) are derived from the inner cell mass of the blastocyst. Despite sharing the common property of pluripotency, hESCs are notably distinct from epiblast cells of the preimplantation blastocyst. Here we use a combination of three small-molecule inhibitors to sustain hESCs in a LIF signaling-dependent hESC state (3iL hESCs) with elevated expression of NANOG and epiblast-enriched genes such as KLF4, DPPA3, and TBX3. Genome-wide transcriptome analysis confirms that the expression signature of 3iL hESCs shares similarities with native preimplantation epiblast cells. We also show that 3iL hESCs have a distinct epigenetic landscape, characterized by derepression of preimplantation epiblast genes. Using genome-wide binding profiles of NANOG and OCT4, we identify enhancers that contribute to rewiring of the regulatory circuitry. In summary, our study identifies a distinct hESC state with defined regulatory circuitry that will facilitate future analysis of human preimplantation embryogenesis and pluripotency.", "author" : [ { "dropping-particle" : "", "family" : "Chan", "given" : "Yun-Shen", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "G\u00f6ke", "given" : "Jonathan", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Ng", "given" : "Jia-Hui", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Lu", "given" : "Xinyi", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Gonzales", "given" : "Kevin Andrew Uy", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Tan", "given" : "Cheng-Peow", "non-dropping-particle" : "", 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Target gene inactivation of Klf5 causes failure of TE and ICM development ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1016/j.stem.2008.09.003", "ISBN" : "1875-9777 (Electronic)", "ISSN" : "1875-9777", "PMID" : "18983969", "abstract" : "The transcription factor Klf4 has demonstrated activity in the reprogramming of somatic cells to a pluripotent state, but the molecular mechanism of this process remains unknown. It is, therefore, of great interest to understand the functional role of Klf4 and related genes in ESC regulation. Here, we show that homozygous disruption of Klf5 results in the failure of ESC derivation from ICM cells and early embryonic lethality due to an implantation defect. Klf5 KO ESCs show increased expression of several differentiation marker genes and frequent, spontaneous differentiation. Conversely, overexpression of Klf5 in ESCs suppressed the expression of differentiation marker genes and maintained pluripotency in the absence of LIF. Our results also suggest that Klf5 regulates ESC proliferation by promoting phosphorylation of Akt1 via induction of Tcl1. These results, therefore, provide new insights into the functional and mechanistic role of Klf5 in regulation of pluripotency.", "author" : [ { "dropping-particle" : "", "family" : "Ema", "given" : "Masatsugu", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Mori", "given" : "Daisuke", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Niwa", "given" : "Hitoshi", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Hasegawa", "given" : "Yoshikazu", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Yamanaka", "given" : "Yojiro", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Hitoshi", "given" : "Seiji", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Mimura", "given" : "Junsei", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Kawabe", "given" : "Yoh-ichi", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Hosoya", "given" : "Tomohiro", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Morita", "given" : "Masanobu", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Shimosato", "given" : "Daisuke", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Uchida", "given" : "Kazuhiko", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Suzuki", "given" : "Norio", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Yanagisawa", "given" : "Jun", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Sogawa", "given" : "Kazuhiro", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Rossant", "given" : "Janet", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Yamamoto", "given" : "Masayuki", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Takahashi", "given" : "Satoru", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Fujii-Kuriyama", "given" : "Yoshiaki", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Cell stem cell", "id" : "ITEM-1", "issue" : "5", "issued" : { "date-parts" : [ [ "2008", "11", "6" ] ] }, "page" : "555-67", "publisher" : "Elsevier Inc.", "title" : "Kr\u00fcppel-like factor 5 is essential for blastocyst development and the normal self-renewal of mouse ESCs.", "type" : "article-journal", "volume" : "3" }, "uris" : [ "" ] }, { "id" : "ITEM-2", "itemData" : { "DOI" : "10.1242/dev.054775", "ISSN" : "1477-9129", "PMID" : "20980403", "abstract" : "Kruppel-like transcription factors (Klfs) are essential for the induction and maintenance of pluripotency of embryonic stem cells (ESCs), yet little is known about their roles in establishing the three lineages of the pre-implantation embryo. Here, we show that Klf5 is required for the formation of the trophectoderm (TE) and the inner cell mass (ICM), and for repressing primitive endoderm (PE) development. Although cell polarity appeared normal, Klf5 mutant embryos arrested at the blastocyst stage and failed to hatch due to defective TE development. Klf5 acted cell-autonomously in the TE, downstream of Fgf4 and upstream of Cdx2, Eomes and Krt8. In the ICM, loss of Klf5 resulted in reduced expression of pluripotency markers Oct4 and Nanog, but led to increased Sox17 expression in the PE, suggesting that Klf5 suppresses the PE lineage. Consistent with this, overexpression of Klf5 in transgenic embryos was sufficient to suppress the Sox17(+) PE lineage in the ICM. Klf5 overexpression led to a dose-dependent decrease in Sox17 promoter activity in reporter assays in cultured cells. Moreover, in chimeric embryos, Klf5(-/-) cells preferentially contributed to the Sox17(+) PE lineage and Cdx2 expression was not rescued in Klf5(-/-) outer cells. Finally, outgrowths from Klf5(-/-) embryos failed to form an ICM/pluripotent colony, had very few Oct4(+) or Cdx2(+) cells, but showed an increase in the percentage of Sox17(+) PE cells. These findings demonstrate that Klf5 is a dynamic regulator of all three lineages in the pre-implantation embryo by promoting the TE and epiblast lineages while suppressing the PE lineage.", "author" : [ { "dropping-particle" : "", "family" : "Lin", "given" : "Suh-Chin J", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Wani", "given" : "Maqsood a", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Whitsett", "given" : "Jeffrey a", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Wells", "given" : "James M", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Development (Cambridge, England)", "id" : "ITEM-2", "issue" : "23", "issued" : { "date-parts" : [ [ "2010", "12" ] ] }, "page" : "3953-63", "title" : "Klf5 regulates lineage formation in the pre-implantation mouse embryo.", "type" : "article-journal", "volume" : "137" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(Ema et al., 2008; Lin et al., 2010)", "plainTextFormattedCitation" : "(Ema et al., 2008; Lin et al., 2010)", "previouslyFormattedCitation" : "(Ema et al., 2008; Lin et al., 2010)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(Ema et al., 2008; Lin et al., 2010), while the molecular mechanism underlying Klf5-regulated ICM development is not well understood.Here, we show that the inner cells of Klf5 KO embryos adopt a PrE lineage fate at the expense of EPI cells, while Klf5 overexpressing (OE) embryos show incomplete lineage segregation as indicated by the persistence of Nanog+/Gata6+ (double-positive; DP) cells. We show that Fgf4 expression is upregulated in Klf5 KO embryos at E3.0, whereas Fgf4 is repressed in Klf5 OE blastocysts. Importantly, single-cell analysis clearly demonstrates that Fgf4 is derepressed in a subset of Fgf4-high inner cells of Klf5 KO embryos. Chromatin immunoprecipitation (ChIP) assays indicate that the Fgf4 locus is occupied by Klf5, suggesting direct regulation of Fgf4 by Klf5. In terms of the emergence of Nanog+ pluripotent EPI cells, the phenotypes of Klf5 KO embryos can be reversed by either Fgfr or ERK inhibition. Taken together, these results provide new insights into the critical interplay between Klf5 and the Fgf4-Fgfr-ERK pathway for the proper lineage segregation.ResultsSkewed EPI and PrE lineage specification in Klf5 KO and OE blastocysts Although Klf5 is indispensable for blastocyst development, the mechanistic functions of Klf5 in ICM development and early lineage segregation remain elusive ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1016/j.stem.2008.09.003", "ISBN" : "1875-9777 (Electronic)", "ISSN" : "1875-9777", "PMID" : "18983969", "abstract" : "The transcription factor Klf4 has demonstrated activity in the reprogramming of somatic cells to a pluripotent state, but the molecular mechanism of this process remains unknown. It is, therefore, of great interest to understand the functional role of Klf4 and related genes in ESC regulation. Here, we show that homozygous disruption of Klf5 results in the failure of ESC derivation from ICM cells and early embryonic lethality due to an implantation defect. Klf5 KO ESCs show increased expression of several differentiation marker genes and frequent, spontaneous differentiation. Conversely, overexpression of Klf5 in ESCs suppressed the expression of differentiation marker genes and maintained pluripotency in the absence of LIF. Our results also suggest that Klf5 regulates ESC proliferation by promoting phosphorylation of Akt1 via induction of Tcl1. These results, therefore, provide new insights into the functional and mechanistic role of Klf5 in regulation of pluripotency.", "author" : [ { "dropping-particle" : "", "family" : "Ema", "given" : "Masatsugu", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Mori", "given" : "Daisuke", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Niwa", "given" : "Hitoshi", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Hasegawa", "given" : "Yoshikazu", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Yamanaka", "given" : "Yojiro", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Hitoshi", "given" : "Seiji", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Mimura", "given" : "Junsei", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Kawabe", "given" : "Yoh-ichi", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Hosoya", "given" : "Tomohiro", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Morita", "given" : "Masanobu", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Shimosato", "given" : "Daisuke", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Uchida", "given" : "Kazuhiko", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Suzuki", "given" : "Norio", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Yanagisawa", "given" : "Jun", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Sogawa", "given" : "Kazuhiro", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Rossant", "given" : "Janet", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Yamamoto", "given" : "Masayuki", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Takahashi", "given" : "Satoru", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Fujii-Kuriyama", "given" : "Yoshiaki", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Cell stem cell", "id" : "ITEM-1", "issue" : "5", "issued" : { "date-parts" : [ [ "2008", "11", "6" ] ] }, "page" : "555-67", "publisher" : "Elsevier Inc.", "title" : "Kr\u00fcppel-like factor 5 is essential for blastocyst development and the normal self-renewal of mouse ESCs.", "type" : "article-journal", "volume" : "3" }, "uris" : [ "" ] }, { "id" : "ITEM-2", "itemData" : { "DOI" : "10.1242/dev.054775", "ISSN" : "1477-9129", "PMID" : "20980403", "abstract" : "Kruppel-like transcription factors (Klfs) are essential for the induction and maintenance of pluripotency of embryonic stem cells (ESCs), yet little is known about their roles in establishing the three lineages of the pre-implantation embryo. Here, we show that Klf5 is required for the formation of the trophectoderm (TE) and the inner cell mass (ICM), and for repressing primitive endoderm (PE) development. Although cell polarity appeared normal, Klf5 mutant embryos arrested at the blastocyst stage and failed to hatch due to defective TE development. Klf5 acted cell-autonomously in the TE, downstream of Fgf4 and upstream of Cdx2, Eomes and Krt8. In the ICM, loss of Klf5 resulted in reduced expression of pluripotency markers Oct4 and Nanog, but led to increased Sox17 expression in the PE, suggesting that Klf5 suppresses the PE lineage. Consistent with this, overexpression of Klf5 in transgenic embryos was sufficient to suppress the Sox17(+) PE lineage in the ICM. Klf5 overexpression led to a dose-dependent decrease in Sox17 promoter activity in reporter assays in cultured cells. Moreover, in chimeric embryos, Klf5(-/-) cells preferentially contributed to the Sox17(+) PE lineage and Cdx2 expression was not rescued in Klf5(-/-) outer cells. Finally, outgrowths from Klf5(-/-) embryos failed to form an ICM/pluripotent colony, had very few Oct4(+) or Cdx2(+) cells, but showed an increase in the percentage of Sox17(+) PE cells. These findings demonstrate that Klf5 is a dynamic regulator of all three lineages in the pre-implantation embryo by promoting the TE and epiblast lineages while suppressing the PE lineage.", "author" : [ { "dropping-particle" : "", "family" : "Lin", "given" : "Suh-Chin J", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Wani", "given" : "Maqsood a", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Whitsett", "given" : "Jeffrey a", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Wells", "given" : "James M", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Development (Cambridge, England)", "id" : "ITEM-2", "issue" : "23", "issued" : { "date-parts" : [ [ "2010", "12" ] ] }, "page" : "3953-63", "title" : "Klf5 regulates lineage formation in the pre-implantation mouse embryo.", "type" : "article-journal", "volume" : "137" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(Ema et al., 2008; Lin et al., 2010)", "plainTextFormattedCitation" : "(Ema et al., 2008; Lin et al., 2010)", "previouslyFormattedCitation" : "(Ema et al., 2008; Lin et al., 2010)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(Ema et al., 2008; Lin et al., 2010). In a previous study, the Klf5 LacZ allele (Ema et al., 2008) was generated by inserting a LacZ cassette into the 2nd exon of Klf5, leaving the rest of the Klf5 locus with the potential to generate a C-terminally truncated protein of approximately 164 amino acids in length (Fig. S1A). To delete almost the entire open reading frame of Klf5, we generated a new KO mouse for Klf5 that removes the two major coding exons (designated as Klf52nd3rd exon) (Fig. S1B). Similar to Klf5 LacZ mice (Fig. S1A) ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1016/j.stem.2008.09.003", "ISBN" : "1875-9777 (Electronic)", "ISSN" : "1875-9777", "PMID" : "18983969", "abstract" : "The transcription factor Klf4 has demonstrated activity in the reprogramming of somatic cells to a pluripotent state, but the molecular mechanism of this process remains unknown. It is, therefore, of great interest to understand the functional role of Klf4 and related genes in ESC regulation. Here, we show that homozygous disruption of Klf5 results in the failure of ESC derivation from ICM cells and early embryonic lethality due to an implantation defect. Klf5 KO ESCs show increased expression of several differentiation marker genes and frequent, spontaneous differentiation. Conversely, overexpression of Klf5 in ESCs suppressed the expression of differentiation marker genes and maintained pluripotency in the absence of LIF. Our results also suggest that Klf5 regulates ESC proliferation by promoting phosphorylation of Akt1 via induction of Tcl1. These results, therefore, provide new insights into the functional and mechanistic role of Klf5 in regulation of pluripotency.", "author" : [ { "dropping-particle" : "", "family" : "Ema", "given" : "Masatsugu", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Mori", "given" : "Daisuke", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Niwa", "given" : "Hitoshi", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Hasegawa", "given" : "Yoshikazu", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Yamanaka", "given" : "Yojiro", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Hitoshi", "given" : "Seiji", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Mimura", "given" : "Junsei", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Kawabe", "given" : "Yoh-ichi", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Hosoya", "given" : "Tomohiro", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Morita", "given" : "Masanobu", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Shimosato", "given" : "Daisuke", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Uchida", "given" : "Kazuhiko", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Suzuki", "given" : "Norio", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Yanagisawa", "given" : "Jun", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Sogawa", "given" : "Kazuhiro", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Rossant", "given" : "Janet", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Yamamoto", "given" : "Masayuki", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Takahashi", "given" : "Satoru", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Fujii-Kuriyama", "given" : "Yoshiaki", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Cell stem cell", "id" : "ITEM-1", "issue" : "5", "issued" : { "date-parts" : [ [ "2008", "11", "6" ] ] }, "page" : "555-67", "publisher" : "Elsevier Inc.", "title" : "Kr\u00fcppel-like factor 5 is essential for blastocyst development and the normal self-renewal of mouse ESCs.", "type" : "article-journal", "volume" : "3" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(Ema et al., 2008)", "plainTextFormattedCitation" : "(Ema et al., 2008)", "previouslyFormattedCitation" : "(Ema et al., 2008)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(Ema et al., 2008), no homozygous pups were obtained from heterozygous inter-crosses of the Klf52nd3rd exon mice, and homozygous Klf52nd3rd exon/ Klf52nd3rd exon embryos showed implantation defects (Fig. S1C). When Klf5 KO morulae (Klf5 LacZ/LacZ or Klf5Δ2nd3rd exon/Δ2nd3rd exon) at E2.5 were stained with antibodies against lineage markers such as Oct3/4 and Cdx2, both types of Klf5 KO embryos showed decreased levels of Cdx2 protein expression and normal levels of Oct3/4 protein expression (Fig. S1D, E). Hereafter, the Klf52nd3rd exon allele was used as the null allele for our study. To obtain insight into the role of Klf5 in early embryogenesis, we established a new ES cell line with Cre-mediated overexpression of the FLAG/HA-Klf5 cassette (Fig. S1F). We used the ES cell line, which expresses GFP prior to Cre-mediated excision, to generate the conditional Klf5 overexpression (OE) mice (Fig. S1G). Upon crossing to the Ayu1-Cre driver line, we confirmed that Klf5 protein is overexpressed 1.5-fold in Klf5 OE blastocysts compared with WT (Fig. S1H).At first, we collected embryos carefully timed every 6?h from E3.25 onwards and found that Klf5 KO embryos had fewer cells than their WT counterparts (Fig. S1I). The total cell number per Klf5 KO embryo never exceeded 64 until E4.25 (Fig. S1I; data not shown). Given that bromodeoxyuridine (BrdU) incorporation was severely affected, it is likely that cell cycle progression had been disturbed (Fig. S1J, K). Co-staining embryos from E3.25 to E3.5 for BrdU incorporation and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) clearly showed that the cells defective in BrdU incorporation were TUNEL+, suggesting that defective cell cycle progression was promoting apoptosis (Fig. S1L). Examination of Nanog and Gata6 expression levels in Klf5 KO embryos revealed that the initial Nanog and Gata6 expression at E3.25 was overall similar to that of WT embryos (Fig. 1A). Analyses of Klf5 KO blastocysts at E3.5 and E3.75 showed that most, if not all, ICM cells were Gata6+, and few cells were Nanog+ (Fig. 1A, C and Fig. S2A, C). Consistent with this result, Nanog protein expression levels decreased in Klf5 KO embryos during the development from E3.25 to E4.0, while the Gata6 protein expression levels increased (Fig. 1B). Since it was reported that Nanog+/Gata6+ (double positive; DP) common precursors differentiate progressively into a Nanog+/Gata6- (Nanog+) EPI or Nanog-/Gata6+ (Gata6+) PrE fate in an asynchronous manner ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1038/ncomms13463", "ISSN" : "2041-1723", "PMID" : "27857135", "abstract" : "Intercellular communication is essential to coordinate the behaviour of individual cells during organismal development. The preimplantation mammalian embryo is a paradigm of tissue self-organization and regulative development; however, the cellular basis of these regulative abilities has not been established. Here we use a quantitative image analysis pipeline to undertake a high-resolution, single-cell level analysis of lineage specification in the inner cell mass (ICM) of the mouse blastocyst. We show that a consistent ratio of epiblast and primitive endoderm lineages is achieved through incremental allocation of cells from a common progenitor pool, and that the lineage composition of the ICM is conserved regardless of its size. Furthermore, timed modulation of the FGF-MAPK pathway shows that individual progenitors commit to either fate asynchronously during blastocyst development. These data indicate that such incremental lineage allocation provides the basis for a tissue size control mechanism that ensures the generation of lineages of appropriate size.", "author" : [ { "dropping-particle" : "", "family" : "Saiz", "given" : "N\u00e9stor", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Williams", "given" : "Kiah M.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Seshan", "given" : "Venkatraman E.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Hadjantonakis", "given" : "Anna-Katerina", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Nature communications", "id" : "ITEM-1", "issued" : { "date-parts" : [ [ "2016", "11", "18" ] ] }, "page" : "13463", "publisher" : "Nature Publishing Group", "title" : "Asynchronous fate decisions by single cells collectively ensure consistent lineage composition in the mouse blastocyst.", "type" : "article-journal", "volume" : "7" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(Saiz et al., 2016)", "plainTextFormattedCitation" : "(Saiz et al., 2016)", "previouslyFormattedCitation" : "(Saiz et al., 2016)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(Saiz et al., 2016), we evaluated the percentages of DP cells, Nanog+ cells and Gata6+ cells from E3.25 to E4.0. The percentage of DP cells in Klf5 KO embryos decreased rapidly, and in turn, the percentage of Gata6+ cells increased, indicating that bipotential DP cells in Klf5 KO embryos prefer to differentiate into Gata6+ PrE cells (Fig. 1C). At the E4.0 late blastocyst stage, most cells in Klf5 KO embryos acquired the Gata6+ PrE fate (Fig. 1A, C). Our finding that only Gata6-expressing cells in Klf5 KO embryos remain at E4.0 could be the consequence of apoptosis of Nanog+ EPI cells rather than more cells differentiating into PrE. To gain insight into the role of apoptosis in the EPI lineage, we analysed the?number of TUNEL+ cells in Klf5 KO and WT embryos at E3.5. We found no significant changes in the percentages of?Nanog+, Gata6+, DP, or double-negative (DN) cells undergoing apoptosis between WT and Klf5 KO embryos (Fig. S1M,?N, O). This finding indicated that the increase in the percentage of Gata6+ cells was not caused by the death of any specific cell lineage, including EPI cells. Because the percentage of DP cells in Klf5 KO embryos was decreased (Fig. 1C), DP cells were likely to have differentiated into PrE. Because the Klf5 KO embryos showed a cell proliferation defect, as shown in Fig. S1I, we categorised embryos by similar total cell numbers (<32 or 32–64) from various days of development and reached the same conclusion (Fig. S2B, C, D).When we investigated Nanog and Gata6 expression in Klf5 OE embryos at E3.25, Klf5 OE blastocysts showed overall co-expression of Nanog and Gata6 (Fig. 1A). The numbers of DP cells per embryo at E3.5 to E4.0 were significantly increased in Klf5 OE embryos (Fig. 1C and Fig. S2C, D). While there were Gata6+ endoderm layers and Nanog+ EPI cells in WT blastocysts at E4.5, there were still significant numbers of DP cells centrally located towards, presumably, uncommitted cells in Klf5 OE blastocysts at E4.5 (Fig. S2A, A). Accelerated PrE lineage specification in Klf5 KO embryosSpecification of the PrE lineage involves Gata6, followed by sequential activation of Pdgfra, Sox17, Gata4 and Sox7 ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1016/j.ydbio.2010.12.007", "ISBN" : "1095-564X (Electronic)\\r0012-1606 (Linking)", "ISSN" : "1095-564X", "PMID" : "21146513", "abstract" : "Cells of the primitive endoderm (PrE) and the pluripotent epiblast (EPI), the two lineages specified within the inner cell mass (ICM) of the mouse blastocyst stage embryo, are segregated into adjacent tissue layers by the end of the preimplantation period. The PrE layer which emerges as a polarized epithelium adjacent to the blastocoel, with a basement membrane separating it from the EPI, has two derivatives, the visceral and parietal endoderm. In this study we have investigated the localization of two transcriptional regulators of the SOX family, SOX17 and SOX7, within the PrE and its derivatives. We noted that SOX17 was first detected in a salt-and-pepper distribution within the ICM, subsequently becoming restricted to the nascent PrE epithelium. This dynamic distribution of SOX17 resembled the localization of GATA6 and GATA4, two other PrE lineage-specific transcription factors. By contrast, SOX7 was only detected in PrE cells positioned in contact with the blastocoel, raising the possibility that these cells are molecularly distinct. Our observations support a model of sequential GATA6 > SOX17 > GATA4 > SOX7 transcription factor activation within the PrE lineage, perhaps correlating with the consecutive periods of cell lineage 'na\u00efvete', commitment and sorting. Furthermore our data suggest that co-expression of SOX17 and SOX7 within sorted PrE cells could account for the absence of a detectable phenotype of Sox17 mutant blastocysts. However, analysis of implantation-delayed blastocysts, revealed a role for SOX17 in the maintenance of PrE epithelial integrity, with the absence of SOX17 leading to premature delamination and migration of parietal endoderm.", "author" : [ { "dropping-particle" : "", "family" : "Artus", "given" : "J\u00e9r\u00f4me", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Piliszek", "given" : "Anna", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Hadjantonakis", "given" : "Anna-Katerina", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Developmental biology", "id" : "ITEM-1", "issue" : "2", "issued" : { "date-parts" : [ [ "2011", "2", "15" ] ] }, "page" : "393-404", "publisher" : "Elsevier B.V.", "title" : "The primitive endoderm lineage of the mouse blastocyst: sequential transcription factor activation and regulation of differentiation by Sox17.", "type" : "article-journal", "volume" : "350" }, "uris" : [ "" ] }, { "id" : "ITEM-2", "itemData" : { "DOI" : "10.1242/dev.084996", "ISSN" : "1477-9129", "PMID" : "23193166", "abstract" : "The emergence of pluripotent epiblast (EPI) and primitive endoderm (PrE) lineages within the inner cell mass (ICM) of the mouse blastocyst involves initial co-expression of lineage-associated markers followed by mutual exclusion and salt-and-pepper distribution of lineage-biased cells. Precisely how EPI and PrE cell fate commitment occurs is not entirely clear; however, previous studies in mice have implicated FGF/ERK signaling in this process. Here, we investigated the phenotype resulting from zygotic and maternal/zygotic inactivation of Fgf4. Fgf4 heterozygous blastocysts exhibited increased numbers of NANOG-positive EPI cells and reduced numbers of GATA6-positive PrE cells, suggesting that FGF signaling is tightly regulated to ensure specification of the appropriate numbers of cells for each lineage. Although the size of the ICM was unaffected in Fgf4 null mutant embryos, it entirely lacked a PrE layer and exclusively comprised NANOG-expressing cells at the time of implantation. An initial period of widespread EPI and PrE marker co-expression was however established even in the absence of FGF4. Thus, Fgf4 mutant embryos initiated the PrE program but exhibited defects in its restriction phase, when lineage bias is acquired. Consistent with this, XEN cells could be derived from Fgf4 mutant embryos in which PrE had been restored and these cells appeared indistinguishable from wild-type cells. Sustained exogenous FGF failed to rescue the mutant phenotype. Instead, depending on concentration, we noted no effect or conversion of all ICM cells to GATA6-positive PrE. We propose that heterogeneities in the availability of FGF produce the salt-and-pepper distribution of lineage-biased cells.", "author" : [ { "dropping-particle" : "", "family" : "Kang", "given" : "Minjung", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Piliszek", "given" : "Anna", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Artus", "given" : "J\u00e9r\u00f4me", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Hadjantonakis", "given" : "Anna-Katerina", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Development (Cambridge, England)", "id" : "ITEM-2", "issue" : "2", "issued" : { "date-parts" : [ [ "2013", "1", "15" ] ] }, "page" : "267-79", "title" : "FGF4 is required for lineage restriction and salt-and-pepper distribution of primitive endoderm factors but not their initial expression in the mouse.", "type" : "article-journal", "volume" : "140" }, "uris" : [ "" ] }, { "id" : "ITEM-3", "itemData" : { "DOI" : "10.1242/dev.021519", "ISSN" : "0950-1991", "PMID" : "18725515", "abstract" : "The first two lineages to differentiate from a pluripotent cell population during mammalian development are the extraembryonic trophectoderm (TE) and the primitive endoderm (PrE). Whereas the mechanisms of TE specification have been extensively studied, segregation of PrE and the pluripotent epiblast (EPI) has received comparatively little attention. A current model of PrE specification suggests PrE precursors exhibit an apparently random distribution within the inner cell mass of the early blastocyst and then segregate to their final position lining the cavity by the late blastocyst. We have identified platelet-derived growth factor receptor alpha (Pdgfralpha) as an early-expressed protein that is also a marker of the later PrE lineage. By combining live imaging of embryos expressing a histone H2B-GFP fusion protein reporter under the control of Pdgfra regulatory elements with the analysis of lineage-specific markers, we investigated the events leading to PrE and EPI lineage segregation in the mouse, and correlated our findings using an embryo staging system based on total cell number. Before blastocyst formation, lineage-specific factors are expressed in an overlapping manner. Subsequently, a gradual progression towards a mutually exclusive expression of PrE- and EPI-specific markers occurs. Finally, cell sorting is achieved by a variety of cell behaviours and by selective apoptosis.", "author" : [ { "dropping-particle" : "", "family" : "Plusa", "given" : "Berenika", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Piliszek", "given" : "Anna", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Frankenberg", "given" : "Stephen", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Artus", "given" : "J\u00e9r\u00f4me", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Hadjantonakis", "given" : "Anna-Katerina", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Development (Cambridge, England)", "id" : "ITEM-3", "issue" : "18", "issued" : { "date-parts" : [ [ "2008", "9" ] ] }, "page" : "3081-91", "title" : "Distinct sequential cell behaviours direct primitive endoderm formation in the mouse blastocyst.", "type" : "article-journal", "volume" : "135" }, "uris" : [ "" ] }, { "id" : "ITEM-4", "itemData" : { "DOI" : "10.1242/dev.050864", "ISSN" : "1477-9129", "PMID" : "20826533", "abstract" : "The inner cell mass (ICM) of the implanting mammalian blastocyst comprises two lineages: the pluripotent epiblast (EPI) and primitive endoderm (PrE). We have identified platelet-derived growth factor receptor alpha (PDGFR\u03b1) as an early marker of the PrE lineage and its derivatives in both mouse embryos and ex vivo paradigms of extra-embryonic endoderm (ExEn). By combining live imaging of embryos and embryo-derived stem cells expressing a histone H2B-GFP fusion reporter under the control of Pdgfra regulatory elements with the analysis of lineage-specific markers, we found that Pdgfra expression coincides with that of GATA6, the earliest expressed transcriptional regulator of the PrE lineage. We show that GATA6 is required for the activation of Pdgfra expression. Using pharmacological inhibition and genetic inactivation we addressed the role of the PDGF pathway in the PrE lineage. Our results demonstrate that PDGF signaling is essential for the establishment, and plays a role in the proliferation, of XEN cells, which are isolated from mouse blastocyst stage embryos and represent the PrE lineage. Implanting Pdgfra mutant blastocysts exhibited a reduced number of PrE cells, an effect that was exacerbated by delaying implantation. Surprisingly, we also noted an increase in the number of EPI cells in implantation-delayed Pdgfra-null mutants. Taken together, our data suggest a role for PDGF signaling in the expansion of the ExEn lineage. Our observations also uncover a possible role for the PrE in regulating the size of the pluripotent EPI compartment.", "author" : [ { "dropping-particle" : "", "family" : "Artus", "given" : "J\u00e9r\u00f4me", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Panthier", "given" : "Jean-Jacques", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Hadjantonakis", "given" : "Anna-Katerina", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Development (Cambridge, England)", "id" : "ITEM-4", "issue" : "20", "issued" : { "date-parts" : [ [ "2010", "10", "28" ] ] }, "page" : "3361-72", "title" : "A role for PDGF signaling in expansion of the extra-embryonic endoderm lineage of the mouse blastocyst.", "type" : "article-journal", "volume" : "137" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(Artus et al., 2010; Artus et al., 2011; Kang et al., 2013; Plusa et al., 2008)", "plainTextFormattedCitation" : "(Artus et al., 2010; Artus et al., 2011; Kang et al., 2013; Plusa et al., 2008)", "previouslyFormattedCitation" : "(Artus et al., 2010; Artus et al., 2011; Kang et al., 2013; Plusa et al., 2008)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(Artus et al., 2010; Artus et al., 2011; Kang et al., 2013; Plusa et al., 2008). Sox17 is activated between the 32- and 64-cell stages ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1016/j.ydbio.2010.12.007", "ISBN" : "1095-564X (Electronic)\\r0012-1606 (Linking)", "ISSN" : "1095-564X", "PMID" : "21146513", "abstract" : "Cells of the primitive endoderm (PrE) and the pluripotent epiblast (EPI), the two lineages specified within the inner cell mass (ICM) of the mouse blastocyst stage embryo, are segregated into adjacent tissue layers by the end of the preimplantation period. The PrE layer which emerges as a polarized epithelium adjacent to the blastocoel, with a basement membrane separating it from the EPI, has two derivatives, the visceral and parietal endoderm. In this study we have investigated the localization of two transcriptional regulators of the SOX family, SOX17 and SOX7, within the PrE and its derivatives. We noted that SOX17 was first detected in a salt-and-pepper distribution within the ICM, subsequently becoming restricted to the nascent PrE epithelium. This dynamic distribution of SOX17 resembled the localization of GATA6 and GATA4, two other PrE lineage-specific transcription factors. By contrast, SOX7 was only detected in PrE cells positioned in contact with the blastocoel, raising the possibility that these cells are molecularly distinct. Our observations support a model of sequential GATA6 > SOX17 > GATA4 > SOX7 transcription factor activation within the PrE lineage, perhaps correlating with the consecutive periods of cell lineage 'na\u00efvete', commitment and sorting. Furthermore our data suggest that co-expression of SOX17 and SOX7 within sorted PrE cells could account for the absence of a detectable phenotype of Sox17 mutant blastocysts. However, analysis of implantation-delayed blastocysts, revealed a role for SOX17 in the maintenance of PrE epithelial integrity, with the absence of SOX17 leading to premature delamination and migration of parietal endoderm.", "author" : [ { "dropping-particle" : "", "family" : "Artus", "given" : "J\u00e9r\u00f4me", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Piliszek", "given" : "Anna", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Hadjantonakis", "given" : "Anna-Katerina", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Developmental biology", "id" : "ITEM-1", "issue" : "2", "issued" : { "date-parts" : [ [ "2011", "2", "15" ] ] }, "page" : "393-404", "publisher" : "Elsevier B.V.", "title" : "The primitive endoderm lineage of the mouse blastocyst: sequential transcription factor activation and regulation of differentiation by Sox17.", "type" : "article-journal", "volume" : "350" }, "uris" : [ "" ] }, { "id" : "ITEM-2", "itemData" : { "DOI" : "10.1073/pnas.0915063107", "ISBN" : "0915063107", "ISSN" : "1091-6490", "PMID" : "20308546", "abstract" : "A crucial question in mammalian development is how cells of the early embryo differentiate into distinct cell types. The first decision is taken when cells undertake waves of asymmetric division that generate one daughter on the inside and one on the outside of the embryo. After this division, some cells on the inside remain pluripotent and give rise to the epiblast, and hence the future body, whereas others develop into the primitive endoderm, an extraembryonic tissue. How the fate of these inside cells is decided is unknown: Is the process random, or is it related to their developmental origins? To address this question, we traced all cells by live-cell imaging in intact, unmanipulated embryos until the epiblast and primitive endoderm became distinct. This analysis revealed that inner cell mass (ICM) cells have unrestricted developmental potential. However, cells internalized by the first wave of asymmetric divisions are biased toward forming pluripotent epiblast, whereas cells internalized in the next two waves of divisions are strongly biased toward forming primitive endoderm. Moreover, we show that cells internalized by the second wave up-regulate expression of Gata6 and Sox17, and changing the expression of these genes determines whether the cells become primitive endoderm. Finally, with our ability to determine the origin of cells, we find that inside cells that are mispositioned when they are born can sort into the correct layer. In conclusion, we propose a model in which the timing of cell internalization, cell position, and cell sorting combine to determine distinct lineages of the preimplantation mouse embryo.", "author" : [ { "dropping-particle" : "", "family" : "Morris", "given" : "Samantha a", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Teo", "given" : "Roy T Y", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Li", "given" : "Huiliang", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Robson", "given" : "Paul", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Glover", "given" : "David M", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Zernicka-Goetz", "given" : "Magdalena", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Proceedings of the National Academy of Sciences of the United States of America", "id" : "ITEM-2", "issue" : "14", "issued" : { "date-parts" : [ [ "2010", "4", "6" ] ] }, "page" : "6364-9", "title" : "Origin and formation of the first two distinct cell types of the inner cell mass in the mouse embryo.", "type" : "article-journal", "volume" : "107" }, "uris" : [ "" ] }, { "id" : "ITEM-3", "itemData" : { "DOI" : "10.1101/gad.1833510", "ISBN" : "1549-5477 (Electronic)\\r0890-9369 (Linking)", "ISSN" : "1549-5477", "PMID" : "20123909", "abstract" : "In embryonic stem (ES) cells, a well-characterized transcriptional network promotes pluripotency and represses gene expression required for differentiation. In comparison, the transcriptional networks that promote differentiation of ES cells and the blastocyst inner cell mass are poorly understood. Here, we show that Sox17 is a transcriptional regulator of differentiation in these pluripotent cells. ES cells deficient in Sox17 fail to differentiate into extraembryonic cell types and maintain expression of pluripotency-associated transcription factors, including Oct4, Nanog, and Sox2. In contrast, forced expression of Sox17 down-regulates ES cell-associated gene expression and directly activates genes functioning in differentiation toward an extraembryonic endoderm cell fate. We show these effects of Sox17 on ES cell gene expression are mediated at least in part through a competition between Sox17 and Nanog for common DNA-binding sites. By elaborating the function of Sox17, our results provide insight into how the transcriptional network promoting ES cell self-renewal is interrupted, allowing cellular differentiation.", "author" : [ { "dropping-particle" : "", "family" : "Niakan", "given" : "Kathy K", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Ji", "given" : "Hongkai", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Maehr", "given" : "Ren\u00e9", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Vokes", "given" : "Steven a", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Rodolfa", "given" : "Kit T", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Sherwood", "given" : "Richard I", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Yamaki", "given" : "Mariko", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Dimos", "given" : "John T", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Chen", "given" : "Alice E", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Melton", "given" : "Douglas a", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "McMahon", "given" : "Andrew P", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Eggan", "given" : "Kevin", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Genes & development", "id" : "ITEM-3", "issue" : "3", "issued" : { "date-parts" : [ [ "2010" ] ] }, "page" : "312-26", "title" : "Sox17 promotes differentiation in mouse embryonic stem cells by directly regulating extraembryonic gene expression and indirectly antagonizing self-renewal.", "type" : "article-journal", "volume" : "24" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(Artus et al., 2011; Morris et al., 2010; Niakan et al., 2010)", "plainTextFormattedCitation" : "(Artus et al., 2011; Morris et al., 2010; Niakan et al., 2010)", "previouslyFormattedCitation" : "(Artus et al., 2011; Morris et al., 2010; Niakan et al., 2010)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(Artus et al., 2011; Morris et al., 2010; Niakan et al., 2010); Gata4 expression marks the onset of the mutual exclusion of Gata6 and Nanog and is activated at the 64-cell stage ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1016/j.ydbio.2010.12.007", "ISBN" : "1095-564X (Electronic)\\r0012-1606 (Linking)", "ISSN" : "1095-564X", "PMID" : "21146513", "abstract" : "Cells of the primitive endoderm (PrE) and the pluripotent epiblast (EPI), the two lineages specified within the inner cell mass (ICM) of the mouse blastocyst stage embryo, are segregated into adjacent tissue layers by the end of the preimplantation period. The PrE layer which emerges as a polarized epithelium adjacent to the blastocoel, with a basement membrane separating it from the EPI, has two derivatives, the visceral and parietal endoderm. In this study we have investigated the localization of two transcriptional regulators of the SOX family, SOX17 and SOX7, within the PrE and its derivatives. We noted that SOX17 was first detected in a salt-and-pepper distribution within the ICM, subsequently becoming restricted to the nascent PrE epithelium. This dynamic distribution of SOX17 resembled the localization of GATA6 and GATA4, two other PrE lineage-specific transcription factors. By contrast, SOX7 was only detected in PrE cells positioned in contact with the blastocoel, raising the possibility that these cells are molecularly distinct. Our observations support a model of sequential GATA6 > SOX17 > GATA4 > SOX7 transcription factor activation within the PrE lineage, perhaps correlating with the consecutive periods of cell lineage 'na\u00efvete', commitment and sorting. Furthermore our data suggest that co-expression of SOX17 and SOX7 within sorted PrE cells could account for the absence of a detectable phenotype of Sox17 mutant blastocysts. However, analysis of implantation-delayed blastocysts, revealed a role for SOX17 in the maintenance of PrE epithelial integrity, with the absence of SOX17 leading to premature delamination and migration of parietal endoderm.", "author" : [ { "dropping-particle" : "", "family" : "Artus", "given" : "J\u00e9r\u00f4me", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Piliszek", "given" : "Anna", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Hadjantonakis", "given" : "Anna-Katerina", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Developmental biology", "id" : "ITEM-1", "issue" : "2", "issued" : { "date-parts" : [ [ "2011", "2", "15" ] ] }, "page" : "393-404", "publisher" : "Elsevier B.V.", "title" : "The primitive endoderm lineage of the mouse blastocyst: sequential transcription factor activation and regulation of differentiation by Sox17.", "type" : "article-journal", "volume" : "350" }, "uris" : [ "" ] }, { "id" : "ITEM-2", "itemData" : { "DOI" : "10.1242/dev.067702", "ISBN" : "1477-9129 (Electronic)\\r0950-1991 (Linking)", "ISSN" : "1477-9129", "PMID" : "22096072", "abstract" : "Cell differentiation during pre-implantation mammalian development involves the formation of two extra-embryonic lineages: trophoblast and primitive endoderm (PrE). A subset of cells within the inner cell mass (ICM) of the blastocyst does not respond to differentiation signals and forms the pluripotent epiblast, which gives rise to all of the tissues in the adult body. How this group of cells is set aside remains unknown. Recent studies documented distinct sequential phases of marker expression during the segregation of epiblast and PrE within the ICM. However, the connection between marker expression and lineage commitment remains unclear. Using a fluorescent reporter for PrE, we investigated the plasticity of epiblast and PrE precursors. Our observations reveal that loss of plasticity does not coincide directly with lineage restriction of epiblast and PrE markers, but rather with exclusion of the pluripotency marker Oct4 from the PrE. We note that individual ICM cells can contribute to all three lineages of the blastocyst until peri-implantation. However, epiblast precursors exhibit less plasticity than precursors of PrE, probably owing to differences in responsiveness to extracellular signalling. We therefore propose that the early embryo environment restricts the fate choice of epiblast but not PrE precursors, thus ensuring the formation and preservation of the pluripotent foetal lineage.", "author" : [ { "dropping-particle" : "", "family" : "Grabarek", "given" : "Joanna B", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Zyzy\u0144ska", "given" : "Krystyna", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Saiz", "given" : "N\u00e9stor", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Piliszek", "given" : "Anna", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Frankenberg", "given" : "Stephen", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Nichols", "given" : "Jennifer", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Hadjantonakis", "given" : "Anna-Katerina", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Plusa", "given" : "Berenika", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Development (Cambridge, England)", "id" : "ITEM-2", "issue" : "1", "issued" : { "date-parts" : [ [ "2012", "1" ] ] }, "page" : "129-39", "publisher" : "Oxford University Press for The Company of Biologists Limited", "title" : "Differential plasticity of epiblast and primitive endoderm precursors within the ICM of the early mouse embryo.", "type" : "article-journal", "volume" : "139" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(Artus et al., 2011; Grabarek et al., 2012)", "plainTextFormattedCitation" : "(Artus et al., 2011; Grabarek et al., 2012)", "previouslyFormattedCitation" : "(Artus et al., 2011; Grabarek et al., 2012)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(Artus et al., 2011; Grabarek et al., 2012). Because we observed a reduction in the percentage of Nanog+ cells and an increase in Gata6+ cells in Klf5 KO embryos at E3.75, we tested whether inner cells at E3.0 already exhibit signs of accelerated PrE lineage specification. Immunohistochemistry with anti-Pdgfra and anti-Sox17 antibodies did not show any detectable Pdgfra or Sox17 protein expression in Klf5 KO morulae at E3.0 (data not shown, Fig. S3A). However, at E3.25, immunohistochemistry showed strong Pdgfra expression in most of the inner cells of Klf5 KO embryos but not WT embryos (Fig. 2A). Quantitation of Pdgfra+ cells indicated that over 80% of the inner cells of Klf5 KO embryos were Pdgfra+, while fewer than 20% of the inner cells of WT embryos were Pdgfra+ (Fig. 2B). It is of note that a small increase in Pdgfra+ cells was observed in the outer cells of Klf5 KO embryos (Fig. 2B). Taken together, the data revealed that the inner cells of Klf5 KO embryos exhibit accelerated PrE lineage specification as early as E3.25 (Fig. 2C). We also found an increase in the percentage of Sox17+ cells in Klf5 KO blastocysts at E3.5 (Fig. S3B, C, D), which was consistent with a previous report ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1242/dev.054775", "ISSN" : "1477-9129", "PMID" : "20980403", "abstract" : "Kruppel-like transcription factors (Klfs) are essential for the induction and maintenance of pluripotency of embryonic stem cells (ESCs), yet little is known about their roles in establishing the three lineages of the pre-implantation embryo. Here, we show that Klf5 is required for the formation of the trophectoderm (TE) and the inner cell mass (ICM), and for repressing primitive endoderm (PE) development. Although cell polarity appeared normal, Klf5 mutant embryos arrested at the blastocyst stage and failed to hatch due to defective TE development. Klf5 acted cell-autonomously in the TE, downstream of Fgf4 and upstream of Cdx2, Eomes and Krt8. In the ICM, loss of Klf5 resulted in reduced expression of pluripotency markers Oct4 and Nanog, but led to increased Sox17 expression in the PE, suggesting that Klf5 suppresses the PE lineage. Consistent with this, overexpression of Klf5 in transgenic embryos was sufficient to suppress the Sox17(+) PE lineage in the ICM. Klf5 overexpression led to a dose-dependent decrease in Sox17 promoter activity in reporter assays in cultured cells. Moreover, in chimeric embryos, Klf5(-/-) cells preferentially contributed to the Sox17(+) PE lineage and Cdx2 expression was not rescued in Klf5(-/-) outer cells. Finally, outgrowths from Klf5(-/-) embryos failed to form an ICM/pluripotent colony, had very few Oct4(+) or Cdx2(+) cells, but showed an increase in the percentage of Sox17(+) PE cells. These findings demonstrate that Klf5 is a dynamic regulator of all three lineages in the pre-implantation embryo by promoting the TE and epiblast lineages while suppressing the PE lineage.", "author" : [ { "dropping-particle" : "", "family" : "Lin", "given" : "Suh-Chin J", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Wani", "given" : "Maqsood a", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Whitsett", "given" : "Jeffrey a", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Wells", "given" : "James M", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Development (Cambridge, England)", "id" : "ITEM-1", "issue" : "23", "issued" : { "date-parts" : [ [ "2010", "12" ] ] }, "page" : "3953-63", "title" : "Klf5 regulates lineage formation in the pre-implantation mouse embryo.", "type" : "article-journal", "volume" : "137" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(Lin et al., 2010)", "plainTextFormattedCitation" : "(Lin et al., 2010)", "previouslyFormattedCitation" : "(Lin et al., 2010)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(Lin et al., 2010). Furthermore, we found an increase in the numbers of Gata4+ cells in Klf5 KO blastocysts at E3.75, which was in sharp contrast with the small number of cells found in Klf5 OE embryos (Fig. 2D, E). Gata4 protein expression levels were increased in Klf5 KO embryos and decreased in Klf5 OE embryos (Fig. 2F). Collectively, these data indicated that Klf5 KO embryos show an accelerated PrE lineage specification in the ICM.During maturation of EPI cells at E4.25-4.5, it has been demonstrated that downregulation of Nanog expression is a hallmark of na?ve to primed transition of EPI cells ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1038/ncomms13463", "ISSN" : "2041-1723", "PMID" : "27857135", "abstract" : "Intercellular communication is essential to coordinate the behaviour of individual cells during organismal development. The preimplantation mammalian embryo is a paradigm of tissue self-organization and regulative development; however, the cellular basis of these regulative abilities has not been established. Here we use a quantitative image analysis pipeline to undertake a high-resolution, single-cell level analysis of lineage specification in the inner cell mass (ICM) of the mouse blastocyst. We show that a consistent ratio of epiblast and primitive endoderm lineages is achieved through incremental allocation of cells from a common progenitor pool, and that the lineage composition of the ICM is conserved regardless of its size. Furthermore, timed modulation of the FGF-MAPK pathway shows that individual progenitors commit to either fate asynchronously during blastocyst development. These data indicate that such incremental lineage allocation provides the basis for a tissue size control mechanism that ensures the generation of lineages of appropriate size.", "author" : [ { "dropping-particle" : "", "family" : "Saiz", "given" : "N\u00e9stor", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Williams", "given" : "Kiah M.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Seshan", "given" : "Venkatraman E.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Hadjantonakis", "given" : "Anna-Katerina", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Nature communications", "id" : "ITEM-1", "issued" : { "date-parts" : [ [ "2016", "11", "18" ] ] }, "page" : "13463", "publisher" : "Nature Publishing Group", "title" : "Asynchronous fate decisions by single cells collectively ensure consistent lineage composition in the mouse blastocyst.", "type" : "article-journal", "volume" : "7" }, "uris" : [ "" ] }, { "id" : "ITEM-2", "itemData" : { "DOI" : "10.1242/dev.142679", "ISSN" : "1477-9129", "PMID" : "28143843", "abstract" : "The regulative capability of single cells to give rise to all primary embryonic lineages is termed pluripotency. Observations of fluctuating gene expression and phenotypic heterogeneity in vitro have fostered a conception of pluripotency as an intrinsically metastable and precarious state. However, in the embryo and in defined culture environments the properties of pluripotent cells change in an orderly sequence. Two phases of pluripotency, called na\u00efve and primed, have previously been described. In this Hypothesis article, a third phase, called formative pluripotency, is proposed to exist as part of a developmental continuum between the na\u00efve and primed phases. The formative phase is hypothesised to be enabling for the execution of pluripotency, entailing remodelling of transcriptional, epigenetic, signalling and metabolic networks to constitute multi-lineage competence and responsiveness to specification cues.", "author" : [ { "dropping-particle" : "", "family" : "Smith", "given" : "Austin", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Development (Cambridge, England)", "id" : "ITEM-2", "issue" : "3", "issued" : { "date-parts" : [ [ "2017", "2", "1" ] ] }, "page" : "365-373", "title" : "Formative pluripotency: the executive phase in a developmental continuum.", "type" : "article-journal", "volume" : "144" }, "uris" : [ "" ] }, { "id" : "ITEM-3", "itemData" : { "DOI" : "10.1016/j.devcel.2017.05.003", "ISSN" : "1878-1551", "PMID" : "28552559", "abstract" : "Fibroblast growth factor 4 (FGF4) is the key signal driving specification of primitive endoderm (PrE) versus pluripotent epiblast (EPI) within the inner cell mass (ICM) of the mouse blastocyst. To gain insight into the receptor(s) responding to FGF4 within ICM cells, we combined single-cell-resolution quantitative imaging with single-cell transcriptomics of wild-type and Fgf receptor (Fgfr) mutant embryos. Despite the PrE-specific expression of FGFR2, it is FGFR1, expressed by all ICM cells, that is critical for establishment of a PrE identity. Signaling through FGFR1 is also required to constrain levels of the pluripotency-associated factor NANOG in EPI cells. However, the activity of both receptors is required for lineage establishment within the ICM. Gene expression profiling of 534 single ICM cells identified distinct downstream targets associated with each receptor. These data lead us to propose a model whereby unique and additive activities of FGFR1 and FGFR2 within the ICM coordinate establishment of two distinct lineages.", "author" : [ { "dropping-particle" : "", "family" : "Kang", "given" : "Minjung", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Garg", "given" : "Vidur", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Hadjantonakis", "given" : "Anna-Katerina", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Developmental cell", "id" : "ITEM-3", "issue" : "5", "issued" : { "date-parts" : [ [ "2017", "6", "5" ] ] }, "page" : "496-510.e5", "publisher" : "Cold Spring Harbor Laboratory Press", "title" : "Lineage Establishment and Progression within the Inner Cell Mass of the Mouse Blastocyst Requires FGFR1 and FGFR2.", "type" : "article-journal", "volume" : "41" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(Kang et al., 2017; Saiz et al., 2016; Smith, 2017)", "plainTextFormattedCitation" : "(Kang et al., 2017; Saiz et al., 2016; Smith, 2017)", "previouslyFormattedCitation" : "(Kang et al., 2017; Saiz et al., 2016; Smith, 2017)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(Kang et al., 2017; Saiz et al., 2016; Smith, 2017). Elevated Nanog expression in Klf5 OE embryos from E3.25 to E4.25 suggests that these cells might remain in a na?ve state and fail to differentiate into mature EPI cells. To examine the consequence of ubiquitous overexpression of Klf5, Klf5 OE embryos at E5.5 (egg cylinder stage) were dissected and subjected to immunohistochemistry. In WT embryos, Klf5 protein was expressed in extra-embryonic ectoderm cells but not in EPI cells; however, Klf5 OE embryos still expressed Klf5 protein in all cell lineages, including the EPI cells (Fig. S4). Thus, the elevated Nanog expression caused by Klf5 OE did not block the differentiation into EPI cells. Yamanaka and colleagues indicated that even though Fgf4 heterozygous embryos exhibit a reduction in the number of PrE cells, the embryos eventually develop normally (Krawchuk et al., 2013). Thus, lower activity of the Fgf4-Fgfr-ERK pathway affects PrE maturation but is restored during development. We presume that the PrE maturation of Klf5 OE embryos is restored as seen in Fgf4 heterozygous embryos. Although Klf5 OE embryos developed normally until E8.0, they died at E11.5 because of unknown reasons, while Klf5 KO embryos showed reduced Cdx2 expression and failed to promote blastocoel expansion, indicating a defect in TE development. Taken together, these results clearly indicated that loss or overexpression of Klf5 results in skewed cell fate alterations in the EPI/PrE lineages during preimplantation development.Over-activation of the Fgf4–Fgfr–ERK pathway in Klf5 KO embryosTo elucidate the molecular mechanism involved in the accelerated PrE lineage specification of Klf5 KO embryos, microarray analyses were performed using amplified cDNAs from WT and Klf5 KO embryos at E3.0 (Fig. S5A) because Klf5 KO embryos at this stage showed no apparent defects and had normal expression levels of Oct3/4, Nanog, Sox2, and Cdx2 mRNAs (Fig. S5B). Bioinformatic analysis indicated that Fgf4 expression was upregulated in Klf5 KO embryos, whereas Spry4, a negative regulator of Fgf-induced ERK activation, was downregulated (Fig. 3A). Pdgfra and Sox17, markers for the PrE lineage, were upregulated in Klf5 KO embryos (Fig. 3A). In agreement with these observations, quantitative reverse transcription polymerase chain reaction (RT–qPCR) analysis confirmed that Fgf4, Pdgfra and Sox17 were significantly upregulated in Klf5 KO embryos at E3.0 (Fig. 3B). Our quantification of Sox17 immunostaining in Klf5 KO embryos showed increased numbers of Sox17+ cells per embryo, as well as increased staining for Sox17 on a per cell level at E3.5 (Figure S3C, D). Therefore, the upregulation of PrE genes in our microarray analysis appears to be a combination of increased PrE cells per embryo, and an increased level of PrE gene expression per cell in some cases, such as Sox17.To examine FGF4 protein expression in Klf5 KO embryos, we validated an anti-FGF4 antibody by staining mouse Fgf4 KO and WT ES cells and found that it could exclusively recognise endogenous FGF4 protein expressed in these cells (Fig. S5C). We further validated the anti-FGF4 antibody by staining the Nanog+ EPI cells of WT blastocysts at E3.75 (Fig. S5D) and found that the staining pattern was consistent with a previous report by Frankenberg et al. ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1016/j.devcel.2011.10.019", "ISSN" : "1878-1551", "PMID" : "22172669", "abstract" : "During preimplantation mouse development, the inner cell mass (ICM) differentiates into two cell lineages--the epiblast and the primitive endoderm (PrE)--whose precursors are identifiable by reciprocal expression of Nanog and Gata6, respectively. PrE formation depends on Nanog by a non-cell-autonomous mechanism. To decipher early cell- and non-cell-autonomous effects, we performed a mosaic knockdown of Nanog and found that this is sufficient to induce a PrE fate cell autonomously. Strikingly, in Nanog null embryos, Gata6 expression is maintained, showing that initiation of the PrE program is Nanog independent. Treatment of Nanog null embryos with pharmacological inhibitors revealed that RTK dependency of Gata6 expression is initially direct but later indirect via Nanog repression. Moreover, we found that subsequent expression of Sox17 and Gata4--later markers of the PrE--depends on the presence of Fgf4 produced by Nanog-expressing cells. Thus, our results reveal three distinct phases in the PrE differentiation program.", "author" : [ { "dropping-particle" : "", "family" : "Frankenberg", "given" : "Stephen", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Gerbe", "given" : "Fran\u00e7ois", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Bessonnard", "given" : "Sylvain", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Belville", "given" : "Corinne", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Pouchin", "given" : "Pierre", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Bardot", "given" : "Olivier", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Chazaud", "given" : "Claire", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Developmental cell", "id" : "ITEM-1", "issue" : "6", "issued" : { "date-parts" : [ [ "2011", "12", "13" ] ] }, "page" : "1005-13", "title" : "Primitive endoderm differentiates via a three-step mechanism involving Nanog and RTK signaling.", "type" : "article-journal", "volume" : "21" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(Frankenberg et al., 2011)", "plainTextFormattedCitation" : "(Frankenberg et al., 2011)", "previouslyFormattedCitation" : "(Frankenberg et al., 2011)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(Frankenberg et al., 2011). Immunohistochemical analysis with this anti-FGF4 antibody confirmed that FGF4 was abundantly expressed in Klf5 KO embryos at E3.0 and E3.25 (Fig. 3C, D, E, F). In contrast, FGF4 expression was significantly reduced in Klf5 OE embryos at E3.25, indicating that Klf5 suppresses Fgf4 (Fig. 3E, F). Since Fgf4 encodes a secreted protein, it is difficult to identify Fgf4-expressing cells. To resolve this issue, we performed single-cell RT-qPCR analysis with amplified cDNA prepared from individual blastomeres of inner cells in WT, Klf5 KO and Klf5 OE embryos at E3.25 using the single-cell mRNA 3-prime end sequencing (SC3-seq) method ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1093/nar/gkv134", "ISSN" : "1362-4962", "PMID" : "25722368", "abstract" : "Single-cell mRNA sequencing (RNA-seq) methods have undergone rapid development in recent years, and transcriptome analysis of relevant cell populations at single-cell resolution has become a key research area of biomedical sciences. We here present single-cell mRNA 3-prime end sequencing (SC3-seq), a practical methodology based on PCR amplification followed by 3-prime-end enrichment for highly quantitative, parallel and cost-effective measurement of gene expression in single cells. The SC3-seq allows excellent quantitative measurement of mRNAs ranging from the 10,000-cell to 1-cell level, and accordingly, allows an accurate estimate of the transcript levels by a regression of the read counts of spike-in RNAs with defined copy numbers. The SC3-seq has clear advantages over other typical single-cell RNA-seq methodologies for the quantitative measurement of transcript levels and at a sequence depth required for the saturation of transcript detection. The SC3-seq distinguishes four distinct cell types in the peri-implantation mouse blastocysts. Furthermore, the SC3-seq reveals the heterogeneity in human-induced pluripotent stem cells (hiPSCs) cultured under on-feeder as well as feeder-free conditions, demonstrating a more homogeneous property of the feeder-free hiPSCs. We propose that SC3-seq might be used as a powerful strategy for single-cell transcriptome analysis in a broad range of investigations in biomedical sciences.", "author" : [ { "dropping-particle" : "", "family" : "Nakamura", "given" : "Tomonori", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Yabuta", "given" : "Yukihiro", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Okamoto", "given" : "Ikuhiro", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Aramaki", "given" : "Shinya", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Yokobayashi", "given" : "Shihori", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Kurimoto", "given" : "Kazuki", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Sekiguchi", "given" : "Kiyotoshi", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Nakagawa", "given" : "Masato", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Yamamoto", "given" : "Takuya", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Saitou", "given" : "Mitinori", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Nucleic acids research", "id" : "ITEM-1", "issue" : "9", "issued" : { "date-parts" : [ [ "2015", "5", "19" ] ] }, "page" : "e60", "publisher" : "Oxford University Press", "title" : "SC3-seq: a method for highly parallel and quantitative measurement of single-cell gene expression.", "type" : "article-journal", "volume" : "43" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(Nakamura et al., 2015)", "plainTextFormattedCitation" : "(Nakamura et al., 2015)", "previouslyFormattedCitation" : "(Nakamura et al., 2015)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(Nakamura et al., 2015) (Fig. 4A). There were two populations: Fgf4-high inner cells and Fgf4-low/negative inner cells. Given that Pdgfra, Fgfr2 and Sox17 are expressed in Fgf4-low/negative inner cells, but not in Fgf4-high inner cells, Fgf4-low/negative inner cells and Fgf4-high inner cells may be PrE- and EPI-cells, respectively (Fig. 4A). There was no significant difference in the expression pattern of major lineage markers, such as Nanog, Gata6, Oct3/4 and Sox2, between WT, Klf5 KO and Klf5 OE embryos (Fig. 4A). Importantly, the proportion of Fgf4-high inner cells to Fgf4-low/negative inner cells was significantly increased in Klf5 KO embryos at E3.25 (Fig. 4B, C). Interestingly, Fgf4 mRNA was significantly upregulated in Fgf4-high inner cells of Klf5 KO embryos compared to those of WT embryos (Fig. 4B, D), whereas Fgf4 expression was downregulated in Fgf4-high inner cells of Klf5 OE embryos compared to those of WT embryos (Fig. 4B, D). These results clearly demonstrated that Klf5 suppresses Fgf4 in Fgf4-high inner cells at E3.0-E3.25. To investigate whether Klf5 directly regulates Fgf4, we surveyed the genomic binding sites of Klf5 by examining public ChIP-seq data and found that three candidate regions in Fgf4 loci were occupied by Klf5 in mouse ES cells (Fig. 4E). To verify this result, we established ES cell lines that overexpressed epitope-tagged Klf5 and confirmed that the epitope-tagged Klf5 binds to the three regions of Fgf4 (Fig. 4F) and to the promoter and enhancer region of Nanog ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1371/journal.pone.0150715", "ISSN" : "1932-6203", "PMID" : "26943822", "abstract" : "Pluripotency is maintained in mouse embryonic stem (ES) cells and is induced from somatic cells by the activation of appropriate transcriptional regulatory networks. Kr\u00fcppel-like factor gene family members, such as Klf2, Klf4 and Klf5, have important roles in maintaining the undifferentiated state of mouse ES cells as well as in cellular reprogramming, yet it is not known whether other Klf family members exert self-renewal and reprogramming functions when overexpressed. In this study, we examined whether overexpression of any representative Klf family member, such as Klf1-Klf10, would be sufficient for the self-renewal of mouse ES cells. We found that only Klf2, Klf4, and Klf5 produced leukemia inhibitory factor (LIF)-independent self-renewal, although most KLF proteins, if not all, have the ability to occupy the regulatory regions of Nanog, a critical Klf target gene. We also examined whether overexpression of any of Klf1-Klf10 would be sufficient to convert epiblast stem cells into a na\u00efve pluripotent state and found that Klf5 had such reprogramming ability, in addition to Klf2 and Klf4. We also delineated the functional domains of the Klf2 protein for LIF-independent self-renewal and reprogramming. Interestingly, we found that both the N-terminal transcriptional activation and C-terminal zinc finger domains were indispensable for this activity. Taken together, our comprehensive analysis provides new insight into the contribution of Klf family members to mouse ES self-renewal and cellular reprogramming.", "author" : [ { "dropping-particle" : "", "family" : "Jeon", "given" : "Hyojung", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Waku", "given" : "Tsuyoshi", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Azami", "given" : "Takuya", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Khoa", "given" : "Le Tran Phuc", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Yanagisawa", "given" : "Jun", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Takahashi", "given" : "Satoru", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Ema", "given" : "Masatsugu", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "PloS one", "editor" : [ { "dropping-particle" : "", "family" : "Rajasingh", "given" : "Johnson", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "id" : "ITEM-1", "issue" : "3", "issued" : { "date-parts" : [ [ "2016", "3", "4" ] ] }, "page" : "e0150715", "title" : "Comprehensive Identification of Kr\u00fcppel-Like Factor Family Members Contributing to the Self-Renewal of Mouse Embryonic Stem Cells and Cellular Reprogramming.", "type" : "article-journal", "volume" : "11" }, "uris" : [ "" ] }, { "id" : "ITEM-2", "itemData" : { "DOI" : "10.1038/ncb1698", "ISSN" : "1476-4679", "PMID" : "18264089", "abstract" : "Embryonic stem (ES) cells are unique in their ability to self-renew indefinitely and maintain pluripotency. These properties require transcription factors that specify the gene expression programme of ES cells. It has been possible to reverse the highly differentiated state of somatic cells back to a pluripotent state with a combination of four transcription factors: Klf4 is one of the reprogramming factors required, in conjunction with Oct4, Sox2 and c-Myc. Maintenance of self-renewal and pluripotency of ES cells requires Oct4, Sox2 and c-Myc, but Klf4 is dispensable. Here, we show that Kr\u00fcppel-like factors are required for the self-renewal of ES cells. Simultaneous depletion of Klf2, Klf4 and Klf5 lead to ES cell differentiation. Chromatin immunoprecipitation coupled to microarray assay reveals that these Klf proteins share many common targets of Nanog, suggesting a close functional relationship between these factors. Expression analysis after triple RNA interference (RNAi) of the Klfs shows that they regulate key pluripotency genes, such as Nanog. Taken together, our study provides new insight into how the core Klf circuitry integrates into the Nanog transcriptional network to specify gene expression that is unique to ES cells.", "author" : [ { "dropping-particle" : "", "family" : "Jiang", "given" : "Jianming", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Chan", "given" : "Yun-Shen", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Loh", "given" : "Yuin-Han", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Cai", "given" : "Jun", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Tong", "given" : "Guo-Qing", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Lim", "given" : "Ching-Aeng", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Robson", "given" : "Paul", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Zhong", "given" : "Sheng", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Ng", "given" : "Huck-Hui", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Nature cell biology", "id" : "ITEM-2", "issue" : "3", "issued" : { "date-parts" : [ [ "2008", "3" ] ] }, "page" : "353-60", "title" : "A core Klf circuitry regulates self-renewal of embryonic stem cells.", "type" : "article-journal", "volume" : "10" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(Jeon et al., 2016; Jiang et al., 2008)", "manualFormatting" : "(Jeon et al., 2016)", "plainTextFormattedCitation" : "(Jeon et al., 2016; Jiang et al., 2008)", "previouslyFormattedCitation" : "(Jeon et al., 2016; Jiang et al., 2008)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(Jeon et al., 2016). These data suggest that Klf5 represses Fgf4 through direct regulation. Although inner cells at E3.25 express Fgfr2 homogeneously, Fgf4 was observed to be expressed at two distinct levels, high expressing and low expressing, in populations of cells ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1016/j.devcel.2010.02.012", "ISSN" : "1878-1551", "PMID" : "20412781", "abstract" : "Three distinct cell types are present within the 64-cell stage mouse blastocyst. We have investigated cellular development up to this stage using single-cell expression analysis of more than 500 cells. The 48 genes analyzed were selected in part based on a whole-embryo analysis of more than 800 transcription factors. We show that in the morula, blastomeres coexpress transcription factors specific to different lineages, but by the 64-cell stage three cell types can be clearly distinguished according to their quantitative expression profiles. We identify Id2 and Sox2 as the earliest markers of outer and inner cells, respectively. This is followed by an inverse correlation in expression for the receptor-ligand pair Fgfr2/Fgf4 in the early inner cell mass. Position and signaling events appear to precede the maturation of the transcriptional program. These results illustrate the power of single-cell expression analysis to provide insight into developmental mechanisms. The technique should be widely applicable to other biological systems.", "author" : [ { "dropping-particle" : "", "family" : "Guo", "given" : "Guoji", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Huss", "given" : "Mikael", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Tong", "given" : "Guo Qing", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Wang", "given" : "Chaoyang", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Li Sun", "given" : "Li", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Clarke", "given" : "Neil D", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Robson", "given" : "Paul", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Developmental cell", "id" : "ITEM-1", "issue" : "4", "issued" : { "date-parts" : [ [ "2010", "4", "20" ] ] }, "page" : "675-85", "publisher" : "Elsevier Ltd", "title" : "Resolution of cell fate decisions revealed by single-cell gene expression analysis from zygote to blastocyst.", "type" : "article-journal", "volume" : "18" }, "uris" : [ "" ] }, { "id" : "ITEM-2", "itemData" : { "DOI" : "10.1093/nar/gkl050", "ISSN" : "1362-4962", "PMID" : "16547197", "abstract" : "A systems-level understanding of a small but essential population of cells in development or adulthood (e.g. somatic stem cells) requires accurate quantitative monitoring of genome-wide gene expression, ideally from single cells. We report here a strategy to globally amplify mRNAs from single cells for highly quantitative high-density oligonucleotide microarray analysis that combines a small number of directional PCR cycles with subsequent linear amplification. Using this strategy, both the representation of gene expression profiles and reproducibility between individual experiments are unambiguously improved from the original method, along with high coverage and accuracy. The immediate application of this method to single cells in the undifferentiated inner cell masses of mouse blastocysts at embryonic day (E) 3.5 revealed the presence of two populations of cells, one with primitive endoderm (PE) expression and the other with pluripotent epiblast-like gene expression. The genes expressed differentially between these two populations were well preserved in morphologically differentiated PE and epiblast in the embryos one day later (E4.5), demonstrating that the method successfully detects subtle but essential differences in gene expression at the single-cell level among seemingly homogeneous cell populations. This study provides a strategy to analyze biophysical events in medicine as well as in neural, stem cell and developmental biology, where small numbers of distinctive or diseased cells play critical roles.", "author" : [ { "dropping-particle" : "", "family" : "Kurimoto", "given" : "Kazuki", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Yabuta", "given" : "Yukihiro", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Ohinata", "given" : "Yasuhide", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Ono", "given" : "Yukiko", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Uno", "given" : "Kenichiro D", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Yamada", "given" : "Rikuhiro G", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Ueda", "given" : "Hiroki R", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Saitou", "given" : "Mitinori", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Nucleic acids research", "id" : "ITEM-2", "issue" : "5", "issued" : { "date-parts" : [ [ "2006", "1" ] ] }, "page" : "e42", "title" : "An improved single-cell cDNA amplification method for efficient high-density oligonucleotide microarray analysis.", "type" : "article-journal", "volume" : "34" }, "uris" : [ "" ] }, { "id" : "ITEM-3", "itemData" : { "DOI" : "10.1038/ncb2881", "ISSN" : "1476-4679", "PMID" : "24292013", "abstract" : "It is now recognized that extensive expression heterogeneities among cells precede the emergence of lineages in the early mammalian embryo. To establish a map of pluripotent epiblast (EPI) versus primitive endoderm (PrE) lineage segregation within the inner cell mass (ICM) of the mouse blastocyst, we characterized the gene expression profiles of individual ICM cells. Clustering analysis of the transcriptomes of 66 cells demonstrated that initially they are non-distinguishable. Early in the segregation, lineage-specific marker expression exhibited no apparent correlation, and a hierarchical relationship was established only in the late blastocyst. Fgf4 exhibited a bimodal expression at the earliest stage analysed, and in its absence, the differentiation of PrE and EPI was halted, indicating that Fgf4 drives, and is required for, ICM lineage segregation. These data lead us to propose a model where stochastic cell-to-cell expression heterogeneity followed by signal reinforcement underlies ICM lineage segregation by antagonistically separating equivalent cells.", "author" : [ { "dropping-particle" : "", "family" : "Ohnishi", "given" : "Yusuke", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Huber", "given" : "Wolfgang", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Tsumura", "given" : "Akiko", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Kang", "given" : "Minjung", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Xenopoulos", "given" : "Panagiotis", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Kurimoto", "given" : "Kazuki", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Ole\u015b", "given" : "Andrzej K.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Ara\u00fazo-Bravo", "given" : "Marcos J.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Saitou", "given" : "Mitinori", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Hadjantonakis", "given" : "Anna-Katerina", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Hiiragi", "given" : "Takashi", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Nature cell biology", "id" : "ITEM-3", "issue" : "1", "issued" : { "date-parts" : [ [ "2014", "1" ] ] }, "page" : "27-37", "publisher" : "Nature Publishing Group", "title" : "Cell-to-cell expression variability followed by signal reinforcement progressively segregates early mouse lineages.", "type" : "article-journal", "volume" : "16" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(Guo et al., 2010; Kurimoto et al., 2006; Ohnishi et al., 2014)", "plainTextFormattedCitation" : "(Guo et al., 2010; Kurimoto et al., 2006; Ohnishi et al., 2014)", "previouslyFormattedCitation" : "(Guo et al., 2010; Kurimoto et al., 2006; Ohnishi et al., 2014)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(Guo et al., 2010; Kurimoto et al., 2006; Ohnishi et al., 2014). However, the regulatory mechanism of Fgf4 at an early stage, such as the morula, is unknown ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1242/dev.128314", "ISSN" : "1477-9129", "PMID" : "27048685", "abstract" : "During mouse preimplantation embryo development, totipotent blastomeres generate the first three cell lineages of the embryo: trophectoderm, epiblast and primitive endoderm. In recent years, studies have shown that this process appears to be regulated by differences in cell-cell interactions, gene expression and the microenvironment of individual cells, rather than the active partitioning of maternal determinants. Precisely how these differences first emerge and how they dictate subsequent molecular and cellular behaviours are key questions in the field. As we review here, recent advances in live imaging, computational modelling and single-cell transcriptome analyses are providing new insights into these questions.", "author" : [ { "dropping-particle" : "", "family" : "Chazaud", "given" : "Claire", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Yamanaka", "given" : "Yojiro", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Development (Cambridge, England)", "id" : "ITEM-1", "issue" : "7", "issued" : { "date-parts" : [ [ "2016", "4", "1" ] ] }, "page" : "1063-74", "publisher" : "Oxford University Press for The Company of Biologists Limited", "title" : "Lineage specification in the mouse preimplantation embryo.", "type" : "article-journal", "volume" : "143" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(Chazaud and Yamanaka, 2016)", "plainTextFormattedCitation" : "(Chazaud and Yamanaka, 2016)", "previouslyFormattedCitation" : "(Chazaud and Yamanaka, 2016)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(Chazaud and Yamanaka, 2016). To the best of our knowledge, we are the first to identify Klf5 as a critical regulator for Fgf4 at E3.0-3.25.Of note, cis-regulatory regions in the Fgf4 promoter and enhancers are occupied by Klf5 in mouse ES cells, as shown in Fig. 4E, F. However, the lack of Klf5 expression did not significantly alter Fgf4 expression in mouse ES cells (Fig. S5E). This finding suggests a minor role for Klf5 in the transcription of Fgf4 in mouse ES cells, in contrast to its strongly suppressive role on Fgf4 at E3.0 or E3.25. Previous work showed that Klf5 is a context-dependent transcription factor and, depending on the cofactor or nuclear environment, behaves as a transcriptional repressor or activator on the same set of genes ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1038/nm1756", "ISBN" : "1546-170X (Electronic)\\n1078-8956 (Linking)", "ISSN" : "1546-170X", "PMID" : "18500350", "abstract" : "Obesity and metabolic syndrome are increasingly recognized as major risk factors for cardiovascular disease. Herein we show that Kr\u00fcppel-like transcription factor 5 (KLF5) is a crucial regulator of energy metabolism. Klf5(+/-) mice were resistant to high fat-induced obesity, hypercholesterolemia and glucose intolerance, despite consuming more food than wild-type mice. This may in part reflect their enhanced energy expenditure. Expression of the genes involved in lipid oxidation and energy uncoupling, including those encoding carnitine-palmitoyl transferase-1b (Cpt1b) and uncoupling proteins 2 and 3 (Ucp2 and Ucp3), was upregulated in the soleus muscles of Klf5(+/-) mice. Under basal conditions, KLF5 modified with small ubiquitin-related modifier (SUMO) proteins was associated with transcriptionally repressive regulatory complexes containing unliganded peroxisome proliferator-activated receptor-delta (PPAR-delta) and co-repressors and thus inhibited Cpt1b, Ucp2 and Ucp3 expression. Upon agonist stimulation of PPAR-delta, KLF5 was deSUMOylated, and became associated with transcriptional activation complexes containing both the liganded PPAR-delta and CREB binding protein (CBP). This activation complex increased the expression of Cpt1b, Ucp2 and Ucp3. Thus, SUMOylation seems to be a molecular switch affecting function of KLF5 and the transcriptional regulatory programs governing lipid metabolism.", "author" : [ { "dropping-particle" : "", "family" : "Oishi", "given" : "Yumiko", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Manabe", "given" : "Ichiro", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Tobe", "given" : "Kazuyuki", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Ohsugi", "given" : "Mitsuru", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Kubota", "given" : "Tetsuya", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Fujiu", "given" : "Katsuhito", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Maemura", "given" : "Koji", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Kubota", "given" : "Naoto", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Kadowaki", "given" : "Takashi", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Nagai", "given" : "Ryozo", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Nature medicine", "id" : "ITEM-1", "issue" : "6", "issued" : { "date-parts" : [ [ "2008", "6" ] ] }, "page" : "656-66", "title" : "SUMOylation of Kr\u00fcppel-like transcription factor 5 acts as a molecular switch in transcriptional programs of lipid metabolism involving PPAR-delta.", "type" : "article-journal", "volume" : "14" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(Oishi et al., 2008)", "plainTextFormattedCitation" : "(Oishi et al., 2008)", "previouslyFormattedCitation" : "(Oishi et al., 2008)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(Oishi et al., 2008). Therefore, the different outcomes of Fgf4 transcription promoted by the absence of Klf5 might result in a different cell type, i.e., inner cells at E3.0-3.25 versus mouse ES cells (equivalent to EPI cells at E4.25) ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1038/ncb2965", "ISBN" : "1476-4679 (Electronic)\\r1465-7392 (Linking)", "ISSN" : "1476-4679", "PMID" : "24859004", "abstract" : "The precise relationship of embryonic stem cells (ESCs) to cells in the mouse embryo remains controversial. We present transcriptional and functional data to identify the embryonic counterpart of ESCs. Marker profiling shows that ESCs are distinct from early inner cell mass (ICM) and closely resemble pre-implantation epiblast. A characteristic feature of mouse ESCs is propagation without ERK signalling. Single-cell culture reveals that cell-autonomous capacity to thrive when the ERK pathway is inhibited arises late during blastocyst development and is lost after implantation. The frequency of deriving clonal ESC lines suggests that all E4.5 epiblast cells can become ESCs. We further show that ICM cells from early blastocysts can progress to ERK independence if provided with a specific laminin substrate. These findings suggest that formation of the epiblast coincides with competence for ERK-independent self-renewal in vitro and consequent propagation as ESC lines.", "author" : [ { "dropping-particle" : "", "family" : "Boroviak", "given" : "Thorsten", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Loos", "given" : "Remco", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Bertone", "given" : "Paul", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Smith", "given" : "Austin", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Nichols", "given" : "Jennifer", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Nature cell biology", "id" : "ITEM-1", "issue" : "6", "issued" : { "date-parts" : [ [ "2014", "6" ] ] }, "page" : "516-28", "title" : "The ability of inner-cell-mass cells to self-renew as embryonic stem cells is acquired following epiblast specification.", "type" : "article-journal", "volume" : "16" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(Boroviak et al., 2014)", "plainTextFormattedCitation" : "(Boroviak et al., 2014)", "previouslyFormattedCitation" : "(Boroviak et al., 2014)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(Boroviak et al., 2014).Fgfr or MEK inhibitors reverse the skewed lineage specification of Klf5 KO blastocystsOur data clearly indicate that Klf5 KO blastocysts show cell proliferation defects and accelerated PrE specification (Fig. S1I, Fig. 1, and Fig. 2), yet it is not clear whether the over-activation of Fgf4-Fgfr-ERK signalling is responsible for cell proliferation defects and accelerated PrE lineage specification in Klf5 KO embryos. In fact, Lin et al. reported no difference in the phosphorylated-ERK (pERK) signal between WT and Klf5 KO blastocysts ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1242/dev.054775", "ISSN" : "1477-9129", "PMID" : "20980403", "abstract" : "Kruppel-like transcription factors (Klfs) are essential for the induction and maintenance of pluripotency of embryonic stem cells (ESCs), yet little is known about their roles in establishing the three lineages of the pre-implantation embryo. Here, we show that Klf5 is required for the formation of the trophectoderm (TE) and the inner cell mass (ICM), and for repressing primitive endoderm (PE) development. Although cell polarity appeared normal, Klf5 mutant embryos arrested at the blastocyst stage and failed to hatch due to defective TE development. Klf5 acted cell-autonomously in the TE, downstream of Fgf4 and upstream of Cdx2, Eomes and Krt8. In the ICM, loss of Klf5 resulted in reduced expression of pluripotency markers Oct4 and Nanog, but led to increased Sox17 expression in the PE, suggesting that Klf5 suppresses the PE lineage. Consistent with this, overexpression of Klf5 in transgenic embryos was sufficient to suppress the Sox17(+) PE lineage in the ICM. Klf5 overexpression led to a dose-dependent decrease in Sox17 promoter activity in reporter assays in cultured cells. Moreover, in chimeric embryos, Klf5(-/-) cells preferentially contributed to the Sox17(+) PE lineage and Cdx2 expression was not rescued in Klf5(-/-) outer cells. Finally, outgrowths from Klf5(-/-) embryos failed to form an ICM/pluripotent colony, had very few Oct4(+) or Cdx2(+) cells, but showed an increase in the percentage of Sox17(+) PE cells. These findings demonstrate that Klf5 is a dynamic regulator of all three lineages in the pre-implantation embryo by promoting the TE and epiblast lineages while suppressing the PE lineage.", "author" : [ { "dropping-particle" : "", "family" : "Lin", "given" : "Suh-Chin J", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Wani", "given" : "Maqsood a", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Whitsett", "given" : "Jeffrey a", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Wells", "given" : "James M", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Development (Cambridge, England)", "id" : "ITEM-1", "issue" : "23", "issued" : { "date-parts" : [ [ "2010", "12" ] ] }, "page" : "3953-63", "title" : "Klf5 regulates lineage formation in the pre-implantation mouse embryo.", "type" : "article-journal", "volume" : "137" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(Lin et al., 2010)", "plainTextFormattedCitation" : "(Lin et al., 2010)", "previouslyFormattedCitation" : "(Lin et al., 2010)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(Lin et al., 2010). However, it is of note that careful and extensive immunohistochemical analyses showed that a strong background signal hampers the significant detection of pERK in preimplantation embryos (Frankenberg et al., 2011), while pERK signals can be observed reproducibly in whole embryos after E5.5 ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1242/dev.00669", "ISSN" : "0950-1991", "PMID" : "12925581", "abstract" : "Signaling between tissues is essential to form the complex, three-dimensional organization of an embryo. Because many receptor tyrosine kinases signal through the RAS-MAPK pathway, phosphorylated ERK can be used as an indicator of when and where signaling is active during development. Using whole-mount immunohistochemistry with antibodies specific to phosphorylated ERK1 and ERK2, we analyzed the location, timing, distribution, duration and intensity of ERK signaling during mouse embryogenesis (5-10.5 days postcoitum). Spatial and temporal domains of ERK activation were discrete with well-defined boundaries, indicating specific regulation of signaling in vivo. Prominent, sustained domains of ERK activation were seen in the ectoplacental cone, extra-embryonic ectoderm, limb buds, branchial arches, frontonasal process, forebrain, midbrain-hindbrain boundary, tailbud, foregut and liver. Transient activation was seen in neural crest, peripheral nervous system, nascent blood vessels, and anlagen of the eye, ear and heart. In the contiguous domains of ERK signaling, phospho-ERK staining was cytoplasmic with no sign of nuclear translocation. With few exceptions, the strongest domains of ERK activation correlated with regions of known or suspected fibroblast growth factor (FGF) signaling, and brief incubation with an inhibitor of the fibroblast growth factor receptor (FGFR) specifically diminished the phospho-ERK staining in these regions. Although many domains of ERK activation were FGFR-dependent, not all domains of FGF signaling were phospho-ERK positive. These studies identify key domains of sustained ERK signaling in the intact mouse embryo, give significant insight into the regulation of this signaling in vivo and pinpoint regions where downstream target genes can be sought.", "author" : [ { "dropping-particle" : "", "family" : "Corson", "given" : "Laura Beth", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Yamanaka", "given" : "Yojiro", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Lai", "given" : "Ka-Man Venus", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Rossant", "given" : "Janet", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Development (Cambridge, England)", "id" : "ITEM-1", "issue" : "19", "issued" : { "date-parts" : [ [ "2003", "10" ] ] }, "page" : "4527-37", "title" : "Spatial and temporal patterns of ERK signaling during mouse embryogenesis.", "type" : "article-journal", "volume" : "130" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(Corson et al., 2003)", "plainTextFormattedCitation" : "(Corson et al., 2003)", "previouslyFormattedCitation" : "(Corson et al., 2003)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(Corson et al., 2003). To examine whether Fgf4-Fgfr-ERK signalling is responsible for the phenotype of Klf5 KO blastocysts, we used chemical inhibitors to block the kinase activities of Fgfr1/2 (SU5402) and MEK (PD0325901) (Fig. 5A). Morulae at E2.75 were collected and cultured for 24 h in the presence or absence of these inhibitors, and we found that Klf5 KO embryos treated with either SU5402 or PD0325901 showed marked phenotypic rescue in terms of a normal morphology with an expanded blastocoel and were indistinguishable from WT blastocysts (Fig. 5B). The numbers of cells per embryo were also increased but were still significantly different from the WT embryos, suggesting that Klf5 regulates cell proliferation in part through a Fgf4-Fgfr-ERK-independent mechanism (Fig. 5C). The numbers of TUNEL+ cells were reduced dramatically in Klf5 KO embryos and were similar to those of WT embryos (Fig. 5C). Notably, inhibitors of the JNK and p38 MAPK pathways did not significantly rescue the cell-cycle defects of Klf5 KO blastocysts (Fig. S6), indicating that JNK and p38 MAPK are not involved in the process. We also attempted to test whether excess FGF4 activity was sufficient to cause the defective cell proliferation in WT embryos and found that culturing in the presence of FGF4 slightly increased the number of TUNEL+ cells but did not significantly change the total cell number (Fig. 5D, E). When Klf5 OE embryos were cultured in the presence of saturated levels of FGF4 (1000 ng/ml) from E2.5 to E3.75 (Fig. S7A), FGF4 stimulation did not change the total cell number (Fig. S7B, C, D); however, Gata6 protein expression was significantly upregulated in FGF4-stimulated Klf5 OE embryos (Fig. S7E). To investigate whether the precocious activation of Fgf4-Fgfr-ERK signalling was responsible for the altered lineage specification of Klf5 KO blastocysts, WT and Klf5 KO morulae at E2.5 were collected and cultured for 24 or 48 h in the presence or absence of PD0325901 and were then subjected to immunohistochemistry (Fig. 6A). Klf5 KO blastocysts cultured in vitro for 24 h from E2.5 showed reduced Nanog and increased Gata6 expression levels, as did the Klf5 KO blastocysts at E3.5 (Fig. 6B, C). MEK inhibitor treatment dramatically reversed the alterations in Nanog and Gata6 expression levels (Fig. 6B, C). Klf5 KO morulae were treated with PD0325901 from E2.5 for 24 h, and most of the ICM cells were Nanog+ EPI-biased cells (Fig. 6B, C). By contrast, when Klf5 KO morulae were treated with vehicle alone for 24 h from E2.5, most of the ICM cells were Gata6+ PrE cells. Importantly, the percentage of Nanog+ cells in the ICM cells of Klf5 KO blastocysts cultured in the presence of PD0325901 was increased compared to WT control but still less than that of PD0325901-treated WT embryos (Fig. 6C). When Klf5 KO morulae were treated with PD0325901 from E2.5 for 48 h (corresponding to E4.5), all the cells in the ICMs were Nanog+ EPI cells (Fig. 6B′). Because our previous results indicated that ES cell lines were not established from Klf5 KO blastocysts and that Klf5 was indispensable for ES derivation from the ICM ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1016/j.stem.2008.09.003", "ISBN" : "1875-9777 (Electronic)", "ISSN" : "1875-9777", "PMID" : "18983969", "abstract" : "The transcription factor Klf4 has demonstrated activity in the reprogramming of somatic cells to a pluripotent state, but the molecular mechanism of this process remains unknown. It is, therefore, of great interest to understand the functional role of Klf4 and related genes in ESC regulation. Here, we show that homozygous disruption of Klf5 results in the failure of ESC derivation from ICM cells and early embryonic lethality due to an implantation defect. Klf5 KO ESCs show increased expression of several differentiation marker genes and frequent, spontaneous differentiation. Conversely, overexpression of Klf5 in ESCs suppressed the expression of differentiation marker genes and maintained pluripotency in the absence of LIF. Our results also suggest that Klf5 regulates ESC proliferation by promoting phosphorylation of Akt1 via induction of Tcl1. These results, therefore, provide new insights into the functional and mechanistic role of Klf5 in regulation of pluripotency.", "author" : [ { "dropping-particle" : "", "family" : "Ema", "given" : "Masatsugu", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Mori", "given" : "Daisuke", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Niwa", "given" : "Hitoshi", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Hasegawa", "given" : "Yoshikazu", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Yamanaka", "given" : "Yojiro", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Hitoshi", "given" : "Seiji", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Mimura", "given" : "Junsei", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Kawabe", "given" : "Yoh-ichi", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Hosoya", "given" : "Tomohiro", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Morita", "given" : "Masanobu", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Shimosato", "given" : "Daisuke", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Uchida", "given" : "Kazuhiko", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Suzuki", "given" : "Norio", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Yanagisawa", "given" : "Jun", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Sogawa", "given" : "Kazuhiro", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Rossant", "given" : "Janet", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Yamamoto", "given" : "Masayuki", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Takahashi", "given" : "Satoru", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Fujii-Kuriyama", "given" : "Yoshiaki", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Cell stem cell", "id" : "ITEM-1", "issue" : "5", "issued" : { "date-parts" : [ [ "2008", "11", "6" ] ] }, "page" : "555-67", "publisher" : "Elsevier Inc.", "title" : "Kr\u00fcppel-like factor 5 is essential for blastocyst development and the normal self-renewal of mouse ESCs.", "type" : "article-journal", "volume" : "3" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(Ema et al., 2008)", "plainTextFormattedCitation" : "(Ema et al., 2008)", "previouslyFormattedCitation" : "(Ema et al., 2008)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(Ema et al., 2008), we attempted to derive ES cell lines from PD0325901-treated Klf5 KO blastocysts; 60 ES cell lines were established and genotyped. Seven Klf5 KO ES cell lines were obtained (data not shown), demonstrating that treatment with this MEK inhibitor rescued the emergence of pluripotent EPI cells in Klf5 KO blastocysts. In contrast, treatment with the MEK or Fgfr inhibitors did not affect the reduced expression of Cdx2 (Fig. S8). This finding suggested that the dysregulation of Cdx2 in Klf5 KO blastocysts was not caused by increased signalling through the Fgf4-Fgfr-ERK pathway but was nonetheless controlled by Klf5. Taken together, our studies clearly showed that the loss of Klf5 results in the induction of Fgf4 in morula at E3.0, followed by the rapid upregulation of Pdgfra in the inner cells at E3.25 and a decrease in Nanog+ cells and DP cells at E3.5, which ultimately led to Gata6-expressing cells only. This skewed EPI/PrE phenotype with Gata6-only ICM cells was clearly reversed by MEK inhibitors. However, overexpression of Klf5 resulted in the reduction of Fgf4 in blastocysts at E3.5 and an increase in Nanog+ cells and DP cells at E3.5 or later, which ultimately led to the presence of DP “uncommitted cells” by E4.0. Therefore, our proposed model is that Klf5 represses Fgf4–Fgfr–ERK signalling to suppress its precocious activation of the PrE specification programme, thus ensuring the emergence of Nanog+ na?ve pluripotent cells during development (Fig. 7).DiscussionPrevious experiments showed that Nanog KO caused a severe reduction in Fgf4 expression in blastocysts at E3.5, demonstrating that Nanog activates Fgf4 expression in EPI precursor cells ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1016/j.devcel.2011.10.019", "ISSN" : "1878-1551", "PMID" : "22172669", "abstract" : "During preimplantation mouse development, the inner cell mass (ICM) differentiates into two cell lineages--the epiblast and the primitive endoderm (PrE)--whose precursors are identifiable by reciprocal expression of Nanog and Gata6, respectively. PrE formation depends on Nanog by a non-cell-autonomous mechanism. To decipher early cell- and non-cell-autonomous effects, we performed a mosaic knockdown of Nanog and found that this is sufficient to induce a PrE fate cell autonomously. Strikingly, in Nanog null embryos, Gata6 expression is maintained, showing that initiation of the PrE program is Nanog independent. Treatment of Nanog null embryos with pharmacological inhibitors revealed that RTK dependency of Gata6 expression is initially direct but later indirect via Nanog repression. Moreover, we found that subsequent expression of Sox17 and Gata4--later markers of the PrE--depends on the presence of Fgf4 produced by Nanog-expressing cells. Thus, our results reveal three distinct phases in the PrE differentiation program.", "author" : [ { "dropping-particle" : "", "family" : "Frankenberg", "given" : "Stephen", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Gerbe", "given" : "Fran\u00e7ois", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Bessonnard", "given" : "Sylvain", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Belville", "given" : "Corinne", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Pouchin", "given" : "Pierre", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Bardot", "given" : "Olivier", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Chazaud", "given" : "Claire", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Developmental cell", "id" : "ITEM-1", "issue" : "6", "issued" : { "date-parts" : [ [ "2011", "12", "13" ] ] }, "page" : "1005-13", "title" : "Primitive endoderm differentiates via a three-step mechanism involving Nanog and RTK signaling.", "type" : "article-journal", "volume" : "21" }, "uris" : [ "" ] }, { "id" : "ITEM-2", "itemData" : { "DOI" : "10.1016/j.ydbio.2010.04.020", "ISSN" : "1095-564X", "PMID" : "20435031", "abstract" : "Early lineage segregation in mouse development results in two, either CDX2- or OCT4/NANOG-positive, cell populations. CDX2-positive cells form the trophectoderm (TE), OCT4/NANOG-positive cells the inner cell mass (ICM). In a second lineage decision ICM cells segregate into Epiblast (EPI) and primitive endoderm (PE). EPI and PE formation depend on the activity of the transcription factors Nanog and Gata4/6. A role for Nanog, a crucial pluripotency factor, in preventing PE differentiation has been proposed, as outgrowths of mutant ICMs result in PE, but not EPI derivatives. We established Nanog-mutant mouse lines and analyzed EPI and PE formation in vivo. Surprisingly, Gata4 expression in mutant ICM cells is absent or strongly decreased, thus loss of Nanog does not result in precocious endoderm differentiation. However, Nanog-deficient embryos retain the capacity to form PE in chimeric embryos and, in contrast to recent reports, in blastocyst outgrowths. Based on our findings we propose a non-cell autonomous requirement of Nanog for proper PE formation in addition to its essential role in EPI determination.", "author" : [ { "dropping-particle" : "", "family" : "Messerschmidt", "given" : "Daniel M", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Kemler", "given" : "Rolf", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Developmental biology", "id" : "ITEM-2", "issue" : "1", "issued" : { "date-parts" : [ [ "2010", "8", "1" ] ] }, "page" : "129-37", "publisher" : "Elsevier Inc.", "title" : "Nanog is required for primitive endoderm formation through a non-cell autonomous mechanism.", "type" : "article-journal", "volume" : "344" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(Frankenberg et al., 2011; Messerschmidt and Kemler, 2010)", "plainTextFormattedCitation" : "(Frankenberg et al., 2011; Messerschmidt and Kemler, 2010)", "previouslyFormattedCitation" : "(Frankenberg et al., 2011; Messerschmidt and Kemler, 2010)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(Frankenberg et al., 2011; Messerschmidt and Kemler, 2010). Moreover, Oct3/4 KO led to reduced Fgf4 expression in blastocysts at E3.5 ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1242/dev.096875", "ISSN" : "1477-9129", "PMID" : "24504341", "abstract" : "The transcription factor Oct4 is required in vitro for establishment and maintenance of embryonic stem cells and for reprogramming somatic cells to pluripotency. In vivo, it prevents the ectopic differentiation of early embryos into trophoblast. Here, we further explore the role of Oct4 in blastocyst formation and specification of epiblast versus primitive endoderm lineages using conditional genetic deletion. Experiments involving mouse embryos deficient for both maternal and zygotic Oct4 suggest that it is dispensable for zygote formation, early cleavage and activation of Nanog expression. Nanog protein is significantly elevated in the presumptive inner cell mass of Oct4 null embryos, suggesting an unexpected role for Oct4 in attenuating the level of Nanog, which might be significant for priming differentiation during epiblast maturation. Induced deletion of Oct4 during the morula to blastocyst transition disrupts the ability of inner cell mass cells to adopt lineage-specific identity and acquire the molecular profile characteristic of either epiblast or primitive endoderm. Sox17, a marker of primitive endoderm, is not detected following prolonged culture of such embryos, but can be rescued by provision of exogenous FGF4. Interestingly, functional primitive endoderm can be rescued in Oct4-deficient embryos in embryonic stem cell complementation assays, but only if the host embryos are at the pre-blastocyst stage. We conclude that cell fate decisions within the inner cell mass are dependent upon Oct4 and that Oct4 is not cell-autonomously required for the differentiation of primitive endoderm derivatives, as long as an appropriate developmental environment is established.", "author" : [ { "dropping-particle" : "", "family" : "Bin", "given" : "Gloryn Chia", "non-dropping-particle" : "Le", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Mu\u00f1oz-Descalzo", "given" : "Silvia", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Kurowski", "given" : "Agata", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Leitch", "given" : "Harry", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Lou", "given" : "Xinghua", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Mansfield", "given" : "William", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Etienne-Dumeau", "given" : "Charles", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Grabole", "given" : "Nils", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Mulas", "given" : "Carla", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Niwa", "given" : "Hitoshi", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Hadjantonakis", "given" : "Anna-Katerina", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Nichols", "given" : "Jennifer", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Development (Cambridge, England)", "id" : "ITEM-1", "issue" : "5", "issued" : { "date-parts" : [ [ "2014", "3" ] ] }, "page" : "1001-10", "title" : "Oct4 is required for lineage priming in the developing inner cell mass of the mouse blastocyst.", "type" : "article-journal", "volume" : "141" }, "uris" : [ "" ] }, { "id" : "ITEM-2", "itemData" : { "DOI" : "10.1016/j.devcel.2013.05.004", "ISBN" : "1878-1551 (Electronic)\\r1534-5807 (Linking)", "ISSN" : "1878-1551", "PMID" : "23747191", "abstract" : "In embryonic stem (ES) cells and in early mouse embryos, the transcription factor Oct4 is an essential regulator of pluripotency. Oct4 transcriptional targets have been described in ES cell lines; however, the molecular mechanisms by which Oct4 regulates establishment of pluripotency in the epiblast (EPI) have not been fully elucidated. Here, we show that neither maternal nor zygotic Oct4 is required for the formation of EPI cells in the blastocyst. Rather, Oct4 is first required for development of the primitive endoderm (PE), an extraembryonic lineage. EPI cells promote PE fate in neighboring cells by secreting Fgf4, and Oct4 is required for expression of Fgf4, but we show that Oct4 promotes PE development cell-autonomously, downstream of Fgf4 and Mapk. Finally, we show that Oct4 is required for the expression of multiple EPI and PE genes as well as multiple metabolic pathways essential for the continued growth of the preimplantation embryo.", "author" : [ { "dropping-particle" : "", "family" : "Frum", "given" : "Tristan", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Halbisen", "given" : "MichaelA A", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Wang", "given" : "Chaoyang", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Amiri", "given" : "Hossein", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Robson", "given" : "Paul", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Ralston", "given" : "Amy", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Developmental cell", "id" : "ITEM-2", "issue" : "6", "issued" : { "date-parts" : [ [ "2013", "6", "24" ] ] }, "page" : "610-22", "publisher" : "Elsevier Inc.", "title" : "Oct4 cell-autonomously promotes primitive endoderm development in the mouse blastocyst.", "type" : "article-journal", "volume" : "25" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(Frum et al., 2013; Le Bin et al., 2014)", "plainTextFormattedCitation" : "(Frum et al., 2013; Le Bin et al., 2014)", "previouslyFormattedCitation" : "(Frum et al., 2013; Le Bin et al., 2014)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(Frum et al., 2013; Le Bin et al., 2014). Furthermore, mutant embryos lacking Sox2, which cooperates with Oct3/4 in the maintenance of ES cell pluripotency, showed reduced Fgf4 expression ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1371/journal.pgen.1004618", "ISSN" : "1553-7404", "PMID" : "25340657", "abstract" : "Pluripotent epiblast (EPI) cells, present in the inner cell mass (ICM) of the mouse blastocyst, are progenitors of both embryonic stem (ES) cells and the fetus. Discovering how pluripotency genes regulate cell fate decisions in the blastocyst provides a valuable way to understand how pluripotency is normally established. EPI cells are specified by two consecutive cell fate decisions. The first decision segregates ICM from trophectoderm (TE), an extraembryonic cell type. The second decision subdivides ICM into EPI and primitive endoderm (PE), another extraembryonic cell type. Here, we investigate the roles and regulation of the pluripotency gene Sox2 during blastocyst formation. First, we investigate the regulation of Sox2 patterning and show that SOX2 is restricted to ICM progenitors prior to blastocyst formation by members of the HIPPO pathway, independent of CDX2, the TE transcription factor that restricts Oct4 and Nanog to the ICM. Second, we investigate the requirement for Sox2 in cell fate specification during blastocyst formation. We show that neither maternal (M) nor zygotic (Z) Sox2 is required for blastocyst formation, nor for initial expression of the pluripotency genes Oct4 or Nanog in the ICM. Rather, Z Sox2 initially promotes development of the primitive endoderm (PE) non cell-autonomously via FGF4, and then later maintains expression of pluripotency genes in the ICM. The significance of these observations is that 1) ICM and TE genes are spatially patterned in parallel prior to blastocyst formation and 2) both the roles and regulation of Sox2 in the blastocyst are unique compared to other pluripotency factors such as Oct4 or Nanog.", "author" : [ { "dropping-particle" : "", "family" : "Wicklow", "given" : "Eryn", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Blij", "given" : "Stephanie", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Frum", "given" : "Tristan", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Hirate", "given" : "Yoshikazu", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Lang", "given" : "Richard A.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Sasaki", "given" : "Hiroshi", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Ralston", "given" : "Amy", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "PLoS genetics", "editor" : [ { "dropping-particle" : "", "family" : "Downs", "given" : "Karen M.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "id" : "ITEM-1", "issue" : "10", "issued" : { "date-parts" : [ [ "2014", "10", "23" ] ] }, "page" : "e1004618", "publisher" : "Public Library of Science", "title" : "HIPPO pathway members restrict SOX2 to the inner cell mass where it promotes ICM fates in the mouse blastocyst.", "type" : "article-journal", "volume" : "10" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(Wicklow et al., 2014)", "plainTextFormattedCitation" : "(Wicklow et al., 2014)", "previouslyFormattedCitation" : "(Wicklow et al., 2014)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(Wicklow et al., 2014). Because Oct3/4-Sox2 complexes can directly induce Fgf4 expression in vitro ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1074/jbc.M000932200", "ISBN" : "0021-9258 (Print)", "ISSN" : "0021-9258", "PMID" : "10801796", "abstract" : "Fibroblast growth factor (FGF)-4 gene expression in the inner cell mass of the blastocyst and in EC cells requires the combined activity of two transcriptional regulators, Sox2 and Oct-3, which bind to adjacent sites on the FGF-4 enhancer DNA and synergistically activate transcription. Sox2 and Oct-3 bind cooperatively to the enhancer DNA through their DNA-binding, high mobility group and POU domains, respectively. These two domains, however, are not sufficient to activate transcription. We have analyzed a number of Sox2 and Oct-3 deletion mutants to identify the domains within each protein that contribute to the activity of the Sox2 x Oct-3 complex. Within Oct-3, we have identified two activation domains, the N-terminal AD1 and the C-terminal AD2, that play a role in the activity of the Sox2 x Oct-3 complex. AD1 also displays transcriptional activation functions in the absence of Sox2 while AD2 function was only detected within the Sox2 x Oct-3 complex. In Sox2, we have identified three activation domains within its C terminus: R1, R2, and R3. R1 and R2 can potentiate weak activation by Sox2 in the absence of Oct-3 but their deletion has no effect on the Sox2 x Oct-3 complex. In contrast, R3 function is only observed when Sox2 is complexed with Oct-3. In addition, analysis of Oct-1/Oct-3 chimeras indicates that the Oct-3 homeodomain also plays a critical role in the formation of a functional Sox2 x Oct-3 complex. Our results are consistent with a model in which the synergistic action of Sox2 and Oct-3 results from two major processes. Cooperative binding of the factors to the enhancer DNA, mediated by their binding domains, stably tethers each factor to DNA and increases the activity of intrinsic activation domains within each protein. Protein-protein and protein-DNA interactions then may lead to reciprocal conformational changes that expose latent activation domains within each protein. These findings define a mechanism that may also be utilized by other Sox x POU protein complexes in gene activation.", "author" : [ { "dropping-particle" : "", "family" : "Ambrosetti", "given" : "Davide Carlo", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Sch\u00f6ler", "given" : "H R", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Dailey", "given" : "Lisa", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Basilico", "given" : "Claudio", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "The Journal of biological chemistry", "id" : "ITEM-1", "issue" : "30", "issued" : { "date-parts" : [ [ "2000", "7", "28" ] ] }, "page" : "23387-97", "title" : "Modulation of the activity of multiple transcriptional activation domains by the DNA binding domains mediates the synergistic action of Sox2 and Oct-3 on the fibroblast growth factor-4 enhancer.", "type" : "article-journal", "volume" : "275" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(Ambrosetti et al., 2000)", "plainTextFormattedCitation" : "(Ambrosetti et al., 2000)", "previouslyFormattedCitation" : "(Ambrosetti et al., 2000)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(Ambrosetti et al., 2000), there is an interplay between Nanog, Oct3/4 and Sox2 to regulate Fgf4 for proper lineage segregation ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1242/dev.128314", "ISSN" : "1477-9129", "PMID" : "27048685", "abstract" : "During mouse preimplantation embryo development, totipotent blastomeres generate the first three cell lineages of the embryo: trophectoderm, epiblast and primitive endoderm. In recent years, studies have shown that this process appears to be regulated by differences in cell-cell interactions, gene expression and the microenvironment of individual cells, rather than the active partitioning of maternal determinants. Precisely how these differences first emerge and how they dictate subsequent molecular and cellular behaviours are key questions in the field. As we review here, recent advances in live imaging, computational modelling and single-cell transcriptome analyses are providing new insights into these questions.", "author" : [ { "dropping-particle" : "", "family" : "Chazaud", "given" : "Claire", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Yamanaka", "given" : "Yojiro", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Development (Cambridge, England)", "id" : "ITEM-1", "issue" : "7", "issued" : { "date-parts" : [ [ "2016", "4", "1" ] ] }, "page" : "1063-74", "publisher" : "Oxford University Press for The Company of Biologists Limited", "title" : "Lineage specification in the mouse preimplantation embryo.", "type" : "article-journal", "volume" : "143" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(Chazaud and Yamanaka, 2016)", "plainTextFormattedCitation" : "(Chazaud and Yamanaka, 2016)", "previouslyFormattedCitation" : "(Chazaud and Yamanaka, 2016)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(Chazaud and Yamanaka, 2016). Thus, these studies showed that Nanog and Oct3/4 activate Fgf4 expression in EPI precursor cells at the E3.5 blastocyst stage; however, these studies did not address the regulatory mechanism of Fgf4 at an earlier stage, such as the morula. The mechanism involved in the induction of Fgf4 expression in a subset of inner cells at this stage is still unknown ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1242/dev.128314", "ISSN" : "1477-9129", "PMID" : "27048685", "abstract" : "During mouse preimplantation embryo development, totipotent blastomeres generate the first three cell lineages of the embryo: trophectoderm, epiblast and primitive endoderm. In recent years, studies have shown that this process appears to be regulated by differences in cell-cell interactions, gene expression and the microenvironment of individual cells, rather than the active partitioning of maternal determinants. Precisely how these differences first emerge and how they dictate subsequent molecular and cellular behaviours are key questions in the field. As we review here, recent advances in live imaging, computational modelling and single-cell transcriptome analyses are providing new insights into these questions.", "author" : [ { "dropping-particle" : "", "family" : "Chazaud", "given" : "Claire", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Yamanaka", "given" : "Yojiro", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Development (Cambridge, England)", "id" : "ITEM-1", "issue" : "7", "issued" : { "date-parts" : [ [ "2016", "4", "1" ] ] }, "page" : "1063-74", "publisher" : "Oxford University Press for The Company of Biologists Limited", "title" : "Lineage specification in the mouse preimplantation embryo.", "type" : "article-journal", "volume" : "143" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(Chazaud and Yamanaka, 2016)", "plainTextFormattedCitation" : "(Chazaud and Yamanaka, 2016)", "previouslyFormattedCitation" : "(Chazaud and Yamanaka, 2016)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(Chazaud and Yamanaka, 2016). Our study clearly demonstrated for the first time that Klf5 is a critical regulator for Fgf4 in the morula at E3.0, before the blastocyst stage. Bimodal Fgf4 expression levels precede the exclusive production of Nanog and Gata6 ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1016/j.devcel.2010.02.012", "ISSN" : "1878-1551", "PMID" : "20412781", "abstract" : "Three distinct cell types are present within the 64-cell stage mouse blastocyst. We have investigated cellular development up to this stage using single-cell expression analysis of more than 500 cells. The 48 genes analyzed were selected in part based on a whole-embryo analysis of more than 800 transcription factors. We show that in the morula, blastomeres coexpress transcription factors specific to different lineages, but by the 64-cell stage three cell types can be clearly distinguished according to their quantitative expression profiles. We identify Id2 and Sox2 as the earliest markers of outer and inner cells, respectively. This is followed by an inverse correlation in expression for the receptor-ligand pair Fgfr2/Fgf4 in the early inner cell mass. Position and signaling events appear to precede the maturation of the transcriptional program. These results illustrate the power of single-cell expression analysis to provide insight into developmental mechanisms. The technique should be widely applicable to other biological systems.", "author" : [ { "dropping-particle" : "", "family" : "Guo", "given" : "Guoji", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Huss", "given" : "Mikael", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Tong", "given" : "Guo Qing", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Wang", "given" : "Chaoyang", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Li Sun", "given" : "Li", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Clarke", "given" : "Neil D", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Robson", "given" : "Paul", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Developmental cell", "id" : "ITEM-1", "issue" : "4", "issued" : { "date-parts" : [ [ "2010", "4", "20" ] ] }, "page" : "675-85", "publisher" : "Elsevier Ltd", "title" : "Resolution of cell fate decisions revealed by single-cell gene expression analysis from zygote to blastocyst.", "type" : "article-journal", "volume" : "18" }, "uris" : [ "" ] }, { "id" : "ITEM-2", "itemData" : { "DOI" : "10.1038/ncb2881", "ISSN" : "1476-4679", "PMID" : "24292013", "abstract" : "It is now recognized that extensive expression heterogeneities among cells precede the emergence of lineages in the early mammalian embryo. To establish a map of pluripotent epiblast (EPI) versus primitive endoderm (PrE) lineage segregation within the inner cell mass (ICM) of the mouse blastocyst, we characterized the gene expression profiles of individual ICM cells. Clustering analysis of the transcriptomes of 66 cells demonstrated that initially they are non-distinguishable. Early in the segregation, lineage-specific marker expression exhibited no apparent correlation, and a hierarchical relationship was established only in the late blastocyst. Fgf4 exhibited a bimodal expression at the earliest stage analysed, and in its absence, the differentiation of PrE and EPI was halted, indicating that Fgf4 drives, and is required for, ICM lineage segregation. These data lead us to propose a model where stochastic cell-to-cell expression heterogeneity followed by signal reinforcement underlies ICM lineage segregation by antagonistically separating equivalent cells.", "author" : [ { "dropping-particle" : "", "family" : "Ohnishi", "given" : "Yusuke", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Huber", "given" : "Wolfgang", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Tsumura", "given" : "Akiko", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Kang", "given" : "Minjung", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Xenopoulos", "given" : "Panagiotis", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Kurimoto", "given" : "Kazuki", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Ole\u015b", "given" : "Andrzej K.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Ara\u00fazo-Bravo", "given" : "Marcos J.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Saitou", "given" : "Mitinori", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Hadjantonakis", "given" : "Anna-Katerina", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Hiiragi", "given" : "Takashi", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Nature cell biology", "id" : "ITEM-2", "issue" : "1", "issued" : { "date-parts" : [ [ "2014", "1" ] ] }, "page" : "27-37", "publisher" : "Nature Publishing Group", "title" : "Cell-to-cell expression variability followed by signal reinforcement progressively segregates early mouse lineages.", "type" : "article-journal", "volume" : "16" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(Guo et al., 2010; Ohnishi et al., 2014)", "plainTextFormattedCitation" : "(Guo et al., 2010; Ohnishi et al., 2014)", "previouslyFormattedCitation" : "(Guo et al., 2010; Ohnishi et al., 2014)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(Guo et al., 2010; Ohnishi et al., 2014) and is the first sign of the segregation of the EPI and PrE lineages. However, what regulates Fgf4 is unknown ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1007/s00018-014-1630-3", "ISBN" : "1420-9071 (Electronic)\\r1420-682X (Linking)", "ISSN" : "1420-9071", "PMID" : "24794628", "abstract" : "During early development, the mammalian embryo undergoes a series of profound changes that lead to the formation of two extraembryonic tissues--the trophectoderm and the primitive endoderm. These tissues encapsulate the pluripotent epiblast at the time of implantation. The current model proposes that the formation of these lineages results from two consecutive binary cell fate decisions. The first controls the formation of the trophectoderm and the inner cell mass, and the second controls the formation of the primitive endoderm and the epiblast within the inner cell mass. While early mammalian embryos develop with extensive plasticity, the embryonic pattern prior to implantation is remarkably reproducible. Here, we review the molecular mechanisms driving the cell fate decision between primitive endoderm and epiblast in the mouse embryo and integrate data from recent studies into the current model of the molecular network regulating the segregation between these lineages and their subsequent differentiation.", "author" : [ { "dropping-particle" : "", "family" : "Artus", "given" : "J\u00e9r\u00f4me", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Chazaud", "given" : "Claire", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Cellular and molecular life sciences : CMLS", "id" : "ITEM-1", "issue" : "17", "issued" : { "date-parts" : [ [ "2014", "9" ] ] }, "page" : "3327-38", "title" : "A close look at the mammalian blastocyst: epiblast and primitive endoderm formation.", "type" : "article-journal", "volume" : "71" }, "uris" : [ "" ] }, { "id" : "ITEM-2", "itemData" : { "DOI" : "10.1242/dev.128314", "ISSN" : "1477-9129", "PMID" : "27048685", "abstract" : "During mouse preimplantation embryo development, totipotent blastomeres generate the first three cell lineages of the embryo: trophectoderm, epiblast and primitive endoderm. In recent years, studies have shown that this process appears to be regulated by differences in cell-cell interactions, gene expression and the microenvironment of individual cells, rather than the active partitioning of maternal determinants. Precisely how these differences first emerge and how they dictate subsequent molecular and cellular behaviours are key questions in the field. As we review here, recent advances in live imaging, computational modelling and single-cell transcriptome analyses are providing new insights into these questions.", "author" : [ { "dropping-particle" : "", "family" : "Chazaud", "given" : "Claire", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Yamanaka", "given" : "Yojiro", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Development (Cambridge, England)", "id" : "ITEM-2", "issue" : "7", "issued" : { "date-parts" : [ [ "2016", "4", "1" ] ] }, "page" : "1063-74", "publisher" : "Oxford University Press for The Company of Biologists Limited", "title" : "Lineage specification in the mouse preimplantation embryo.", "type" : "article-journal", "volume" : "143" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(Artus and Chazaud, 2014; Chazaud and Yamanaka, 2016)", "plainTextFormattedCitation" : "(Artus and Chazaud, 2014; Chazaud and Yamanaka, 2016)", "previouslyFormattedCitation" : "(Artus and Chazaud, 2014; Chazaud and Yamanaka, 2016)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(Artus and Chazaud, 2014; Chazaud and Yamanaka, 2016). Yamanaka and colleagues clearly demonstrated that all ICM cells acquired a PrE fate when they were cultured in the presence of saturated levels of FGF4, and these authors proposed that the local concentration of FGF4 in the inner cells is important for the establishment of its salt-and-pepper distribution at the blastocyst stage ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1242/dev.043471", "ISSN" : "1477-9129", "PMID" : "20147376", "abstract" : "Primitive endoderm (PE) and epiblast (EPI) are two lineages derived from the inner cell mass (ICM) of the E3.5 blastocyst. Recent studies showed that EPI and PE progenitors expressing the lineage-specific transcriptional factors Nanog and Gata6, respectively, arise progressively as the ICM develops. Subsequent sorting of the two progenitors during blastocyst maturation results in the ormation of morphologically distinct EPI and PE layers at E4.5. It is, however, unknown how the initial differences between the two populations become established in the E3.5 blastocyst. Because the ICM cells are derived from two distinct rounds of polarized cell divisions during cleavage, a possible role for cell lineage history in promoting EPI versus PE fate has been proposed. We followed cell lineage from the eight-cell stage by live cell tracing and could find no clear linkage between developmental history of individual ICM cells and later cell fate. However, modulating FGF signaling levels by inhibition of the receptor/MAP kinase pathway or by addition of exogenous FGF shifted the fate of ICM cells to become either EPI or PE, respectively. Nanog- or Gata6-expressing progenitors could still be shifted towards the alternative fate by modulating FGF signaling during blastocyst maturation, suggesting that the ICM progenitors are not fully committed to their final fate at the time that initial segregation of gene expression occurs. In conclusion, we propose a model in which stochastic and progressive specification of EPI and PE lineages occurs during maturation of the blastocyst in an FGF/MAP kinase signal-dependent manner.", "author" : [ { "dropping-particle" : "", "family" : "Yamanaka", "given" : "Yojiro", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Lanner", "given" : "Fredrik", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Rossant", "given" : "Janet", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Development (Cambridge, England)", "id" : "ITEM-1", "issue" : "5", "issued" : { "date-parts" : [ [ "2010", "3" ] ] }, "page" : "715-24", "title" : "FGF signal-dependent segregation of primitive endoderm and epiblast in the mouse blastocyst.", "type" : "article-journal", "volume" : "137" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(Yamanaka et al., 2010)", "plainTextFormattedCitation" : "(Yamanaka et al., 2010)", "previouslyFormattedCitation" : "(Yamanaka et al., 2010)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(Yamanaka et al., 2010). At E3.0-E3.25, DP inner cells exist as a common precursor pool and have the potential to commit to either fate asynchronously ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1038/ncomms13463", "ISSN" : "2041-1723", "PMID" : "27857135", "abstract" : "Intercellular communication is essential to coordinate the behaviour of individual cells during organismal development. The preimplantation mammalian embryo is a paradigm of tissue self-organization and regulative development; however, the cellular basis of these regulative abilities has not been established. Here we use a quantitative image analysis pipeline to undertake a high-resolution, single-cell level analysis of lineage specification in the inner cell mass (ICM) of the mouse blastocyst. We show that a consistent ratio of epiblast and primitive endoderm lineages is achieved through incremental allocation of cells from a common progenitor pool, and that the lineage composition of the ICM is conserved regardless of its size. Furthermore, timed modulation of the FGF-MAPK pathway shows that individual progenitors commit to either fate asynchronously during blastocyst development. These data indicate that such incremental lineage allocation provides the basis for a tissue size control mechanism that ensures the generation of lineages of appropriate size.", "author" : [ { "dropping-particle" : "", "family" : "Saiz", "given" : "N\u00e9stor", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Williams", "given" : "Kiah M.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Seshan", "given" : "Venkatraman E.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Hadjantonakis", "given" : "Anna-Katerina", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Nature communications", "id" : "ITEM-1", "issued" : { "date-parts" : [ [ "2016", "11", "18" ] ] }, "page" : "13463", "publisher" : "Nature Publishing Group", "title" : "Asynchronous fate decisions by single cells collectively ensure consistent lineage composition in the mouse blastocyst.", "type" : "article-journal", "volume" : "7" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(Saiz et al., 2016)", "plainTextFormattedCitation" : "(Saiz et al., 2016)", "previouslyFormattedCitation" : "(Saiz et al., 2016)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(Saiz et al., 2016). Activity of the Fgf4-Fgfr-ERK pathway regulates the balance of EPI and PrE differentiation from common precursors. We demonstrated that Fgf4 expression is induced in a subset of Fgf4-high inner cells of Klf5 KO embryos at E3.0-E3.25. At that time, the number of DP cells was reduced in Klf5 KO embryos. Furthermore, the skewed cell fate of Klf5 KO embryos was markedly reversed by inhibitors of MEK and Fgfr. These results demonstrated that Klf5 is involved in the segregation of the EPI and PrE lineages by suppressing precocious activation of the Fgf4–Fgfr-Fgfr-ERK pathway. Further examination of the mechanisms involved in the transcription of Klf5 and the transcriptional activity of Klf5 protein could allow us to understand how the bimodal expression of Fgf4 is generated.Given an elevation in GATA6 protein and Sox17 mRNA expression in?Klf5 KO embryos at E3.0 or E3.25 (Fig. 1B, 3B, and 4D), one may think of a cell-autonomous role for Klf5 in the PrE lineage. However, the mRNA expression level of Sox17 at E3.0 was much lower than that of other transcription factors such as Nanog, Sox2 and Gata6 (Sox17/Nanog ratio = 0.0000056, Sox17/Sox2 = 0.0000528, Sox17/Gata6 = 0.00125), suggesting that Sox17 mRNA expression is very low. Furthermore, we found no detectable Sox17 protein expression (Fig. S3A). This is consistent with previous reports that Sox17 is activated between the 32- and 64-cell stages in mouse embryos ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1016/j.ydbio.2010.12.007", "ISBN" : "1095-564X (Electronic)\\r0012-1606 (Linking)", "ISSN" : "1095-564X", "PMID" : "21146513", "abstract" : "Cells of the primitive endoderm (PrE) and the pluripotent epiblast (EPI), the two lineages specified within the inner cell mass (ICM) of the mouse blastocyst stage embryo, are segregated into adjacent tissue layers by the end of the preimplantation period. The PrE layer which emerges as a polarized epithelium adjacent to the blastocoel, with a basement membrane separating it from the EPI, has two derivatives, the visceral and parietal endoderm. In this study we have investigated the localization of two transcriptional regulators of the SOX family, SOX17 and SOX7, within the PrE and its derivatives. We noted that SOX17 was first detected in a salt-and-pepper distribution within the ICM, subsequently becoming restricted to the nascent PrE epithelium. This dynamic distribution of SOX17 resembled the localization of GATA6 and GATA4, two other PrE lineage-specific transcription factors. By contrast, SOX7 was only detected in PrE cells positioned in contact with the blastocoel, raising the possibility that these cells are molecularly distinct. Our observations support a model of sequential GATA6 > SOX17 > GATA4 > SOX7 transcription factor activation within the PrE lineage, perhaps correlating with the consecutive periods of cell lineage 'na\u00efvete', commitment and sorting. Furthermore our data suggest that co-expression of SOX17 and SOX7 within sorted PrE cells could account for the absence of a detectable phenotype of Sox17 mutant blastocysts. However, analysis of implantation-delayed blastocysts, revealed a role for SOX17 in the maintenance of PrE epithelial integrity, with the absence of SOX17 leading to premature delamination and migration of parietal endoderm.", "author" : [ { "dropping-particle" : "", "family" : "Artus", "given" : "J\u00e9r\u00f4me", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Piliszek", "given" : "Anna", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Hadjantonakis", "given" : "Anna-Katerina", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Developmental biology", "id" : "ITEM-1", "issue" : "2", "issued" : { "date-parts" : [ [ "2011", "2", "15" ] ] }, "page" : "393-404", "publisher" : "Elsevier B.V.", "title" : "The primitive endoderm lineage of the mouse blastocyst: sequential transcription factor activation and regulation of differentiation by Sox17.", "type" : "article-journal", "volume" : "350" }, "uris" : [ "" ] }, { "id" : "ITEM-2", "itemData" : { "DOI" : "10.1073/pnas.0915063107", "ISBN" : "0915063107", "ISSN" : "1091-6490", "PMID" : "20308546", "abstract" : "A crucial question in mammalian development is how cells of the early embryo differentiate into distinct cell types. The first decision is taken when cells undertake waves of asymmetric division that generate one daughter on the inside and one on the outside of the embryo. After this division, some cells on the inside remain pluripotent and give rise to the epiblast, and hence the future body, whereas others develop into the primitive endoderm, an extraembryonic tissue. How the fate of these inside cells is decided is unknown: Is the process random, or is it related to their developmental origins? To address this question, we traced all cells by live-cell imaging in intact, unmanipulated embryos until the epiblast and primitive endoderm became distinct. This analysis revealed that inner cell mass (ICM) cells have unrestricted developmental potential. However, cells internalized by the first wave of asymmetric divisions are biased toward forming pluripotent epiblast, whereas cells internalized in the next two waves of divisions are strongly biased toward forming primitive endoderm. Moreover, we show that cells internalized by the second wave up-regulate expression of Gata6 and Sox17, and changing the expression of these genes determines whether the cells become primitive endoderm. Finally, with our ability to determine the origin of cells, we find that inside cells that are mispositioned when they are born can sort into the correct layer. In conclusion, we propose a model in which the timing of cell internalization, cell position, and cell sorting combine to determine distinct lineages of the preimplantation mouse embryo.", "author" : [ { "dropping-particle" : "", "family" : "Morris", "given" : "Samantha a", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Teo", "given" : "Roy T Y", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Li", "given" : "Huiliang", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Robson", "given" : "Paul", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Glover", "given" : "David M", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Zernicka-Goetz", "given" : "Magdalena", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Proceedings of the National Academy of Sciences of the United States of America", "id" : "ITEM-2", "issue" : "14", "issued" : { "date-parts" : [ [ "2010", "4", "6" ] ] }, "page" : "6364-9", "title" : "Origin and formation of the first two distinct cell types of the inner cell mass in the mouse embryo.", "type" : "article-journal", "volume" : "107" }, "uris" : [ "" ] }, { "id" : "ITEM-3", "itemData" : { "DOI" : "10.1101/gad.1833510", "ISBN" : "1549-5477 (Electronic)\\r0890-9369 (Linking)", "ISSN" : "1549-5477", "PMID" : "20123909", "abstract" : "In embryonic stem (ES) cells, a well-characterized transcriptional network promotes pluripotency and represses gene expression required for differentiation. In comparison, the transcriptional networks that promote differentiation of ES cells and the blastocyst inner cell mass are poorly understood. Here, we show that Sox17 is a transcriptional regulator of differentiation in these pluripotent cells. ES cells deficient in Sox17 fail to differentiate into extraembryonic cell types and maintain expression of pluripotency-associated transcription factors, including Oct4, Nanog, and Sox2. In contrast, forced expression of Sox17 down-regulates ES cell-associated gene expression and directly activates genes functioning in differentiation toward an extraembryonic endoderm cell fate. We show these effects of Sox17 on ES cell gene expression are mediated at least in part through a competition between Sox17 and Nanog for common DNA-binding sites. By elaborating the function of Sox17, our results provide insight into how the transcriptional network promoting ES cell self-renewal is interrupted, allowing cellular differentiation.", "author" : [ { "dropping-particle" : "", "family" : "Niakan", "given" : "Kathy K", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Ji", "given" : "Hongkai", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Maehr", "given" : "Ren\u00e9", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Vokes", "given" : "Steven a", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Rodolfa", "given" : "Kit T", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Sherwood", "given" : "Richard I", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Yamaki", "given" : "Mariko", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Dimos", "given" : "John T", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Chen", "given" : "Alice E", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Melton", "given" : "Douglas a", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "McMahon", "given" : "Andrew P", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Eggan", "given" : "Kevin", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Genes & development", "id" : "ITEM-3", "issue" : "3", "issued" : { "date-parts" : [ [ "2010" ] ] }, "page" : "312-26", "title" : "Sox17 promotes differentiation in mouse embryonic stem cells by directly regulating extraembryonic gene expression and indirectly antagonizing self-renewal.", "type" : "article-journal", "volume" : "24" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(Artus et al., 2011; Morris et al., 2010; Niakan et al., 2010)", "plainTextFormattedCitation" : "(Artus et al., 2011; Morris et al., 2010; Niakan et al., 2010)", "previouslyFormattedCitation" : "(Artus et al., 2011; Morris et al., 2010; Niakan et al., 2010)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(Artus et al., 2011; Morris et al., 2010; Niakan et al., 2010). Thus, the elevation in Sox17 mRNA expression in?Klf5 KO ICMs is still too low to cause PrE-differentiation. Gata6 protein expression level also showed a slight increase (29%) at E3.25 (Fig. 1B). But, the mRNA expression level of Gata6 was not changed significantly between WT and Klf5 KO embryos at E3.0 (Fig. S5B) and E3.25 (Fig. 4D), indicating that Gata6 is not regulated by Klf5 at the transcriptional level around E3.0–E3.25. Taken together, these data suggest a regulation of Fgf4 inducing PrE differentiation rather than Sox17 or a Gata6-mediated cell-autonomous mechanism.Previous studies have clearly demonstrated that Klf5 regulates lineage specification in TE and ICM ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1016/j.stem.2008.09.003", "ISBN" : "1875-9777 (Electronic)", "ISSN" : "1875-9777", "PMID" : "18983969", "abstract" : "The transcription factor Klf4 has demonstrated activity in the reprogramming of somatic cells to a pluripotent state, but the molecular mechanism of this process remains unknown. It is, therefore, of great interest to understand the functional role of Klf4 and related genes in ESC regulation. Here, we show that homozygous disruption of Klf5 results in the failure of ESC derivation from ICM cells and early embryonic lethality due to an implantation defect. Klf5 KO ESCs show increased expression of several differentiation marker genes and frequent, spontaneous differentiation. Conversely, overexpression of Klf5 in ESCs suppressed the expression of differentiation marker genes and maintained pluripotency in the absence of LIF. Our results also suggest that Klf5 regulates ESC proliferation by promoting phosphorylation of Akt1 via induction of Tcl1. These results, therefore, provide new insights into the functional and mechanistic role of Klf5 in regulation of pluripotency.", "author" : [ { "dropping-particle" : "", "family" : "Ema", "given" : "Masatsugu", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Mori", "given" : "Daisuke", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Niwa", "given" : "Hitoshi", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Hasegawa", "given" : "Yoshikazu", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Yamanaka", "given" : "Yojiro", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Hitoshi", "given" : "Seiji", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Mimura", "given" : "Junsei", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Kawabe", "given" : "Yoh-ichi", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Hosoya", "given" : "Tomohiro", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Morita", "given" : "Masanobu", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Shimosato", "given" : "Daisuke", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Uchida", "given" : "Kazuhiko", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Suzuki", "given" : "Norio", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Yanagisawa", "given" : "Jun", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Sogawa", "given" : "Kazuhiro", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Rossant", "given" : "Janet", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Yamamoto", "given" : "Masayuki", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Takahashi", "given" : "Satoru", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Fujii-Kuriyama", "given" : "Yoshiaki", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Cell stem cell", "id" : "ITEM-1", "issue" : "5", "issued" : { "date-parts" : [ [ "2008", "11", "6" ] ] }, "page" : "555-67", "publisher" : "Elsevier Inc.", "title" : "Kr\u00fcppel-like factor 5 is essential for blastocyst development and the normal self-renewal of mouse ESCs.", "type" : "article-journal", "volume" : "3" }, "uris" : [ "" ] }, { "id" : "ITEM-2", "itemData" : { "DOI" : "10.1242/dev.054775", "ISSN" : "1477-9129", "PMID" : "20980403", "abstract" : "Kruppel-like transcription factors (Klfs) are essential for the induction and maintenance of pluripotency of embryonic stem cells (ESCs), yet little is known about their roles in establishing the three lineages of the pre-implantation embryo. Here, we show that Klf5 is required for the formation of the trophectoderm (TE) and the inner cell mass (ICM), and for repressing primitive endoderm (PE) development. Although cell polarity appeared normal, Klf5 mutant embryos arrested at the blastocyst stage and failed to hatch due to defective TE development. Klf5 acted cell-autonomously in the TE, downstream of Fgf4 and upstream of Cdx2, Eomes and Krt8. In the ICM, loss of Klf5 resulted in reduced expression of pluripotency markers Oct4 and Nanog, but led to increased Sox17 expression in the PE, suggesting that Klf5 suppresses the PE lineage. Consistent with this, overexpression of Klf5 in transgenic embryos was sufficient to suppress the Sox17(+) PE lineage in the ICM. Klf5 overexpression led to a dose-dependent decrease in Sox17 promoter activity in reporter assays in cultured cells. Moreover, in chimeric embryos, Klf5(-/-) cells preferentially contributed to the Sox17(+) PE lineage and Cdx2 expression was not rescued in Klf5(-/-) outer cells. Finally, outgrowths from Klf5(-/-) embryos failed to form an ICM/pluripotent colony, had very few Oct4(+) or Cdx2(+) cells, but showed an increase in the percentage of Sox17(+) PE cells. These findings demonstrate that Klf5 is a dynamic regulator of all three lineages in the pre-implantation embryo by promoting the TE and epiblast lineages while suppressing the PE lineage.", "author" : [ { "dropping-particle" : "", "family" : "Lin", "given" : "Suh-Chin J", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Wani", "given" : "Maqsood a", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Whitsett", "given" : "Jeffrey a", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Wells", "given" : "James M", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Development (Cambridge, England)", "id" : "ITEM-2", "issue" : "23", "issued" : { "date-parts" : [ [ "2010", "12" ] ] }, "page" : "3953-63", "title" : "Klf5 regulates lineage formation in the pre-implantation mouse embryo.", "type" : "article-journal", "volume" : "137" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(Ema et al., 2008; Lin et al., 2010)", "plainTextFormattedCitation" : "(Ema et al., 2008; Lin et al., 2010)", "previouslyFormattedCitation" : "(Ema et al., 2008; Lin et al., 2010)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(Ema et al., 2008; Lin et al., 2010). While Klf5 directly regulates Sox17 (Lin et al., 2010), it is still unclear how Klf5 regulates the balance of EPI and PrE. Our results clearly indicate that Klf5 is required to suppress Fgf4 in morula at E3.0, but it is not clear whether it continues to suppress Fgf4 later in development because Klf5 KO ES cells expressed normal levels of Fgf4 (Fig. S5E available online). Fgf4 and Klf5 are expressed abundantly in such cells and are important for their differentiation and normal self-renewal, respectively ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1016/j.stem.2008.09.003", "ISBN" : "1875-9777 (Electronic)", "ISSN" : "1875-9777", "PMID" : "18983969", "abstract" : "The transcription factor Klf4 has demonstrated activity in the reprogramming of somatic cells to a pluripotent state, but the molecular mechanism of this process remains unknown. It is, therefore, of great interest to understand the functional role of Klf4 and related genes in ESC regulation. Here, we show that homozygous disruption of Klf5 results in the failure of ESC derivation from ICM cells and early embryonic lethality due to an implantation defect. Klf5 KO ESCs show increased expression of several differentiation marker genes and frequent, spontaneous differentiation. Conversely, overexpression of Klf5 in ESCs suppressed the expression of differentiation marker genes and maintained pluripotency in the absence of LIF. Our results also suggest that Klf5 regulates ESC proliferation by promoting phosphorylation of Akt1 via induction of Tcl1. These results, therefore, provide new insights into the functional and mechanistic role of Klf5 in regulation of pluripotency.", "author" : [ { "dropping-particle" : "", "family" : "Ema", "given" : "Masatsugu", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Mori", "given" : "Daisuke", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Niwa", "given" : "Hitoshi", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Hasegawa", "given" : "Yoshikazu", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Yamanaka", "given" : "Yojiro", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Hitoshi", "given" : "Seiji", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Mimura", "given" : "Junsei", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Kawabe", "given" : "Yoh-ichi", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Hosoya", "given" : "Tomohiro", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Morita", "given" : "Masanobu", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Shimosato", "given" : "Daisuke", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Uchida", "given" : "Kazuhiko", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Suzuki", "given" : "Norio", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Yanagisawa", "given" : "Jun", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Sogawa", "given" : "Kazuhiro", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Rossant", "given" : "Janet", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Yamamoto", "given" : "Masayuki", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Takahashi", "given" : "Satoru", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Fujii-Kuriyama", "given" : "Yoshiaki", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Cell stem cell", "id" : "ITEM-1", "issue" : "5", "issued" : { "date-parts" : [ [ "2008", "11", "6" ] ] }, "page" : "555-67", "publisher" : "Elsevier Inc.", "title" : "Kr\u00fcppel-like factor 5 is essential for blastocyst development and the normal self-renewal of mouse ESCs.", "type" : "article-journal", "volume" : "3" }, "uris" : [ "" ] }, { "id" : "ITEM-2", "itemData" : { "DOI" : "10.1242/dev.02880", "ISSN" : "0950-1991", "PMID" : "17660198", "abstract" : "Pluripotent embryonic stem (ES) cells must select between alternative fates of self-replication and lineage commitment during continuous proliferation. Here, we delineate the role of autocrine production of fibroblast growth factor 4 (Fgf4) and associated activation of the Erk1/2 (Mapk3/1) signalling cascade. Fgf4 is the major stimulus activating Erk in mouse ES cells. Interference with FGF or Erk activity using chemical inhibitors or genetic ablations does not impede propagation of undifferentiated ES cells. Instead, such manipulations restrict the ability of ES cells to commit to differentiation. ES cells lacking Fgf4 or treated with FGF receptor inhibitors resist neural and mesodermal induction, and are refractory to BMP-induced non-neural differentiation. Lineage commitment potential of Fgf4-null cells is restored by provision of FGF protein. Thus, FGF enables rather than antagonises the differentiation activity of BMP. The key downstream role of Erk signalling is revealed by examination of Erk2-null ES cells, which fail to undergo either neural or mesodermal differentiation in adherent culture, and retain expression of pluripotency markers Oct4, Nanog and Rex1. These findings establish that Fgf4 stimulation of Erk1/2 is an autoinductive stimulus for na\u00efve ES cells to exit the self-renewal programme. We propose that the Erk cascade directs transition to a state that is responsive to inductive cues for germ layer segregation. Consideration of Erk signalling as a primary trigger that potentiates lineage commitment provides a context for reconciling disparate views on the contribution of FGF and BMP pathways during germ layer specification in vertebrate embryos.", "author" : [ { "dropping-particle" : "", "family" : "Kunath", "given" : "Tilo", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Saba-El-Leil", "given" : "Marc K", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Almousailleakh", "given" : "Marwa", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Wray", "given" : "Jason", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Meloche", "given" : "Sylvain", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Smith", "given" : "Austin", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Development (Cambridge, England)", "id" : "ITEM-2", "issue" : "16", "issued" : { "date-parts" : [ [ "2007", "8" ] ] }, "page" : "2895-902", "title" : "FGF stimulation of the Erk1/2 signalling cascade triggers transition of pluripotent embryonic stem cells from self-renewal to lineage commitment.", "type" : "article-journal", "volume" : "134" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(Ema et al., 2008; Kunath et al., 2007)", "plainTextFormattedCitation" : "(Ema et al., 2008; Kunath et al., 2007)", "previouslyFormattedCitation" : "(Ema et al., 2008; Kunath et al., 2007)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(Ema et al., 2008; Kunath et al., 2007). Cis-regulatory regions in the Fgf4 promoter and enhancers are occupied by Klf5 in mouse ES cells, as shown in Fig. 4F. Nevertheless, the lack of Klf5 expression did not significantly alter Fgf4 expression (Fig. S5E). Therefore, we speculate that the transcriptional repression of Fgf4 by Klf5 occurs within a brief developmental window, such that the induction of Fgf4 does not hamper the normal segregation of EPI and PrE lineages, and later Klf5 expression does not repress Fgf4 by the time the mature EPI cells arise. This is consistent with the observation that aggregation of Klf5 KO ES cells with WT tetraploid embryos generates normal Klf5 KO embryos at E8.5, indicating that Klf5 is not required for normal development once the epiblast is established (Ema et al., 2008).Activation of the Fgf4–Fgfr–ERK pathway destabilises a na?ve pluripotent state in mouse ES cells and promotes a primed state, while a reduction in ERK activity strongly promotes na?ve pluripotency ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1371/journal.pone.0060907", "ISSN" : "1932-6203", "PMID" : "23613754", "abstract" : "Activation of the FGF-ERK pathway is necessary for na\u00efve mouse embryonic stem (ES) cells to exit self-renewal and commit to early differentiated lineages. Here we show that genetic ablation of Erk2, the predominant ERK isozyme expressed in ES cells, results in hyper-phosphorylation of ERK1, but an overall decrease in total ERK activity as judged by substrate phosphorylation and immediate-early gene (IEG) induction. Normal induction of this subset of canonical ERK targets, as well as p90RSK phosphorylation, was rescued by transgenic expression of either ERK1 or ERK2 indicating a degree of functional redundancy. In contrast to previously published work, Erk2-null ES cells exhibited no detectable defect in lineage specification to any of the three germ layers when induced to differentiate in either embryoid bodies or in defined neural induction conditions. However, under self-renewing conditions Erk2-null ES cells express increased levels of the pluripotency-associated transcripts, Nanog and Tbx3, a decrease in Nanog-GFP heterogeneity, and exhibit enhanced self-renewal in colony forming assays. Transgenic add-back of ERK2 is capable of restoring normal pluripotent gene expression and self-renewal capacity. We show that ERK2 contributes to the destabilization of ES cell self-renewal by reducing expression of pluripotency genes, such as Nanog, but is not specifically required for the early stages of germ layer specification.", "author" : [ { "dropping-particle" : "", "family" : "Hamilton", "given" : "William B", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Kaji", "given" : "Keisuke", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Kunath", "given" : "Tilo", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "PloS one", "id" : "ITEM-1", "issue" : "4", "issued" : { "date-parts" : [ [ "2013", "1" ] ] }, "page" : "e60907", "title" : "ERK2 suppresses self-renewal capacity of embryonic stem cells, but is not required for multi-lineage commitment.", "type" : "article-journal", "volume" : "8" }, "uris" : [ "" ] }, { "id" : "ITEM-2", "itemData" : { "DOI" : "10.1242/dev.02880", "ISSN" : "0950-1991", "PMID" : "17660198", "abstract" : "Pluripotent embryonic stem (ES) cells must select between alternative fates of self-replication and lineage commitment during continuous proliferation. Here, we delineate the role of autocrine production of fibroblast growth factor 4 (Fgf4) and associated activation of the Erk1/2 (Mapk3/1) signalling cascade. Fgf4 is the major stimulus activating Erk in mouse ES cells. Interference with FGF or Erk activity using chemical inhibitors or genetic ablations does not impede propagation of undifferentiated ES cells. Instead, such manipulations restrict the ability of ES cells to commit to differentiation. ES cells lacking Fgf4 or treated with FGF receptor inhibitors resist neural and mesodermal induction, and are refractory to BMP-induced non-neural differentiation. Lineage commitment potential of Fgf4-null cells is restored by provision of FGF protein. Thus, FGF enables rather than antagonises the differentiation activity of BMP. The key downstream role of Erk signalling is revealed by examination of Erk2-null ES cells, which fail to undergo either neural or mesodermal differentiation in adherent culture, and retain expression of pluripotency markers Oct4, Nanog and Rex1. These findings establish that Fgf4 stimulation of Erk1/2 is an autoinductive stimulus for na\u00efve ES cells to exit the self-renewal programme. We propose that the Erk cascade directs transition to a state that is responsive to inductive cues for germ layer segregation. Consideration of Erk signalling as a primary trigger that potentiates lineage commitment provides a context for reconciling disparate views on the contribution of FGF and BMP pathways during germ layer specification in vertebrate embryos.", "author" : [ { "dropping-particle" : "", "family" : "Kunath", "given" : "Tilo", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Saba-El-Leil", "given" : "Marc K", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Almousailleakh", "given" : "Marwa", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Wray", "given" : "Jason", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Meloche", "given" : "Sylvain", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Smith", "given" : "Austin", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Development (Cambridge, England)", "id" : "ITEM-2", "issue" : "16", "issued" : { "date-parts" : [ [ "2007", "8" ] ] }, "page" : "2895-902", "title" : "FGF stimulation of the Erk1/2 signalling cascade triggers transition of pluripotent embryonic stem cells from self-renewal to lineage commitment.", "type" : "article-journal", "volume" : "134" }, "uris" : [ "" ] }, { "id" : "ITEM-3", "itemData" : { "DOI" : "10.1038/nature06968", "ISSN" : "1476-4687", "PMID" : "18497825", "abstract" : "In the three decades since pluripotent mouse embryonic stem (ES) cells were first described they have been derived and maintained by using various empirical combinations of feeder cells, conditioned media, cytokines, growth factors, hormones, fetal calf serum, and serum extracts. Consequently ES-cell self-renewal is generally considered to be dependent on multifactorial stimulation of dedicated transcriptional circuitries, pre-eminent among which is the activation of STAT3 by cytokines (ref. 8). Here we show, however, that extrinsic stimuli are dispensable for the derivation, propagation and pluripotency of ES cells. Self-renewal is enabled by the elimination of differentiation-inducing signalling from mitogen-activated protein kinase. Additional inhibition of glycogen synthase kinase 3 consolidates biosynthetic capacity and suppresses residual differentiation. Complete bypass of cytokine signalling is confirmed by isolating ES cells genetically devoid of STAT3. These findings reveal that ES cells have an innate programme for self-replication that does not require extrinsic instruction. This property may account for their latent tumorigenicity. The delineation of minimal requirements for self-renewal now provides a defined platform for the precise description and dissection of the pluripotent state.", "author" : [ { "dropping-particle" : "", "family" : "Ying", "given" : "Qi-Long", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Wray", "given" : "Jason", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Nichols", "given" : "Jennifer", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Batlle-Morera", "given" : "Laura", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Doble", "given" : "Bradley", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Woodgett", "given" : "James", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Cohen", "given" : "Philip", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Smith", "given" : "Austin", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Nature", "id" : "ITEM-3", "issue" : "7194", "issued" : { "date-parts" : [ [ "2008", "5", "22" ] ] }, "page" : "519-23", "title" : "The ground state of embryonic stem cell self-renewal.", "type" : "article-journal", "volume" : "453" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(Hamilton et al., 2013; Kunath et al., 2007; Ying et al., 2008)", "plainTextFormattedCitation" : "(Hamilton et al., 2013; Kunath et al., 2007; Ying et al., 2008)", "previouslyFormattedCitation" : "(Hamilton et al., 2013; Kunath et al., 2007; Ying et al., 2008)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(Hamilton et al., 2013; Kunath et al., 2007; Ying et al., 2008). However, the precise molecular mechanisms of self-renewal promoted by ERK inhibition remain elusive. Although ERK inhibition contributes to self-renewal in part through the stabilisation of Klf2 and Klf4 proteins that undergo proteasome-dependent degradation of ERK-phosphorylated forms in mouse ES cells ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1038/nsmb.2217", "ISSN" : "1545-9985", "PMID" : "22307056", "abstract" : "Understanding and controlling the mechanism by which stem cells balance self-renewal versus differentiation is of great importance for stem cell therapeutics. Klf4 promotes the self-renewal of embryonic stem cells, but the precise mechanism regulating this role of Klf4 is unclear. We found that ERK1 or ERK2 binds the activation domain of Klf4 and directly phosphorylates Klf4 at Ser123. This phosphorylation suppresses Klf4 activity, inducing embryonic stem cell differentiation. Conversely, inhibition of Klf4 phosphorylation enhances Klf4 activity and suppresses embryonic stem cell differentiation. Notably, phosphorylation of Klf4 by ERKs causes recruitment and binding of the F-box proteins \u03b2TrCP1 or \u03b2TrCP2 (components of an ubiquitin E3 ligase) to the Klf4 N-terminal domain, which results in Klf4 ubiquitination and degradation. Overall, our data provide a molecular basis for the role of ERK1 and ERK2 in regulating Klf4-mediated mouse embryonic stem cell self-renewal.", "author" : [ { "dropping-particle" : "", "family" : "Kim", "given" : "Myoung Ok", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Kim", "given" : "Sung-Hyun", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Cho", "given" : "Yong-Yeon", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Nadas", "given" : "Janos", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Jeong", "given" : "Chul-Ho", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Yao", "given" : "Ke", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Kim", "given" : "Dong Joon", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Yu", "given" : "Dong-Hoon", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Keum", "given" : "Young-Sam", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Lee", "given" : "Kun-Yeong", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Huang", "given" : "Zunnan", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Bode", "given" : "Ann M", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Dong", "given" : "Zigang", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Nature structural & molecular biology", "id" : "ITEM-1", "issue" : "3", "issued" : { "date-parts" : [ [ "2012", "3", "5" ] ] }, "page" : "283-90", "publisher" : "Nature Publishing Group", "title" : "ERK1 and ERK2 regulate embryonic stem cell self-renewal through phosphorylation of Klf4.", "type" : "article-journal", "volume" : "19" }, "uris" : [ "" ] }, { "id" : "ITEM-2", "itemData" : { "DOI" : "10.1016/j.stem.2014.04.015", "ISSN" : "1875-9777", "PMID" : "24905170", "abstract" : "The maintenance of mouse embryonic stem cells (mESCs) requires LIF and serum. However, a pluripotent \"ground state,\" bearing resemblance to preimplantation mouse epiblasts, can be established through dual inhibition (2i) of both prodifferentiation Mek/Erk and Gsk3/Tcf3 pathways. While Gsk3 inhibition has been attributed to the transcriptional derepression of Esrrb, the molecular mechanism mediated by Mek inhibition remains unclear. In this study, we show that Kr\u00fcppel-like factor 2 (Klf2) is phosphorylated by Erk2 and that phospho-Klf2 is proteosomally degraded. Mek inhibition hence prevents Klf2 protein phosphodegradation to sustain pluripotency. Indeed, while Klf2-null mESCs can survive under LIF/Serum, they are not viable under 2i, demonstrating that Klf2 is essential for ground state pluripotency. Importantly, we also show that ectopic Klf2 expression can replace Mek inhibition in mESCs, allowing the culture of Klf2-null mESCs under Gsk3 inhibition alone. Collectively, our study defines the Mek/Erk/Klf2 axis that cooperates with the Gsk3/Tcf3/Esrrb pathway in mediating ground state pluripotency.", "author" : [ { "dropping-particle" : "", "family" : "Yeo", "given" : "Jia-Chi", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Jiang", "given" : "Jianming", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Tan", "given" : "Zi-Ying", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Yim", "given" : "Guo-Rong", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Ng", "given" : "Jia-Hui", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "G\u00f6ke", "given" : "Jonathan", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Kraus", "given" : "Petra", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Liang", "given" : "Hongqing", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Gonzales", "given" : "Kevin Andrew Uy", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Chong", "given" : "Han-Chung", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Tan", "given" : "Cheng-Peow", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Lim", "given" : "Yee-Siang", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Tan", "given" : "Nguan-Soon", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Lufkin", "given" : "Thomas", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Ng", "given" : "Huck-Hui", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Cell stem cell", "id" : "ITEM-2", "issue" : "6", "issued" : { "date-parts" : [ [ "2014", "6", "5" ] ] }, "page" : "864-72", "title" : "Klf2 is an essential factor that sustains ground state pluripotency.", "type" : "article-journal", "volume" : "14" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(Kim et al., 2012; Yeo et al., 2014)", "plainTextFormattedCitation" : "(Kim et al., 2012; Yeo et al., 2014)", "previouslyFormattedCitation" : "(Kim et al., 2012; Yeo et al., 2014)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(Kim et al., 2012; Yeo et al., 2014), it is interesting to note that Klf5 modulates the level of pERK in mouse ES cells (Azami, unpublished observations). ERK inhibition also facilitates the emergence of na?ve pluripotent cells in the blastocyst during murine development ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1242/dev.038893", "ISSN" : "1477-9129", "PMID" : "19710168", "abstract" : "Embryonic stem (ES) cells can be derived and propagated from multiple strains of mouse and rat through application of small-molecule inhibitors of the fibroblast growth factor (FGF)/Erk pathway and of glycogen synthase kinase 3. These conditions shield pluripotent cells from differentiation-inducing stimuli. We investigate the effect of these inhibitors on the development of pluripotent epiblast in intact pre-implantation embryos. We find that blockade of Erk signalling from the 8-cell stage does not impede blastocyst formation but suppresses development of the hypoblast. The size of the inner cell mass (ICM) compartment is not reduced, however. Throughout the ICM, the epiblast-specific marker Nanog is expressed, and in XX embryos epigenetic silencing of the paternal X chromosome is erased. Epiblast identity and pluripotency were confirmed by contribution to chimaeras with germline transmission. These observations indicate that segregation of hypoblast from the bipotent ICM is dependent on FGF/Erk signalling and that in the absence of this signal, the entire ICM can acquire pluripotency. Furthermore, the epiblast does not require paracrine support from the hypoblast. Thus, na\u00efve epiblast and ES cells are in a similar ground state, with an autonomous capacity for survival and replication, and high vulnerability to Erk signalling. We probed directly the relationship between na\u00efve epiblast and ES cells. Dissociated ICM cells from freshly harvested late blastocysts gave rise to up to 12 ES cell clones per embryo when plated in the presence of inhibitors. We propose that ES cells are not a tissue culture creation, but are essentially identical to pre-implantation epiblast cells.", "author" : [ { "dropping-particle" : "", "family" : "Nichols", "given" : "Jennifer", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Silva", "given" : "Jose", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Roode", "given" : "Mila", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Smith", "given" : "Austin", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Development (Cambridge, England)", "id" : "ITEM-1", "issue" : "19", "issued" : { "date-parts" : [ [ "2009", "10" ] ] }, "page" : "3215-22", "title" : "Suppression of Erk signalling promotes ground state pluripotency in the mouse embryo.", "type" : "article-journal", "volume" : "136" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(Nichols et al., 2009)", "plainTextFormattedCitation" : "(Nichols et al., 2009)", "previouslyFormattedCitation" : "(Nichols et al., 2009)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(Nichols et al., 2009). Although it remains unclear whether there is a specific physiological mechanism that mediates ERK inhibition to promote pluripotency in vivo, Klf5 might be a key genetic component in this regard. Further investigation of the functions of Klf5 might allow us to understand how the symmetric expression of Fgf4 and Fgfr2 breaks during early development.Materials and MethodsGeneration of Klf5 KO mice and overexpressing transgenic mice. Klf5 lacZ/lacZ mice were generated as previously described ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1016/j.stem.2008.09.003", "ISBN" : "1875-9777 (Electronic)", "ISSN" : "1875-9777", "PMID" : "18983969", "abstract" : "The transcription factor Klf4 has demonstrated activity in the reprogramming of somatic cells to a pluripotent state, but the molecular mechanism of this process remains unknown. It is, therefore, of great interest to understand the functional role of Klf4 and related genes in ESC regulation. Here, we show that homozygous disruption of Klf5 results in the failure of ESC derivation from ICM cells and early embryonic lethality due to an implantation defect. Klf5 KO ESCs show increased expression of several differentiation marker genes and frequent, spontaneous differentiation. Conversely, overexpression of Klf5 in ESCs suppressed the expression of differentiation marker genes and maintained pluripotency in the absence of LIF. Our results also suggest that Klf5 regulates ESC proliferation by promoting phosphorylation of Akt1 via induction of Tcl1. These results, therefore, provide new insights into the functional and mechanistic role of Klf5 in regulation of pluripotency.", "author" : [ { "dropping-particle" : "", "family" : "Ema", "given" : "Masatsugu", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Mori", "given" : "Daisuke", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Niwa", "given" : "Hitoshi", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Hasegawa", "given" : "Yoshikazu", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Yamanaka", "given" : "Yojiro", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Hitoshi", "given" : "Seiji", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Mimura", "given" : "Junsei", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Kawabe", "given" : "Yoh-ichi", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Hosoya", "given" : "Tomohiro", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Morita", "given" : "Masanobu", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Shimosato", "given" : "Daisuke", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Uchida", "given" : "Kazuhiko", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Suzuki", "given" : "Norio", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Yanagisawa", "given" : "Jun", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Sogawa", "given" : "Kazuhiro", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Rossant", "given" : "Janet", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Yamamoto", "given" : "Masayuki", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Takahashi", "given" : "Satoru", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Fujii-Kuriyama", "given" : "Yoshiaki", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Cell stem cell", "id" : "ITEM-1", "issue" : "5", "issued" : { "date-parts" : [ [ "2008", "11", "6" ] ] }, "page" : "555-67", "publisher" : "Elsevier Inc.", "title" : "Kr\u00fcppel-like factor 5 is essential for blastocyst development and the normal self-renewal of mouse ESCs.", "type" : "article-journal", "volume" : "3" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(Ema et al., 2008)", "plainTextFormattedCitation" : "(Ema et al., 2008)", "previouslyFormattedCitation" : "(Ema et al., 2008)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(Ema et al., 2008). The LacZ cassette was inserted into the 2nd exon of the Klf5 gene. Conditional KO and OE alleles for Klf5 were generated as described in the Supplementary Materials and Methods section. Primers used for the genotyping of the conditional KO and OE alleles for Klf5 mice are described in Table S1. Mouse embryos were recovered at noon on the day in which the vaginal plug is discovered (considered E0.5).Pluripotent stem cells. Mouse Klf5+/+ (WT)::Oct3/4-ireszeocinR ES cells, Klf5 lacZ/lacZ (KO)::Oct3/4-ireszeocinR ES cells and Klf5+/+ (WT)::Oct3/4-ireszeocinR:: Klf5 overexpressing ES cells were generated as previously described ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1016/j.stem.2008.09.003", "ISBN" : "1875-9777 (Electronic)", "ISSN" : "1875-9777", "PMID" : "18983969", "abstract" : "The transcription factor Klf4 has demonstrated activity in the reprogramming of somatic cells to a pluripotent state, but the molecular mechanism of this process remains unknown. It is, therefore, of great interest to understand the functional role of Klf4 and related genes in ESC regulation. Here, we show that homozygous disruption of Klf5 results in the failure of ESC derivation from ICM cells and early embryonic lethality due to an implantation defect. Klf5 KO ESCs show increased expression of several differentiation marker genes and frequent, spontaneous differentiation. Conversely, overexpression of Klf5 in ESCs suppressed the expression of differentiation marker genes and maintained pluripotency in the absence of LIF. Our results also suggest that Klf5 regulates ESC proliferation by promoting phosphorylation of Akt1 via induction of Tcl1. These results, therefore, provide new insights into the functional and mechanistic role of Klf5 in regulation of pluripotency.", "author" : [ { "dropping-particle" : "", "family" : "Ema", "given" : "Masatsugu", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Mori", "given" : "Daisuke", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Niwa", "given" : "Hitoshi", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Hasegawa", "given" : "Yoshikazu", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Yamanaka", "given" : "Yojiro", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Hitoshi", "given" : "Seiji", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Mimura", "given" : "Junsei", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Kawabe", "given" : "Yoh-ichi", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Hosoya", "given" : "Tomohiro", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Morita", "given" : "Masanobu", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Shimosato", "given" : "Daisuke", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Uchida", "given" : "Kazuhiko", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Suzuki", "given" : "Norio", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Yanagisawa", "given" : "Jun", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Sogawa", "given" : "Kazuhiro", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Rossant", "given" : "Janet", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Yamamoto", "given" : "Masayuki", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Takahashi", "given" : "Satoru", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Fujii-Kuriyama", "given" : "Yoshiaki", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Cell stem cell", "id" : "ITEM-1", "issue" : "5", "issued" : { "date-parts" : [ [ "2008", "11", "6" ] ] }, "page" : "555-67", "publisher" : "Elsevier Inc.", "title" : "Kr\u00fcppel-like factor 5 is essential for blastocyst development and the normal self-renewal of mouse ESCs.", "type" : "article-journal", "volume" : "3" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(Ema et al., 2008)", "plainTextFormattedCitation" : "(Ema et al., 2008)", "previouslyFormattedCitation" : "(Ema et al., 2008)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(Ema et al., 2008) and were cultured in DMEM + 15% Knockout Serum Replacement (KSR; Invitrogen). The detail was described in the Supplementary Materials and Methods section.Manipulation of early embryos. Embryo manipulations were performed according to Nagy et al. ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "ISBN" : "9781936113019", "abstract" : "Fourth edition. Genetics and embryology of the mouse: past, present, and future -- Summary of mouse development -- A mouse colony for the production of transgenic and chimeric animals -- Recovery and in vitro culture of preimplantation embryos -- Isolation, culture, and manipulation of postimplantation embryos -- Surgical procedures -- Production of transgenic mice by pronuclear microinjection -- Embryo-derived stem cell lines -- Germline-competent stem cells derived from adult mice -- Vector designs for pluripotent stem cell-based transgenesis and genome alterations -- Introduction of foreign DNA into embryonic stem cells -- Production of chimeras -- Genotyping -- Parthenogenesis, pronuclear transfer, and mouse cloning -- Assisted reproduction: ovary transplantation, in vitro fertilization, artificial insemination, and intracytoplasmic sperm injection -- Cryopreservation, rederivation, and transport of mouse strains -- Techniques for visualizing gene products, cells, tissues, and organ systems -- Setting up a micromanipulation lab.", "author" : [ { "dropping-particle" : "", "family" : "Nagy", "given" : "Andras", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Gertsenstein", "given" : "Marina", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Vintersten", "given" : "Kristina", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Behringer", "given" : "Richard", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press", "id" : "ITEM-1", "issued" : { "date-parts" : [ [ "2003" ] ] }, "number-of-pages" : "814", "publisher" : "Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY", "title" : "Manipulating the Mouse Embryo", "type" : "book" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(Nagy et al., 2003)", "plainTextFormattedCitation" : "(Nagy et al., 2003)", "previouslyFormattedCitation" : "(Nagy et al., 2003)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(Nagy et al., 2003). For immunosurgery followed by ES derivation, blastocysts were incubated with rabbit anti-mouse red blood cell antibody (Inter-Cell Technologies) for 10 min. After the blastocysts were briefly washed twice in M2, they were incubated with guinea pig serum (Calbiochem) for 15 min. After removal of the zona pellucidae with acidic Tyrode’s solution (Sigma-Aldrich), the cells were cultured in ES cell medium for two weeks on a gelatine-coated dish. For in vitro culture of early mouse embryos, embryos at the 2-cell stage or later were incubated in KSOM (Millipore) in the presence or absence of PD0325901 (1 M; Wako), SU5402 (2 M; Wako), CHIR99021 (Chiron; 3 M; Wako), JNK inhibitor II (5 M; Calbiochem), SB203508 (10 M; Calbiochem), or LY924002 (10 M; Calbiochem). To activate the Fgf4-Fgfr-ERK pathway, recombinant hFGF4 (R&D) was added at a saturated concentration (1000 ng/ml) prepared in KSOM. In total, 1 ?g/ml Heparin (Sigma) was added together with FGF4 and control. BrdU was added (10 M) for 2 h, and BrdU Flow kits (BD Pharmingen) were used for detection.Immunohistochemistry. Embryos were fixed in 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) for 15 min, permeabilised in 0.5% Triton X-100 for 15 min and incubated in a blocking reagent (PBS + 10% donkey serum + 0.1% BSA + 0.01% Tween 20) for 1 h. Embryos were incubated at 4 °C overnight with primary antibodies prepared in blocking reagent. After the embryos were washed with PBS + 0.5% Triton X-100, they were incubated with Alexa 488-conjugated donkey anti-mouse IgG antibody (Molecular Probes) and Cy3-conjugated donkey anti-rabbit IgG antibody (Jackson Immunologicals) for 3 h at 4 °C. Nuclei were stained with Hoechst 33342 (10 g ml?1, Molecular Probes). Antibodies used for the immunohistochemitry are described in Table S2. Immunohistochemistry with anti-FGF4 antibody was performed in the Supplementary Materials and Methods section.Confocal microscopy analysis and image data acquisition. Embryos were mounted in drops of 30% glycerol on glass-bottom dishes. Confocal images were acquired using Leica TCS-SP5 or SP8 cameras. Fluorescence was excited with a 405-nm UV laser for Hoechst33342, a 638-nm laser for Cy5 or Alexa Fluor633, a 552-nm laser for Cy3, and a 488-nm laser for Alexa Fluor488. Images were acquired using an HC PL APO CS2 40 /1.30 oil immersion objective lens (Leica), with optical sections of 2–2.5 m. A Hybrid Detector system was used for the acquisition of raw images, which were processed using Leica software or Photoshop CS6 (Adobe). Cell nuclei were counted manually using ImageJ image analysis software (National Institutes of Health, Bethesda, MD). Protein expression levels were analysed as described ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1242/dev.003798", "ISBN" : "0950-1991 (Print)\\r0950-1991 (Linking)", "ISSN" : "0950-1991", "PMID" : "17978007", "abstract" : "Mouse pre-implantation development gives rise to the blastocyst, which is made up of at least three distinct cell types: the trophectoderm (TE) that surrounds a cavity, and an inner cell mass (ICM) comprising the primitive endoderm (PE) and epiblast (EPI). However, the underlying mechanisms involved in patterning the cleavage-stage embryo are still unresolved. By analyzing the distribution of the transcription factors Oct4 (Pou5f1), Cdx2 and Nanog at precisely defined stages in pre-implantation development, we were able to identify critical events leading to the divergence of TE, EPI and PE lineages. We found that Oct4 is present in all cells until late blastocyst, gradually disappearing from the TE thereafter. The expression patterns of both Cdx2 and Nanog exhibit two specific phases, culminating in their restriction to TE and EPI, respectively. In the first phase, starting after compaction, blastomeres show highly variable Cdx2 and Nanog protein levels. Importantly, the variability in Nanog levels is independent of position within the morula, whereas Cdx2 variability may originate from asymmetric cell divisions at the 8-cell stage in a non-stereotypic way. Furthermore, there is initially no reciprocal relationship between Cdx2 and Oct4 or between Cdx2 and Nanog protein levels. In the second phase, a definite pattern is established, possibly by a sorting process that accommodates intrinsic and extrinsic cues. Based on these results, we propose a model in which early embryonic mouse patterning includes stochastic processes, consistent with the highly regulative capacity of the embryo. This may represent a feature unique to early mammalian development.", "author" : [ { "dropping-particle" : "", "family" : "Dietrich", "given" : "Jens-Erik", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Hiiragi", "given" : "Takashi", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Development (Cambridge, England)", "id" : "ITEM-1", "issue" : "23", "issued" : { "date-parts" : [ [ "2007", "12" ] ] }, "page" : "4219-31", "title" : "Stochastic patterning in the mouse pre-implantation embryo.", "type" : "article-journal", "volume" : "134" }, "uris" : [ "" ] }, { "id" : "ITEM-2", "itemData" : { "DOI" : "10.1242/dev.084996", "ISSN" : "1477-9129", "PMID" : "23193166", "abstract" : "The emergence of pluripotent epiblast (EPI) and primitive endoderm (PrE) lineages within the inner cell mass (ICM) of the mouse blastocyst involves initial co-expression of lineage-associated markers followed by mutual exclusion and salt-and-pepper distribution of lineage-biased cells. Precisely how EPI and PrE cell fate commitment occurs is not entirely clear; however, previous studies in mice have implicated FGF/ERK signaling in this process. Here, we investigated the phenotype resulting from zygotic and maternal/zygotic inactivation of Fgf4. Fgf4 heterozygous blastocysts exhibited increased numbers of NANOG-positive EPI cells and reduced numbers of GATA6-positive PrE cells, suggesting that FGF signaling is tightly regulated to ensure specification of the appropriate numbers of cells for each lineage. Although the size of the ICM was unaffected in Fgf4 null mutant embryos, it entirely lacked a PrE layer and exclusively comprised NANOG-expressing cells at the time of implantation. An initial period of widespread EPI and PrE marker co-expression was however established even in the absence of FGF4. Thus, Fgf4 mutant embryos initiated the PrE program but exhibited defects in its restriction phase, when lineage bias is acquired. Consistent with this, XEN cells could be derived from Fgf4 mutant embryos in which PrE had been restored and these cells appeared indistinguishable from wild-type cells. Sustained exogenous FGF failed to rescue the mutant phenotype. Instead, depending on concentration, we noted no effect or conversion of all ICM cells to GATA6-positive PrE. We propose that heterogeneities in the availability of FGF produce the salt-and-pepper distribution of lineage-biased cells.", "author" : [ { "dropping-particle" : "", "family" : "Kang", "given" : "Minjung", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Piliszek", "given" : "Anna", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Artus", "given" : "J\u00e9r\u00f4me", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Hadjantonakis", "given" : "Anna-Katerina", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Development (Cambridge, England)", "id" : "ITEM-2", "issue" : "2", "issued" : { "date-parts" : [ [ "2013", "1", "15" ] ] }, "page" : "267-79", "title" : "FGF4 is required for lineage restriction and salt-and-pepper distribution of primitive endoderm factors but not their initial expression in the mouse.", "type" : "article-journal", "volume" : "140" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(Dietrich and Hiiragi, 2007; Kang et al., 2013)", "plainTextFormattedCitation" : "(Dietrich and Hiiragi, 2007; Kang et al., 2013)", "previouslyFormattedCitation" : "(Dietrich and Hiiragi, 2007; Kang et al., 2013)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(Dietrich and Hiiragi, 2007; Kang et al., 2013). Briefly, mean fluorescence intensities inside regions of interest (for example, nuclei) were measured, and subtracted from background signals, which were defined as the average of the mean fluorescence intensities of randomly chosen cytoplasmic signals, and were then normalised against the mean fluorescence intensity in the Hoechst channel using ImageJ ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1242/dev.003798", "ISBN" : "0950-1991 (Print)\\r0950-1991 (Linking)", "ISSN" : "0950-1991", "PMID" : "17978007", "abstract" : "Mouse pre-implantation development gives rise to the blastocyst, which is made up of at least three distinct cell types: the trophectoderm (TE) that surrounds a cavity, and an inner cell mass (ICM) comprising the primitive endoderm (PE) and epiblast (EPI). However, the underlying mechanisms involved in patterning the cleavage-stage embryo are still unresolved. By analyzing the distribution of the transcription factors Oct4 (Pou5f1), Cdx2 and Nanog at precisely defined stages in pre-implantation development, we were able to identify critical events leading to the divergence of TE, EPI and PE lineages. We found that Oct4 is present in all cells until late blastocyst, gradually disappearing from the TE thereafter. The expression patterns of both Cdx2 and Nanog exhibit two specific phases, culminating in their restriction to TE and EPI, respectively. In the first phase, starting after compaction, blastomeres show highly variable Cdx2 and Nanog protein levels. Importantly, the variability in Nanog levels is independent of position within the morula, whereas Cdx2 variability may originate from asymmetric cell divisions at the 8-cell stage in a non-stereotypic way. Furthermore, there is initially no reciprocal relationship between Cdx2 and Oct4 or between Cdx2 and Nanog protein levels. In the second phase, a definite pattern is established, possibly by a sorting process that accommodates intrinsic and extrinsic cues. Based on these results, we propose a model in which early embryonic mouse patterning includes stochastic processes, consistent with the highly regulative capacity of the embryo. This may represent a feature unique to early mammalian development.", "author" : [ { "dropping-particle" : "", "family" : "Dietrich", "given" : "Jens-Erik", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Hiiragi", "given" : "Takashi", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Development (Cambridge, England)", "id" : "ITEM-1", "issue" : "23", "issued" : { "date-parts" : [ [ "2007", "12" ] ] }, "page" : "4219-31", "title" : "Stochastic patterning in the mouse pre-implantation embryo.", "type" : "article-journal", "volume" : "134" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(Dietrich and Hiiragi, 2007)", "plainTextFormattedCitation" : "(Dietrich and Hiiragi, 2007)", "previouslyFormattedCitation" : "(Dietrich and Hiiragi, 2007)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(Dietrich and Hiiragi, 2007). We defined a cell as positive if it showed a higher fluorescence signal compared with the background. To quantify Nanog and Gata6 protein expression levels, we measured Nanog expression in both Nanog+ /Gata6- and Nanog+/Gata6+ (DP) cells, and Gata6 expression in both Gata6+ /Nanog- and DP cells. For the quantification of FGF4 expression in preimplantation embryos, background signals were subtracted from mean fluorescence intensities inside regions of interest in the cytoplasm of FGF4+ cells. Background signals were defined as the average of the mean fluorescence intensities of randomly chosen cytoplasmic regions in without-antibody negative controls. FGF4 expression values were then normalised against the mean fluorescence intensity in the Hoechst channel using ImageJ. Individual cells were distinguished using intercellular gaps seen in differential interference contrast (DIC) images from the same focal plane as the corresponding confocal microscopy image.TUNEL assay. Apoptotic cells were identified using the DeadEnd? Fluorometric TUNEL System (Promega). For counting TUNEL+ cells, individual cells were identified using the intercellular gaps seen in DIC images merged with images of nuclear staining and TUNEL staining. Fragmented or pyknotic nuclei bounded by the same intercellular gap were counted as one cell.Chromatin immunoprecipitation (ChIP) assays. The ChIP assays were performed as described ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1172/JCI67804", "ISSN" : "1558-8238", "PMID" : "24135137", "abstract" : "The TGF-\u03b2 superfamily comprises pleiotropic cytokines that regulate SMAD and non-SMAD signaling. TGF-\u03b2-SMAD signal transduction is known to be involved in tissue fibrosis, including renal fibrosis. Here, we found that 1,25-dihydroxyvitamin D3-bound [1,25(OH)2D3-bound] vitamin D receptor (VDR) specifically inhibits TGF-\u03b2-SMAD signal transduction through direct interaction with SMAD3. In mouse models of tissue fibrosis, 1,25(OH)2D3 treatment prevented renal fibrosis through the suppression of TGF-\u03b2-SMAD signal transduction. Based on the structure of the VDR-ligand complex, we generated 2 synthetic ligands. These ligands selectively inhibited TGF-\u03b2-SMAD signal transduction without activating VDR-mediated transcription and significantly attenuated renal fibrosis in mice. These results indicate that 1,25(OH)2D3-dependent suppression of TGF-\u03b2-SMAD signal transduction is independent of VDR-mediated transcriptional activity. In addition, these ligands did not cause hypercalcemia resulting from stimulation of the transcriptional activity of the VDR. Thus, our study provides a new strategy for generating chemical compounds that specifically inhibit TGF-\u03b2-SMAD signal transduction. Since TGF-\u03b2-SMAD signal transduction is reportedly involved in several disorders, our results will aid in the development of new drugs that do not cause detectable adverse effects, such as hypercalcemia.", "author" : [ { "dropping-particle" : "", "family" : "Ito", "given" : "Ichiaki", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Waku", "given" : "Tsuyoshi", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Aoki", "given" : "Masato", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Abe", "given" : "Rumi", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Nagai", "given" : "Yu", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Watanabe", "given" : "Tatsuya", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Nakajima", "given" : "Yuka", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Ohkido", "given" : "Ichiro", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Yokoyama", "given" : "Keitaro", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Miyachi", "given" : "Hiroyuki", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Shimizu", "given" : "Toshiyuki", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Murayama", "given" : "Akiko", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Kishimoto", "given" : "Hiroyuki", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Nagasawa", "given" : "Kazuo", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Yanagisawa", "given" : "Junn", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "The Journal of clinical investigation", "id" : "ITEM-1", "issue" : "11", "issued" : { "date-parts" : [ [ "2013", "11", "1" ] ] }, "page" : "4579-94", "title" : "A nonclassical vitamin D receptor pathway suppresses renal fibrosis.", "type" : "article-journal", "volume" : "123" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(Ito et al., 2013)", "plainTextFormattedCitation" : "(Ito et al., 2013)", "previouslyFormattedCitation" : "(Ito et al., 2013)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(Ito et al., 2013). Cells were fixed with 1% formaldehyde and sonicated. The samples were incubated with 4 g of normal mouse IgG (Santa Cruz Biochemicals) and anti-FLAG-M2 antibody (Sigma-Aldrich). The detail was described in in the Supplementary Materials and Methods section. The sequences of the primers used for ChIP-qPCR are listed in Table S3.Chromatin immunoprecipitation (ChIP) assays and ChIP-sequencing (ChIP-seq) data analysis. The ChIP assays were performed as previously described ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1172/JCI67804", "ISSN" : "1558-8238", "PMID" : "24135137", "abstract" : "The TGF-\u03b2 superfamily comprises pleiotropic cytokines that regulate SMAD and non-SMAD signaling. TGF-\u03b2-SMAD signal transduction is known to be involved in tissue fibrosis, including renal fibrosis. Here, we found that 1,25-dihydroxyvitamin D3-bound [1,25(OH)2D3-bound] vitamin D receptor (VDR) specifically inhibits TGF-\u03b2-SMAD signal transduction through direct interaction with SMAD3. In mouse models of tissue fibrosis, 1,25(OH)2D3 treatment prevented renal fibrosis through the suppression of TGF-\u03b2-SMAD signal transduction. Based on the structure of the VDR-ligand complex, we generated 2 synthetic ligands. These ligands selectively inhibited TGF-\u03b2-SMAD signal transduction without activating VDR-mediated transcription and significantly attenuated renal fibrosis in mice. These results indicate that 1,25(OH)2D3-dependent suppression of TGF-\u03b2-SMAD signal transduction is independent of VDR-mediated transcriptional activity. In addition, these ligands did not cause hypercalcemia resulting from stimulation of the transcriptional activity of the VDR. Thus, our study provides a new strategy for generating chemical compounds that specifically inhibit TGF-\u03b2-SMAD signal transduction. Since TGF-\u03b2-SMAD signal transduction is reportedly involved in several disorders, our results will aid in the development of new drugs that do not cause detectable adverse effects, such as hypercalcemia.", "author" : [ { "dropping-particle" : "", "family" : "Ito", "given" : "Ichiaki", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Waku", "given" : "Tsuyoshi", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Aoki", "given" : "Masato", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Abe", "given" : "Rumi", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Nagai", "given" : "Yu", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Watanabe", "given" : "Tatsuya", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Nakajima", "given" : "Yuka", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Ohkido", "given" : "Ichiro", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Yokoyama", "given" : "Keitaro", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Miyachi", "given" : "Hiroyuki", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Shimizu", "given" : "Toshiyuki", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Murayama", "given" : "Akiko", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Kishimoto", "given" : "Hiroyuki", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Nagasawa", "given" : "Kazuo", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Yanagisawa", "given" : "Junn", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "The Journal of clinical investigation", "id" : "ITEM-1", "issue" : "11", "issued" : { "date-parts" : [ [ "2013", "11", "1" ] ] }, "page" : "4579-94", "title" : "A nonclassical vitamin D receptor pathway suppresses renal fibrosis.", "type" : "article-journal", "volume" : "123" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(Ito et al., 2013)", "plainTextFormattedCitation" : "(Ito et al., 2013)", "previouslyFormattedCitation" : "(Ito et al., 2013)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(Ito et al., 2013). Details are described in the Supplementary Materials and Methods section.Microarray analysis. cDNAs were synthesised from individual WT and Klf5 KO embryos at E3.0 as previously described ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1038/nprot.2007.79", "ISBN" : "1750-2799 (Electronic) 1750-2799 (Linking)", "ISSN" : "1750-2799", "PMID" : "17406636", "abstract" : "We describe here a protocol for the representative amplification of global mRNAs from typical single mammalian cells to provide a template for high-density oligonucleotide microarray analysis. A single cell is lysed in a tube without purification and first-strand cDNAs are synthesized using a poly(dT)-tailed primer. Unreacted primer is specifically eliminated by exonuclease treatment and second strands are generated with a second poly(dT)-tailed primer after poly(dA) tailing of the first-strand cDNAs. The cDNAs are split into four tubes, which are independently directionally amplified by PCR, and then recombined. The amplified products (approximately 100 ng) show superior representation and reproducibility of original gene expression, especially for genes expressed in more than 20 copies per cell, compared with those obtained by a conventional PCR protocol, and can effectively be used for quantitative PCR and EST analyses. The cDNAs are then subjected to another PCR amplification with primers bearing the T7 promoter sequence. The resultant cDNA products are gel purified, amplified by one final cycle and used for isothermal linear amplification by T7 RNA polymerase to synthesize cRNAs for microarray hybridization. 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We here present single-cell mRNA 3-prime end sequencing (SC3-seq), a practical methodology based on PCR amplification followed by 3-prime-end enrichment for highly quantitative, parallel and cost-effective measurement of gene expression in single cells. The SC3-seq allows excellent quantitative measurement of mRNAs ranging from the 10,000-cell to 1-cell level, and accordingly, allows an accurate estimate of the transcript levels by a regression of the read counts of spike-in RNAs with defined copy numbers. The SC3-seq has clear advantages over other typical single-cell RNA-seq methodologies for the quantitative measurement of transcript levels and at a sequence depth required for the saturation of transcript detection. The SC3-seq distinguishes four distinct cell types in the peri-implantation mouse blastocysts. Furthermore, the SC3-seq reveals the heterogeneity in human-induced pluripotent stem cells (hiPSCs) cultured under on-feeder as well as feeder-free conditions, demonstrating a more homogeneous property of the feeder-free hiPSCs. 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The cDNAs were amplified further in a linear fashion and labelled with a Cy3- or Cy5-conjugated nucleotide. The hybridisation procedures were performed by TaKaRa Bio Inc. The data have been deposited in the Gene Expression Omnibus database under accession number GEO65020.Isolation of single cells for cDNA amplification. Outer cells of E3.25 embryos were removed by immunosurgery. Inner cells were incubated in a 1:1 mixture of Accutase (Nakalai Tesque) and 0.25% trypsin-EDTA (Invitrogen) for approximately 5 min at 37°C and then dissociated into single cells by pipetting. cDNAs were synthesised from the isolated single cell using the single-cell mRNA 3-prime sequencing (SC3-seq) method as previously described ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1093/nar/gkv134", "ISSN" : "1362-4962", "PMID" : "25722368", "abstract" : "Single-cell mRNA sequencing (RNA-seq) methods have undergone rapid development in recent years, and transcriptome analysis of relevant cell populations at single-cell resolution has become a key research area of biomedical sciences. We here present single-cell mRNA 3-prime end sequencing (SC3-seq), a practical methodology based on PCR amplification followed by 3-prime-end enrichment for highly quantitative, parallel and cost-effective measurement of gene expression in single cells. 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For the RT–qPCR analysis, first-strand cDNA was synthesised from total RNA using a QuantiTect Reverse Transcription Kit (QIAGEN). Real-time PCR was performed with SYBR Premix Ex Taq II (TaKaRa) and analysed on a Thermal Cycler Dice Real Time System (TP850; TaKaRa). The amount of target RNA was estimated using an appropriate standard curve and divided by the estimated amount of -actin for normalisation. The sequences of the primers used for quantitative PCR are listed in Table S3.Statistical analysis. Statistical analyses were performed using the nonparametric Mann–Whitney U-test or Student’s t-test. The data are expressed as the mean and standard error. Differences were considered significant at P < 0.05. Statistical analyses were performed using Graph Pad Prism6 (Graph Pad Software) for the nonparametric Mann–Whitney U-test and EXCEL (Microsoft) for Student’s t-test. Single-cell qPCR data analysis was performed using R software with gplots (ver. 3.0.1) and ggplot2 (ver. 2.2.1) and EXCEL (Microsoft).Author contributionsT. A., H. J., S. T., and J. Y. performed the biochemical and cell biological experiments. T. W. performed the ChIP experiments. K. M. and A. K. established the ES cell lines. M. M. performed bioinformatics analysis. T. A. and T. N. performed single-cell RT-qPCR analysis. H. N., I. M., R. N., T. K., and J. Y. provided essential materials. K. K. and M. S. contributed to microarray analysis. T. A. and M. E. contributed to the development of the hypothesis, experimental design, and interpretation of the data. M. E. wrote the peting financial interestsThe authors declare no competing financial interests.AcknowledgementsWe thank Drs. Hitoshi Niwa, Yojiro Yamanaka and Tomoyuki Tsukiyama for helpful discussions and reagents. M. E. thanks Dr. Vincent Kelly for critical readings of the manuscript. 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FGF signal-dependent segregation of primitive endoderm and epiblast in the mouse blastocyst. Development 137, 715–24.Yeo, J.-C., Jiang, J., Tan, Z.-Y., Yim, G.-R., Ng, J.-H., G?ke, J., Kraus, P., Liang, H., Gonzales, K. A. U., Chong, H.-C., et al. (2014). Klf2 is an essential factor that sustains ground state pluripotency. Cell Stem Cell 14, 864–72.Ying, Q.-L., Wray, J., Nichols, J., Batlle-Morera, L., Doble, B., Woodgett, J., Cohen, P. and Smith, A. (2008). The ground state of embryonic stem cell self-renewal. Nature 453, 519–23.Figure legendsFigure 1 Skewed EPI and PrE lineage specification in Klf5 KO and OE blastocysts. (A) Expression levels of Nanog and Gata6 in WT, Klf5 KO and Klf5 OE embryos from E3.25–4.0. Confocal microscopy cross-sections are shown. Note that Nanog expression was reduced in Klf5 KO blastocysts at E3.25 and was dramatically reduced after E3.5. Gata6 expression was induced at E3.25–E4.0. C, cell number. (B) Tukey box plots of Nanog and Gata6 protein expression levels. Embryo numbers are shown in brackets. A.U., arbitrary unit. (C) Percentages of the number of Nanog+ (Nanog+/Gata6-), Gata6+ (Nanog-/Gata6+) and DP cells per embryo. Asterisks in (B) and (C) indicate statistical significance compared to WT: *P < 0.01; ** P < 0.001; Mann–Whitney U-test. Embryo numbers are shown in brackets. Figure 2 Accelerated PrE lineage specification in Klf5 KO embryos. (A) Upregulation of Pdgfra in the inner cells of the Klf5 KO embryos at E3.25. Note that the inner cells of the Klf5 KO embryos were DP cells that strongly express Pdgfra. Arrowheads and arrows indicate inner cells and outer cells, respectively. Confocal microscopy cross-sections for E3.25 are shown. (B) Absolute number of Pdgfra+ and Pdgfra- cells in inner or outer cells. (C) Graphical image of WT and Klf5 KO embryos at E3.25. More than 80% of inner cells in Klf5 KO embryos strongly expressed Pdgfra (indicated by the orange outline of the cell membrane) but not WT embryos, indicating the accelerated PrE specification of the inner cells. (D) Expression of Nanog and Gata4 in WT and Klf5 KO blastocysts from E3.75. Note that Gata4 expression levels were dramatically upregulated in Klf5 KO blastocysts. Confocal microscopy cross-sections are shown. C, cell number. (E) Percentages of the number of Gata4+ cells per embryo. (F) Tukey box plots of Gata4 protein expression levels. Embryo numbers are shown in brackets. A.U., arbitrary unit. Asterisks in (B), (E), and (F) indicate statistical significance: *P < 0.01; ** P < 0.001; n.s., not significant.Figure 3 Klf5 is required for the repression of Fgf4 expression in morulae at E3.0. (A) Heat map for representative up- and downregulated genes. (B) Average of relative gene expression in Klf5 KO embryos (n = 14) compared to WT embryos (n = 14) at E3.0. β-Actin was used for the normalisation of mRNA expression: *P < 0.05, **P < 0.01; Mann–Whitney U-test. (C) Increased expression levels of FGF4 protein in Klf5 KO morulae at E3.0. Confocal cross-sections are shown. Insets indicate high-magnification images of FGF4+ cells. The data are shown from a negative control experiment on WT morulae without the anti-FGF4 antibody (-Ab). C, cell number. Scale bars represent 20 ?m. (D) Box plots showing FGF4 protein expression levels in FGF4+ cells of WT and Klf5 KO morulae at E3.0. The data are represented as Tukey box plots. The central mark is the median, the edges of the box are the 25th and 75th percentiles, and the whiskers extend to 1.5 the interquartile range. Asterisks indicate statistical significance: *P < 0.01; Mann–Whitney U-test. A. U., arbitrary unit. (E) FGF4 protein expression levels in FGF4+ cells of WT, Klf5 KO and Klf5 OE blastocysts at E3.25. Confocal cross-sections are shown. C, cell number. Scale bars represent 20 ?m. (F) Tukey box plots showing FGF4 protein expression levels in FGF4+ cells of WT, Klf5 KO, and Klf5 OE embryos at E3.25. Asterisks indicate statistical significance: *P < 0.05, ** P < 0.01; Mann–Whitney U-test. A. U., arbitrary unit.Figure 4 Klf5 is required to suppress Fgf4 in a subset of inner cells at E3.0-E3.25.(A, B, C) Single-cell RT-qPCR analysis of inner cells from WT, Klf5 KO, and Klf5 OE embryos at E3.25. cDNAs extracted from 84 cells from WT embryos (n = 14), 54 cells from Klf5 KO embryos (n = 7), and 53 cells from Klf5 OE embryos (n = 8) were synthesised using the SC3-seq method. (A) The ΔCt values from the average Ct value of β-Actin are shown as heat maps and are used for clustering. The genotype of each sample is shown above　heat map. (B) Violin plot of single-cell mRNA expression levels of Fgf4 in WT, Klf5 KO, and Klf5 OE inner cells at E3.25. The ΔCt values from the average Ct value of β-Actin are shown as a violin plot. There were two cell populations with different Fgf4 expression levels: Fgf4-high cells and low/negative cells. Fgf4-high cells and low/negative cells were determined by a ΔCt value of 4.3 that marks the 25th percentile expression levels in WT embryos. Asterisks indicate statistical significance: *P < 0.01; ** P < 0.001; Mann–Whitney U-test. (C) Numbers of Fgf4-high cells in the inner cell of WT, Klf5 KO, and Klf5 OE embryos at E3.25 from single cell RT-qPCR are shown in (A). (D) The ΔΔCt values normalised to the average WT ΔCt value of Fgf4-high and -low/negative cells in WT, Klf5 KO, and Klf5 OE embryos are shown as box plots. Fgf4-high cells were determined by ΔCt values of 4.3 that marks the 25th percentile expression levels in WT embryos. Sample number is indicated above each boxplot. Asterisks indicate statistical significance: *P < 0.01; ** P < 0.001; Mann–Whitney U-test. Percentages are shown in brackets. Asterisks indicate statistical significance against to WT: *P < 0.01; Fishers exact test. (E) Binding peaks of Klf5 to Fgf4 genes in mouse ES cells. The number below the binding peaks indicates regions designated for ChIP primers used in Figure 4F. (F) ChIP analysis of the binding of Klf5 to Fgf4 in mouse ES cells. The promoter region of Vegfr2, which is not regulated by Klf5, was analysed as a negative control region. Asterisks indicate statistical significance: *P < 0.01; Student’s t-test.Figure 5 Inhibition of Fgfr–ERK signalling rescues the cell-cycle defects of Klf5 KO blastocysts. (A) Experimental outline for assessing the role of the Fgf4–Fgfr–MAPK pathway in Klf5 KO blastocysts. (B) Effects of inhibitors of MEK (MEKi) and Fgfr (Fgfri) in WT and Klf5 KO blastocysts. Embryos at E2.75 were recovered and incubated in the presence or absence of inhibitors of MEK (PD0325901, 1 ?M) and Fgfr (SU5402, 2 ?M) for 24 h, and then subjected to TUNEL assay and immunohistochemistry. Z-projected confocal microscopy cross-sections are shown. C, cell number. Scale bars represent 25 ?m. (C) The numbers of cells (left) and percentages of TUNEL+ (apoptotic) cells per embryo (right). Embryo numbers are shown in brackets. (D) Bright field and fluorescent images of WT embryos cultured in the presence of FGF4 (1000 ng/ml) from E2.75 to E3.75 and of Klf5 KO embryos at E3.75. Maximum intensity projections of confocal microscopy images are shown. C, cell number. Scale bars represent 20 ?m. (E) WT embryos cultured in the presence of FGF4 showed a slight but not significant reduction in cell numbers (left) and increase in the percentages of TUNEL+ cells (right). Embryo numbers are shown in brackets. Asterisks in (C) and (E) indicate statistical significance: *P < 0.01; ** P < 0.001; Mann–Whitney U-test.Figure 6 Treatment with Fgfr or MEK inhibitors reversed the skewed lineage specification of Klf5 KO blastocysts. (A) Experimental outline to assess the role of the Fgf–Fgfr–ERK pathway in Klf5 KO embryos. Embryos were collected from Klf5 heterozygous inter-crosses at E2.5 and cultured in the presence or absence of MEKi (PD0325901, 1 ?M) for 24 h and 48 h. (B, B) Effects of MEKi on skewed lineage specification of Klf5 KO blastocysts at E3.5 (B) and E4.5 (B). Confocal microscopy cross-sections are shown. C, cell number. (C) Tukey box plots of Nanog and Gata6 protein expression levels per embryo (left). Percentages of the numbers of Nanog+ and Gata6+ cells per embryo (right). Embryo numbers are shown in brackets. Plotted results are the mean and SEM of three independent experiments. A.U., arbitrary unit. Asterisks indicate statistical significance: *P < 0.01; ** P < 0.001; Mann–Whitney U-test.Figure 7 Model of the possible role of Klf5 in ICM lineage specification. At E3.0, the inner cells are DP precursors. At E3.5–3.75, the salt-and-pepper distribution of Nanog and Gata6 is evident, and cells are considered Nanog+ EPI and Gata6+ PrE lineage-precursor cells. At E4.0–4.25, the lineages have been determined. While Fgf4–ERK signalling is not involved in the initial co-expression of Nanog and Gata6, it regulates EPI/PrE specification by inhibiting Nanog and maintaining Gata6 expression. In the Klf5 KO embryos, Fgf4 is overexpressed because of derepression in the absence of Klf5, leading to PrE lineage specification. Gata4, a late marker for PrE lineage specification, is seen from the 64-cell stage in Klf5 KO embryos but only in a few Klf5 OE embryos, indicating accelerated differentiation into the PrE lineage. At E3.5, Fgf4 is activated in EPI-precursor cells in a Nanog- and Oct3/4-dependent manner according to Messerschmidt and Kemler, Frankenberg et al., Frum et al., and Le Bin et al. ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1016/j.devcel.2011.10.019", "ISSN" : "1878-1551", "PMID" : "22172669", "abstract" : "During preimplantation mouse development, the inner cell mass (ICM) differentiates into two cell lineages--the epiblast and the primitive endoderm (PrE)--whose precursors are identifiable by reciprocal expression of Nanog and Gata6, respectively. PrE formation depends on Nanog by a non-cell-autonomous mechanism. To decipher early cell- and non-cell-autonomous effects, we performed a mosaic knockdown of Nanog and found that this is sufficient to induce a PrE fate cell autonomously. Strikingly, in Nanog null embryos, Gata6 expression is maintained, showing that initiation of the PrE program is Nanog independent. Treatment of Nanog null embryos with pharmacological inhibitors revealed that RTK dependency of Gata6 expression is initially direct but later indirect via Nanog repression. Moreover, we found that subsequent expression of Sox17 and Gata4--later markers of the PrE--depends on the presence of Fgf4 produced by Nanog-expressing cells. Thus, our results reveal three distinct phases in the PrE differentiation program.", "author" : [ { "dropping-particle" : "", "family" : "Frankenberg", "given" : "Stephen", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Gerbe", "given" : "Fran\u00e7ois", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Bessonnard", "given" : "Sylvain", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Belville", "given" : "Corinne", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Pouchin", "given" : "Pierre", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Bardot", "given" : "Olivier", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Chazaud", "given" : "Claire", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Developmental cell", "id" : "ITEM-1", "issue" : "6", "issued" : { "date-parts" : [ [ "2011", "12", "13" ] ] }, "page" : "1005-13", "title" : "Primitive endoderm differentiates via a three-step mechanism involving Nanog and RTK signaling.", "type" : "article-journal", "volume" : "21" }, "uris" : [ "" ] }, { "id" : "ITEM-2", "itemData" : { "DOI" : "10.1016/j.devcel.2013.05.004", "ISBN" : "1878-1551 (Electronic)\\r1534-5807 (Linking)", "ISSN" : "1878-1551", "PMID" : "23747191", "abstract" : "In embryonic stem (ES) cells and in early mouse embryos, the transcription factor Oct4 is an essential regulator of pluripotency. Oct4 transcriptional targets have been described in ES cell lines; however, the molecular mechanisms by which Oct4 regulates establishment of pluripotency in the epiblast (EPI) have not been fully elucidated. Here, we show that neither maternal nor zygotic Oct4 is required for the formation of EPI cells in the blastocyst. Rather, Oct4 is first required for development of the primitive endoderm (PE), an extraembryonic lineage. EPI cells promote PE fate in neighboring cells by secreting Fgf4, and Oct4 is required for expression of Fgf4, but we show that Oct4 promotes PE development cell-autonomously, downstream of Fgf4 and Mapk. Finally, we show that Oct4 is required for the expression of multiple EPI and PE genes as well as multiple metabolic pathways essential for the continued growth of the preimplantation embryo.", "author" : [ { "dropping-particle" : "", "family" : "Frum", "given" : "Tristan", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Halbisen", "given" : "MichaelA A", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Wang", "given" : "Chaoyang", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Amiri", "given" : "Hossein", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Robson", "given" : "Paul", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Ralston", "given" : "Amy", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Developmental cell", "id" : "ITEM-2", "issue" : "6", "issued" : { "date-parts" : [ [ "2013", "6", "24" ] ] }, "page" : "610-22", "publisher" : "Elsevier Inc.", "title" : "Oct4 cell-autonomously promotes primitive endoderm development in the mouse blastocyst.", "type" : "article-journal", "volume" : "25" }, "uris" : [ "" ] }, { "id" : "ITEM-3", "itemData" : { "DOI" : "10.1242/dev.096875", "ISSN" : "1477-9129", "PMID" : "24504341", "abstract" : "The transcription factor Oct4 is required in vitro for establishment and maintenance of embryonic stem cells and for reprogramming somatic cells to pluripotency. In vivo, it prevents the ectopic differentiation of early embryos into trophoblast. Here, we further explore the role of Oct4 in blastocyst formation and specification of epiblast versus primitive endoderm lineages using conditional genetic deletion. Experiments involving mouse embryos deficient for both maternal and zygotic Oct4 suggest that it is dispensable for zygote formation, early cleavage and activation of Nanog expression. Nanog protein is significantly elevated in the presumptive inner cell mass of Oct4 null embryos, suggesting an unexpected role for Oct4 in attenuating the level of Nanog, which might be significant for priming differentiation during epiblast maturation. Induced deletion of Oct4 during the morula to blastocyst transition disrupts the ability of inner cell mass cells to adopt lineage-specific identity and acquire the molecular profile characteristic of either epiblast or primitive endoderm. Sox17, a marker of primitive endoderm, is not detected following prolonged culture of such embryos, but can be rescued by provision of exogenous FGF4. Interestingly, functional primitive endoderm can be rescued in Oct4-deficient embryos in embryonic stem cell complementation assays, but only if the host embryos are at the pre-blastocyst stage. We conclude that cell fate decisions within the inner cell mass are dependent upon Oct4 and that Oct4 is not cell-autonomously required for the differentiation of primitive endoderm derivatives, as long as an appropriate developmental environment is established.", "author" : [ { "dropping-particle" : "", "family" : "Bin", "given" : "Gloryn Chia", "non-dropping-particle" : "Le", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Mu\u00f1oz-Descalzo", "given" : "Silvia", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Kurowski", "given" : "Agata", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Leitch", "given" : "Harry", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Lou", "given" : "Xinghua", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Mansfield", "given" : "William", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Etienne-Dumeau", "given" : "Charles", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Grabole", "given" : "Nils", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Mulas", "given" : "Carla", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Niwa", "given" : "Hitoshi", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Hadjantonakis", "given" : "Anna-Katerina", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Nichols", "given" : "Jennifer", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Development (Cambridge, England)", "id" : "ITEM-3", "issue" : "5", "issued" : { "date-parts" : [ [ "2014", "3" ] ] }, "page" : "1001-10", "title" : "Oct4 is required for lineage priming in the developing inner cell mass of the mouse blastocyst.", "type" : "article-journal", "volume" : "141" }, "uris" : [ "" ] }, { "id" : "ITEM-4", "itemData" : { "DOI" : "10.1016/j.ydbio.2010.04.020", "ISSN" : "1095-564X", "PMID" : "20435031", "abstract" : "Early lineage segregation in mouse development results in two, either CDX2- or OCT4/NANOG-positive, cell populations. CDX2-positive cells form the trophectoderm (TE), OCT4/NANOG-positive cells the inner cell mass (ICM). In a second lineage decision ICM cells segregate into Epiblast (EPI) and primitive endoderm (PE). EPI and PE formation depend on the activity of the transcription factors Nanog and Gata4/6. A role for Nanog, a crucial pluripotency factor, in preventing PE differentiation has been proposed, as outgrowths of mutant ICMs result in PE, but not EPI derivatives. We established Nanog-mutant mouse lines and analyzed EPI and PE formation in vivo. Surprisingly, Gata4 expression in mutant ICM cells is absent or strongly decreased, thus loss of Nanog does not result in precocious endoderm differentiation. However, Nanog-deficient embryos retain the capacity to form PE in chimeric embryos and, in contrast to recent reports, in blastocyst outgrowths. Based on our findings we propose a non-cell autonomous requirement of Nanog for proper PE formation in addition to its essential role in EPI determination.", "author" : [ { "dropping-particle" : "", "family" : "Messerschmidt", "given" : "Daniel M", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Kemler", "given" : "Rolf", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Developmental biology", "id" : "ITEM-4", "issue" : "1", "issued" : { "date-parts" : [ [ "2010", "8", "1" ] ] }, "page" : "129-37", "publisher" : "Elsevier Inc.", "title" : "Nanog is required for primitive endoderm formation through a non-cell autonomous mechanism.", "type" : "article-journal", "volume" : "344" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(Frankenberg et al., 2011; Frum et al., 2013; Le Bin et al., 2014; Messerschmidt and Kemler, 2010)", "plainTextFormattedCitation" : "(Frankenberg et al., 2011; Frum et al., 2013; Le Bin et al., 2014; Messerschmidt and Kemler, 2010)", "previouslyFormattedCitation" : "(Frankenberg et al., 2011; Frum et al., 2013; Le Bin et al., 2014; Messerschmidt and Kemler, 2010)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(Frankenberg et al., 2011; Frum et al., 2013; Le Bin et al., 2014; Messerschmidt and Kemler, 2010). Klf5 represses precocious Fgf4 transcription to suppress the Fgf4–Fgfr–ERK pathway, thereby ensuring Nanog+ pluripotent epiblast development. HYPERLINK "" HYPERLINK "" ................
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