O2 deprivation (hypoxia) occurs in a number of ...



Supplementary Figure 1. Hif-1α-/- phenotypes in the SvEvTac background.

(A) The Hif-1α-/- null allele (Compernolle et al. 2003) was backcrossed into the SvEvTac genetic background. The resulting heterozygous Hif-1α+/- mice were intercrossed to generate E9.5 embryos. Hif-1α-/- embryos displayed developmental arrest and morphological phenotypes identical to those previously reported for other genetic backgrounds. (B) E9.5 Hif-1α-/- embryos were recovered in expected Mendelian ratios. (C) In contrast to Hif-2α KI/KI embryos, E7.5 Hif-1α-/- embryos were phenotypically normal. (D-E) Whole mount RNA in situ hybridization on E7.5 embryos using probes for the mesodermal marker Brachyury (Bry) (D) and visceral endodermal marker Alpha-fetoprotein (AFP) (E). Compared to WT littermate controls, Hif-1α-/- embryos also display normal mesoderm migration through the primitive streak and AFP expression is restricted to the extraembryonic visceral endoderm. (F) Homozygous Hif-1α KI ES cells were injected into E3.5 blastocysts to generate chimeric embryos. Examples of highly chimeric E9.5 Hif-1α KI embryos that were recovered are shown. Hif-1α KI embryos appear normal at these stages, whereas homozygous Hif-2α KI embryos were not recovered at this stage.

Supplementary Figure 2. Generation and characterization of HIF-1α knock-in control.

(A) Targeting strategy showing cDNA encoding c-Myc epitope-tagged HIF-1α fused to the SV40 polyadenylation sequence, the neomycin resistance gene cassette (NeoR), and loxP recombination sites (black triangles). The 4.7 kb 5’ homology arm and 6 kb 3’ homology arm are designated by thick lines. The targeting vector was electroporated into ES cells, where the properly targeted Hif-1α KI allele resulted from homologous recombination. No Hif-1α genomic sequences were deleted. The Neo-R cassette was removed by Cre recombinase via transient transfection, producing the final Hif-1α KI allele. This targeting is nearly identical to that described in Covello et. al (2005), except that a HIF-1α cDNA was inserted, and a slightly longer 5’ homology arm was used. (B) Southern blot analysis of HpaI-digested DNA from ES cell clones heterozygous or homozygous for the Hif-1α KI allele verifying correct targeting. Probe derived from sequences 5’ to the homology arm (black box) distinguishes the Hif-1α KI allele from wild type. Southern blot analysis of heterozygous and homozygous ES cells following Cre-mediated deletion of the NeoR cassette. Hpa I-digested genomic DNA was hybridized to an allele-specific probe derived from the SV40 polyadenylation sequence fused to the HIF-1α cDNA (white box)

Supplementary Figure 3. Oct-4 knockdown by RNAi.

(A) Western blot analysis for Oct-4 indicating knockdown of Oct-4 protein up to 75% of WT in homozygous Hif-2α KI/KI 3.5 day embryoid bodies. AKT, loading control.

(B) Real-time PCR analysis demonstrating increased trophectoderm markers Cdx2 and Hand1 in homozygous Hif-2α KI/KI cells that displayed Oct-4 levels lower than WT.

Supplementary Figure 4. Altered differentiation in Hif-2α KI/KI teratomas with Oct-4 expression reduced.

(A-C) Hematoxylin and eosin (H&E) staining of WT (A), Hif-2α KI/KI (B), and Hif-2α KI/KI-Oct-4 shRNA teratomas (C). (A) Primitive neuroectoderm in WT teratomas. (B) Hif-2α KI/KI teratomas contained regions exhibiting highly undifferentiated cells (not shown) or highly differentiated tissues such as cartilage, Schwann cells and ganglia. (C) When Oct-4 levels are reduced in Hif-2α KI/KI-Oct-4 shRNA tumors, H&E staining reveals more primitive neuoectoderm compared to the Hif-2α KI/KI teratomas. ne, neuroectoderm; c, cartilage; s, Schwann cells; g, ganglia. (D) Quantitation of differentiated tissues in the teratomas. Oct-4 knockdown in the Hif-2α KI/KI teratomas decreased cartilage, bone, Schwann cell, and ganglia content, but not the number of fat cells or hemangiomas. (E) Quantitation of Nanog positive cells (undifferentiated cells) in the teratomas.

Supplementary Figure 5. Disrupted development in HIF-2α transgenic embryos.

(A,B) HIF-2α transgenic embryos with normal (A) and increased (B) Hif-2α and Oct-4 expression.

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