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The Role of Vacuolar Type H+-ATPase (V-ATPase) in Hair Cell Development A Thesis Project Submitted in Partial Fulfillment of the Requirements of the Renée Crown University Honors Program at Syracuse University Victoria-Marie Berlandi-Short Candidate for B.S. in Nutrition Science Candidate for B.S. in Neuroscience and Renée Crown University HonorsMay 2020Honors Thesis Project in Developmental Biology and Neuroscience Thesis Project Advisor: _________________________Thesis Project Reader:_________________________Honors Director: __________________________Date: April 24, 2020RUNNING HEAD: The role of V-ATPase in Hair Cell DevelopmentThe Role of Vacuolar Type H+-ATPase (V-ATPase) in Hair Cell Development Victoria Berlandi-Short Syracuse University AbstractThe vacuolar type H+-ATPase (V-ATPase) is a membrane-bound proton (H+) pump that exists in several isoforms, allowing it to perform many critical cellular processes such as acidifying intracellular domains, receptor-mediated endocytosis, vesicle trafficking, protein degradation, prohormone processing, sperm maturation, small molecule uptake (e.g. neurotransmitters), and many others (Jefferies et al., 2008; Toei et al., 2010). Current literature suggests that V-ATPase activity is important in pH regulation of certain organelles, but functions of V-ATPase during embryonic development are poorly understood. Interestingly, patients with mutations in V-ATPase have been reported to develop sensorineural deafness among other related phenotypes (Colacurcio et al., 2016). To investigate potential underlying mechanisms for this phenotype, we have started characterizing the effects of V-ATPase loss via genetic knockout on the hair cells within two zebrafish auditory regions, the inner ear macula and lateral line neuromast. We have observed a significant reduction of hair cells in the neuromast of mutant embryos, but this relationship is not as pronounced in the inner ear macula. We have also found significant reductions in the amount and length of hair bundles in the mutant lines (featuring V-ATPase loss) in both the inner ear macula and neuromast. Furthermore, we tested the presence of mitotic support cells and found that there was also no statistical significance between actively dividing support cells and V-ATPase presence in the wild type and mutant lines at both the inner ear macula and neuromast. The purpose of these experiments was to study the role of V-ATPase in both the inner ear macula and lateral line neuromast hair cells, then observe differences in hair cell development between these two regions of zebrafish morphology. The results of these experiments suggest that V-ATPase inhibition may have more influence over the survival of the neuromast hair cells, and hair bundles of both the neuromast and inner ear macula. though it may not influence other structures, such as support cells, to the same magnitude. The results presented here alongside the clinical significance of hair cell damage from V-ATPase inhibition suggest that there is much about V-ATPase functionality and hair cell development that needs to be investigated, though a negative correlation between V-ATPase, hair cell survival, and mechanotransduction is plausible. Keywords: zebrafish, V-ATPase, hair cell, inner ear, macula, neuromast, lateral line, mechanotransduction, developmentExecutive SummaryThis cell and developmental biology thesis project explored the relationship between a well-known, proton-dependent and pH regulating transmembrane protein, known as V-ATPase, and its influence on the development, proliferation, and survival of a specific cell type, the auditory hair cell. The hair cells we studied were in two locations across the zebrafish- the organism used to perform our experiments- the inner ear macula (IE) and lateral line neuromast (NM). Researching the effects of V-ATPase loss in the same cell type, but in different locations, allowed us to make further comparisons between development, proliferation, and survival between the wild type (with V-ATPase) and mutant (V-ATPase loss) groups. Clinically, patients with mutations, or adverse genetic alterations to the DNA, of the V-ATPase protein within inner ear hair cell membranes exhibit sensorineural hearing loss. As of 2014, hearing loss was the most common sensory deficit in humans (Stawicki et al., 2014). Since hair cells are the beginning of the pathway in the auditory system in which sound is converted to a neural signal, loss of functional hair cells would prevent the auditory stimulus from being sent to the nerve. The clinical presentation of this mutation provides significance and reason to study this protein; the more we understand about its functionality, the more likely we are to someday be able to treat those with this specific type of hearing loss more effectively. Furthermore, V-ATPase is a very common protein across complex organisms, such as humans, mammals, amphibians, and reptiles, and is seen in the structure of insects, plants, and fungi (Finbow et al., 1997). While it is found in multiple areas of the same cell in an organism, it can also be present across different cell types of that same organism. For example, in humans V-ATPase is found in the IE as mentioned, but also lies in kidney tubules, osteoclasts (bone cells responsible for breaking down and releasing calcium, phosphate, and vitamin D from bone), and the male reproductive tract for sperm maturation (Toei et al., 2010). Mutations in various subunits of V-ATPase lead to renal tubular acidosis (Chen et al., 2020), osteopetrosis (a rare condition in which bones harden, making them more prone to fracture), depression, (Duan et al., 2018) and even in processes such as tumor metastasis, among a plethora of others (Toie et al., 2010). To provide further clarification on hair cell anatomy, it should be noted that there are several structures included within the hair cell that are relevant for discussion. First, hair cells develop in bundles, and are observed in this fashion in both the inner ear and neuromasts. Second, each hair cell contains up to 100 projections extending from the top, or apical, side of the cell; these are known as stereocilia, (Bear et al., 2007; Gillespie et al., 2005), and are grouped in hair cell bundles. Third, there is one additional projection at the end of the stereocilia stalk, called the kinocilium. It is longer than the stereocilia, and during sound conduction, all stereocilia bend towards this kinocilium in response to the auditory stimulus in order to send the sound signal to the brain (Bear et al., 2007; Gillespie et al., 2005). Lastly, there are supporting cells that surround the hair cell bundles, and research has shown that they develop into hair cells and can also replace hair cells in the case of damage and/or death (Thomas et al., 2014). In this thesis three experiments to characterize the development, proliferation, and survival of hair cells, containing V-ATPase, in the inner ear macula and lateral line neuromast are presented. All three experiments provide two comparisons: the first investigates hair cell survival and proliferation of the IE and NM between the wild type (WT) and mutants (v1f). The second compares the development of WT and v1f hair cells by calculating the amount and length of hair bundles that protrude from the hair cells. The third experiment examines a route of cell differentiation by studying the amount of mitotic, or dividing support cells between the WT and v1f of both regions. Overall, the results suggested that V-ATPase has a much greater role in hair cell survival in the NM than in the IE. Also, it appears to significantly affect hair bundle amount and length; the mutants show reductions in both of these characteristics at both regions. V-ATPase was shown to not be influential in the mitotic activity of support cells, suggesting that its effect on cell differentiation is minimal. To test for proliferation, more experiments are necessary at earlier times in embryonic development. Most importantly however, the more that we know about V-ATPase and its functionality across multiple cell and tissue types, the more effectively we can provide treatment for clinical presentations of its damage in a patient. Table of ContentsAbstract…………………………………………………………………………………...3Executive Summary……………………………………………………………………...4Acknowledgments………………………………………………………………………...8Introduction……………………………………………………………………...……….10Methods and Materials…………………...……………………………………………...19Results…………………………………………………………………………………….24 Discussion………………………………………………………………………………...32Conclusion………………………………………………………………………………..35List of Abbreviations………………………………………………………………….....36Appendix………………………………………………………………………………….37References………………………………………………………………………………...38Acknowledgements I would like to give a huge thanks to the Principal Investigator of the lab I studied in, Dr. Jeffrey Amack, and to PhD candidate, my mentor, Peu Santra. Your constant encouragement and willingness to answer questions and spur critical thinking for the preparation of these experiments developed me so much as a student and as a researcher. There is something purely special about being in an academic environment in which all you can do is learn every day and find the challenge consistently humbling. Working in a lab taught me a great deal of what methodic, benchwork science is really like, and a lot about life that I would not learn anywhere else. My appreciation for that is endless. Thank you to Alexis Whellan and Justin Cox for showing me the way around the lab, teaching me valuable protocols, several times over. I wish you both the most exciting and well-rounded careers out there. Thank you to Dr. Robin Jones, who introduced me to neuroscience several years ago and through her classes, reminded me of my potential and competence in the field. Thank you for the real and true dialogue, and for taking on the arduous task of being my thesis reader amidst your hectic schedule. Knowing that your door is always open, and that you are always in my corner means the most. Lastly, thank you to Dr. Margaret Voss, I genuinely believe that I would not be in this position if it were not for you. Your passion for the sciences is radiant and authentic; it has helped me persevere regardless of the discipline I am studying. I find myself actively seeking knowledge purely for curiosity over any other reason that’s often found in academia. My ambition to learn for the sake of learning is wholly inspired by you. As a senior, I am ecstatic knowing that I am graduating with two degrees from Syracuse University, and that is in no small part due to your encouragement and faith in me over the past three years. My professional and personal goal to be as well-rounded, literate, and grounded as possible is because of you. Thank you, truly, for everything. I would be remiss if I did not mention the pivotal contributions to this thesis from my mentor, Peu, whom I worked closely over the past year and a half to collect and analyze data for this project. She assisted with immunostaining, confocal microscopy, image analysis, statistics and analyses. Thank you for introducing me to the start of my professional career. IntroductionV-ATPase V-ATPase (Figure 1) is an enzyme complex divided into two regions: V0 on the extracellular or luminal side of the membrane, and V1 on the cytoplasmic side (Jefferies et al., 2008). V1 comprises a minimum of eight subunits: A through H (Jefferies et al., 2008; Horng et al., 2007) while V0 has six subunits responsible for proton translocation (Toei et al., 2010). When the V1 region hydrolyzes ATP (adenosine triphosphate) into ADP (adenosine diphosphate) and Pi (inorganic phosphate), a structural change in the A subunit occurs that propels rotation of the central stalk. Upon this rotation, cytoplasmic H+ are translocated into the lumens of organelles, or into the extracellular space in the case of plasma membrane V-ATPases (Horng et al., 2007). The resulting pH of V-ATPase-containing organelles or the extracellular space decreases, thus increasing acidity. Figure 1. V-ATPase and subunits. The superior portion, or V1 is cytoplasmic, while the inferior portion, V0 is extracellular. (, 2019). Prior literature has shown that V-ATPase pumps are pivotal for maintaining intracellular and intra-organellar pH in a plethora of systems across eukaryotes (Lin et al., 2019; Stawicki et al., 2014). V-ATPase pumps are found within the membranes of organelles such as vacuoles, lysosomes, and vesicles budding from the Golgi apparatus (Horng et al., 2007). Though ubiquitous across differentiated cell types, the specific concentration of V-ATPases within organelles, cell and tissue types depends on the necessity of pH regulation for that region (Duan et al., 2018). Their role in pH regulation is associated with that cells’ individual survival and ability to operate normally. Any deviation in the structure, and thus function of V-ATPase will likely result in impaired pH regulation of the organelle or cell it is embedded in. This will result in a major impairment of a multitude of pH-dependent processes occurring in that space. At the organismal level, such loss of V-ATPase functionality affects tissues and the corresponding organ, thus altering the overall quality of life and the ability of the organism to survive. Mechanotransduction and the Inner Ear V-ATPase, in regard to both the mammalian and fish auditory systems, is associated with mechanotransduction. At its foundation, this is the process by which external auditory stimuli, including gravity and head orientation (Kawashima et al., 2011; Paluch et al., 2015), are mechanically propagated to the inner ear, where it is then converted to an electrochemical signal and transmitted to the brain via afferent neurons for perception and response (Iskratsch et al., 2014; Olt et al., 2016). While this definition includes both the auditory and vestibular systems, the focus of the research presented here is on the auditory system. In humans, all stimuli are funneled into the ear via the external auditory meatus, through the air-filled auditory canal, and reverberate off the tympanic membrane of the middle ear (Ballachanda et al., 2013; Bear et al., 2007). Vibrations from the tympanic membrane move through the three auditory ossicles of the middle ear: the malleus, incus, and stapes (Figure 2). While all three ossicles participate in amplifying the external signal as it moves towards the inner ear, the stapes specifically contains a piston-like apparatus that vibrates against the membrane of the oval window, at which point the vibrations reach the inner ear (Bear et al., 2007; Bekesy 2017). Figure 2: A view of the external and middle ear regions in the pathway of auditory mechanotransduction (Jones, 2017). The fluid-filled, cochlea and the labyrinth comprise the inner ear; the former serves its role predominantly in audition while the latter participates in the vestibular system, to maintain equilibrium. The oval window resides at the base of the cochlea, and the round window is just inferior. Within the cochlea are three chambers: the scala vestibuli, scala media, and scala tympani, from superior to inferior (Figure 3a). Two membranes separate the regions: the superior Reissner’s membrane and the inferior basilar membrane. Fluid waves advance through the spiraled cochlea until they reach the apex of the basilar membrane, upon which rests the Organ of Corti. The two fluids in this region include perilymph, an ionic fluid with low [K+] (potassium) and high [Na+] (sodium; within the scala vestibuli and scala tympani), and endolymph, with opposing high [K+] and low [Na+] (within the scala media). Due to active transport, conductance, and concentration gradients in the scala media endothelium, an endolymph electrical potential, or endocochlear potential, results that is about 80mV greater than the potential of the perilymph fluid (Figure 4). Once vibrations reach the basilar membrane, the mechanical stimuli shift to an electrochemical, or neural signal (Bear et al., 2007).Figure 3 (left). a) cross-sectional diagram of human cochlea. b) cross-sectional diagram of Organ of Corti. (Pearson Education, 2004). Figure 4 (right). Cross-sectional diagram of cochlea, featuring endolymph and perilymph electrical potentials (Treuting et al., 2018). The aforementioned Organ of Corti consists of hair cell bundles, rods of Corti, and supporting cells surrounding each hair cell bundle. Hair cells are the beginning of the neural segment of mechanotransduction of sound in the inner ear, and have an estimate of 100 projections out of the apical surface, called stereocilia, which are made primarily of actin. (Bear et al., 2007; Gillespie et al., 2005). There are two types of cochlear hair cells: inner and outer hair cells (Figure 3b). The inner hair cells are afferent, or sensory, and send the signal to the brain, while the outer hair cells are efferent, or motor, and aid in the cochlea’s ability to receive frequencies by contracting the tectorial membrane (Purves et al., 2001). The incoming fluid waves reach the stereocilia of inner hair cells and, if strong enough, will deflect the stereocilia towards the single longest projecting cilia, known as the kinocilium, which is microtubule based (Gillespie et al., 2005). All cilia are connected by tip links at the very end of their projections (Figure 5). These tip links are connected to TRPA1 (transient receptor potential A1) channels on either side that will open in response to stereocilia deflecting from the fluid wave, and allow endolymph K+ ions to flow into the stereocilia and travel down towards the hair cell (Corey et al., 2004). If this depolarization reaches the membrane threshold, then voltage-gated calcium channels open, mobilizing neurotransmitter-containing vesicles to migrate to the basal end of the hair cell (Xiong 2018). This is known as an action potential, in which the signal is propagated via neurotransmitter release at the basal end of the hair cells and synapses with spiral ganglion cells below the basilar membrane (Figure 3b). These neurons converge and synapse with the auditory nerve, (i.e. auditory-vestibular nerve or Cranial Nerve VIII) which sends the signal to the medullary cochlear nuclei (Bear et al., 2007), for later sound processing, conscious perception, and response. Thus, hair cells are pivotal in the process of audition. Figure 5. Apical surface of one hair cell with protruding stereocilia and kinocilia. Tip links appear across the top of each cilium, and are attached to a mechanical TRPA1 channel, not pictured (Gillespie et al., 2005). Zebrafish as a Model System to Study HearingThere are a few anatomical differences between human and zebrafish auditory-vestibular system. The zebrafish lacks the outer and middle ear structures, specifically the auditory ossicles featured in humans, and instead has two otolith organs, a saccular and a utricular otolith, that are attached to a posterior and anterior macula, respectively, on either side (Baxendale et al., 2014; Pais-Roldán et al., 2016). The otoliths are made of calcium carbonate and other otolithic proteins, and are prefaced by the Weberian ossicles that aid in funneling sound towards the otoliths (Baxendale et al., 2014; Stooke-Vaughan, et al., 2015). Hair cells appear in bundles in the macula of the zebrafish inner ear, as opposed to an Organ of Corti within the inner ear cochlea observed in humans. These otolith organs serve vestibular functions in the zebrafish, and are equivalent to human otoconia within the otolithic membrane. Lastly, the zebrafish otoliths respond to acceleration and sound waves moving through water and synapse at the macula hair cells. The process of depolarization then propagates to the zebrafish brain (Pais-Roldán et al., 2016).Neuromast and the Lateral Line The lateral line is a facet of anatomy that is specific only to aquatic vertebrates and amphibians (Thomas et al., 2014; Whitfield et al., 1996). Humans do not have a lateral line system, but researching the effects of V-ATPase loss in this region allows further insight into the overall qualities of both V-ATPase and hair cell development. Furthermore, other research has suggested that there is an association between ideal pH and functionality of neuromast hair cells (Lin et al., 2019), which also provides a basis to perform further experiments. The lateral line (Figure 6) extends both anteriorly and posteriorly on the surfaces of the zebrafish (Gompel et al., 2001), and is responsible for sensing changes in water flow and water vibrations with a range of up to 300Hz (Thomas et al., 2014; Olt et al., 2016). Behaviorally, the sensory information from the lateral line neuromasts influences the response of the organism- for schooling, mating purposes, and prey and predator detection (Olt et al., 2016; Pujol-Martí et al., 2013). Furthermore, the lateral line is somatotopically organized in the brain to allow complex processing and timely responses to external stimuli (Pujol-Martí et al., 2013; Baxendale et al., 2014). Very early in embryonic development, a cluster of cells known as the primordium establishes multiple neuromasts along the lateral line (Thomas et al., 2014). Similar to the inner ear, the hair cell bundles within each neuromast are also surrounded by support cells (Gompel et al., 2014) and undergo mechanotransduction (Thomas et al., 2014). The apical ends of each hair cell bundle face the water within an enclosed membrane known as the cupula (Stawicki et al., 2014). Research of this region from the past several years has suggested that hair cell bundles within the neuromasts may have some ability to regenerate following damage, though this is specific to the lateral line of aquatic and amphibians systems (Thomas et al., 2014). Figure 6: Epifluorescence of neuromast locations.. Adapted from Whitfield et al., 1996, unpublished. (). Experiment Overview The goal of this project was to investigate the relationship between V-ATPase activity and hair cell development. Previous results from our lab, particularly from Peu Santra, have identified hair cell defects in neuromasts in V-ATPase mutant zebrafish. In atp6v1f mutants the overall number of hair cells in the neuromasts is reduced. To determine whether this defect is specific to hair cells in neuromasts or if it also includes the inner ear, I wanted to test the effect of loss of V-ATPase function on hair cells in this region as well. In the first set of experiments, I compared the number of inner ear hair cells in V-ATPase mutant and wild-type embryos. Second, I measured the length of the stereocilia and quantified the amount of hair bundles in the inner ear. Third, I tested the hypothesis that loss of V-ATPase activity alters cell proliferation by utilizing the mitotic marker PH3B (phosphohistone 3B) to calculate the number of proliferating cells. Methods and MaterialsAnimalAll experiments were performed on Danio rerio, approved by the IACUC (Institutional Animal Care and Use Committee). Adult fishes were raised in tanks supported by an automated system that circulated UV treated water throughout the system. All the experiments utilized embryos of 4 dpf unless stated otherwise.Zebrafish, Danio rerio, are a well-known model organism used in developmental biology research. They have a short breeding time, are externally fertilized, and develop rapidly (Pais-Roldán et al., 2016), which tends to decrease the overall length of experiments. Their embryos are transparent, making them very easy to observe in the first few day’s post-fertilization, which is the timeline for all experiments conducted. Furthermore, they are easy to impose genetic and chemical alterations on (Baxendale et al., 2014), allowing for a wide range of potential experiments. While mammalian systems contain bones that encapsulate the inner ear, making it more difficult to study, zebrafish hair cell bundles do not have the same difficulty at either the inner ear or neuromast regions; thus, they are relatively more accessible. In the first few day’s post-fertilization, ossicles have not developed to obstruct any view or protocol (Olt et al., 2016; Baxendale et al., 2014). Lastly, since hair cell structure and many of the genes influencing otic development and hearing loss diseases are highly conserved between the two species, it is acceptable to use zebrafish for these experiments since the results pertaining to the inner ear macula will be generalizable to the theoretical results of the same experiments in humans (Baxendale et al., 2014). Genetic Knockout of V-ATPaseTo create a comparison of hair cell development with and without functioning V-ATPase, a genetic knockout was employed to characterize the effects. We used a zebrafish strain known as atp6v1f, which denoted a mutation in the F subunit of the V1 region of V-ATPase, rendering the entire pump dysfunctional. We crossed this line with the WT strain to yield our two testing groups: homozygous WT and homozygous atp6v1f. At about 36hpf (hours post-fertilization), hypopigmentation is visible to denote the mutants, as this is a marked defect also resulting from V-ATPase inhibition (Duan et al., 2018). Since the v1f mutation is lethal, these embryos do not survive past 5dpf (days post-fertilization). ProcedureAll offspring were raised in embryo water and incubated at 29.5℃. Once the target incubation time for inner ear and neuromast development was reached (either 2dpf or 4dpf), embryos were sorted into Eppendorf tubes depending on their genotype: WT or v1f. Following this step, embryo water was removed and replaced with PFA1 (paraformaldehyde) to begin the immunohistochemistry protocol (see next section). For the first two experiments discussed, WT and v1f embryos were raised to 4dpf without any further intervention. They were fixed with PFA and imaged following IHC. The third experiment was the only one with embryos fixed at 2dpf, to provide a comparison between 2dpf and 4dpf. Once IHC for experiments was completed, embryos were imaged with a spinning disk microscope and analyzed using the computer software Fiji to quantify cell types and/or measure cilia length. Immunohistochemistry Protocol Immunohistochemistry, or IHC, is a very common application that allows researchers to tag a specific cell or tissue type to track its appearance, movement, and/or disappearance over stages of an experiment. IHC encompasses the strategic use of mono or polyclonal antibodies to label a desired antigen (Kaliyappan et al., 2012). The specific location of antibody binding is observed visually through the use of an immunofluorescent tag, that is seen at a specific wavelength under the proper microscope. The following is the standard IHC protocol used for these experiments: Day 1Fix embryos with 4% PFA containing 1% Tween20 in PBS Day 2Remove fix by washing the embryos in PBS containing 1% Tween20, Permeabilization in Acetone @ 20℃ for 8 minutes. Wash embryos once with PBS-1% Tween20 for 15 minutes at room temperature (RT).Block with 10% BSA (bovine serum albumin) in 1X PBS at RT for an hour, ~200uL. Add primary antibody/ies to the blocking solution at a 1:200uL ratio (10% BSA in 1X PBS). Incubate at 4℃ overnight. Day 3Wash the embryos with PBS-1% Tween20, 8 times for 15 minutes each at RT. Block with 10% BSA in 1X PBS for an hour at RT. Add secondary antibody/ies and/or dyes, at a 1:200uL ratio. Incubate at 4℃ overnight. Day 4Wash the embryos with PBS-1% Tween20, 8 times for 15 minutes each at RT.Image with spinning disk microscope, operating with three wavelengths for red, green, and blue. Antibodies and Dyes Primary antibodies used: Mouse anti acetylated tubulin (Sigma Aldrich-T7451) - 1:200Rabbit anti phosphohistone H3 (Cell signaling technology-9701) - 1:200Secondary antibodies used: Goat anti rabbit alexa fluor 488 (Abcam-ab150077) - 1:200Goat anti mouse alexa fluor 569 (ThermoFisher scientific-A11004) - 1:200Secondary stains and dyes used: Rhodamine Phalloidin (ThermoFisher scientific-R415) - 1:400DAPI (ThermoFisher scientific-62248) - 1:500Functions: In the first two experiments, phalloidin was used specifically to stain F-actin based stereocilia, while acetylated tubulin was used to bind to microtubule based kinocilia. In the third experiment, acetylated tubulin and PH3B were used as primaries; acetylated tubulin was used to tag microtubule-based structures and PH3B was used to label support cells that were actively dividing at that stage in development, also known as mitotic. Secondary antibodies used included Goat anti rabbit alexa fluor 488 and Goat anti mouse alexa fluor 569 for all experiments. DAPI, a nuclear dye, was also added here to provide a view of nuclei in both regions.Imaging and Analysis The embryos were mounted on 2% melting point agarose in a mattek dish and imaged with 40X water objective using a Perkin Elmer Spinning disk confocal microscope. All images were analyzed using Fiji/ImageJ. Statistical analyses were done and graphs were prepared using Graphpad Prism. All experiments were tested for statistical significance using Student’s T-test with Welch’s corrections, except experiment-3;fig.12 where we used One way ANOVA ResultsExperiment 1In the first set of experiments, I tested the amount of hair cells in the neuromast and inner ear, between WT and v1f lines. Since V-ATPase in the v1f lines is not functional, we expected there to be a significant difference in HC quantity between the WT and v1f lines at both the IE and NM regions; the v1f embryos would have fewer HCs than the WT. A decrease in HC quantity would most likely poorly affect mechanotransduction, as there would be fewer cells to receive the stimulus and convert it to a neuronal signal. The results suggested that there was a significant difference in the NM, and the v1f line had a lesser amount of HCs. Figure 7 shows a comparison of neuromast hair cells of WT (top row) and v1f (bottom row). Using acetylated tubulin to stain for kinocilia, a notable difference is observed between the WT and v1f (left column) kinocilia surrounding the center of the NM. The center column also shows more HC nuclei between WT and v1f. Figure 8A quantitatively shows a significant increase in pyknotic nuclei (PN) in the v1f line and Figure 8B shows a significant decrease in HCs in the v1f line. Using phalloidin and DAPI to stain the hair bundles and HC nuclei respectively, Figure 9 shows the images of hair bundle (left column) and hair cell nuclei (center column) between WT and v1f. From the images, no significant qualitative data can be obtained to suggest differences between the groups. Figure 10 also used PN as a potential marker for HC death; there was no significant difference in the amount of PN between either group after 4dpf. Overall, the results show that in the NM, significantly fewer HCs are surviving in the v1f embryos than in the WT after 4dpf. PN data suggests that HCs appear to be developing in the NM, but are dying more frequently in the mutants than in the WT. A rationale for this may be that V-ATPase inhibition occurs at 4dpf in the mutants, leading to this outcome. PN data for the IE was not significant, proposing that V-ATPase loss in HCs has a greater effect in the NM than it does in the IE; it may not be highly important in this region for HC survival. Figure 7. Images of neuromast acetylated-tubulin as a marker of hair cells. (Left column) Acetylated tubulin binding to kinocilia around the NM. (Center column) DAPI staining to HC nuclei of NM. (Right column) Combined image of left and center. Acetylated tubulin (pink) and DAPI (blue). A BFigure 8. (A) Results of pyknotic nuclei (PN) as a potential marker of hair cell death in the neuromast. The v1f line shows a significant increase in PN after 3 experiments. (B) Results show that the WT had significantly more HCs in the NM region than the mutant after 3 experiments. 4dpf. Figure 9. (Left column) Images of hair bundles (white) via phalloidin staining of the IE in WT (top) and v1f (bottom). (Center column) Images of IE HC nuclei following DAPI staining. (Right column) Combined image of phalloidin stained hair bundles (pink) and DAPI stained HC nuclei (blue). 4dpf. Figure 10. A measure of pyknotic nuclei in the IE after 2 experiments. There was no significant difference in PN proliferation between WT and v1f after 4dpf. Experiment 2In the second set of experiments I quantified both the amount and length of hair bundles (i.e. stereocilia) in the IE. As mentioned, each hair cell has about 100 stereocilia protruding from the apical side, denoted as a hair bundle. Thus, each hair bundle suggests one hair cell below it, and becomes another marker to quantify HCs in the WT and v1f lines. The hypothesis was that there would be a decreased amount of hair bundles in the mutant embryos as well as decreased length, due to V-ATPase knockout. Both of these outcomes may adversely affect mechanotransduction, and potentially result in the altered phenotype observed clinically. Phalloidin and DAPI staining were used in Figure 11 to qualitatively depict the amount of hair bundles (left column) and HC nuclei (center column), respectively. The images suggest that there are more hair bundles and HC nuclei in the WT than in the v1f. Figure 12 confirms these qualitative results; after quantitative analysis there were significantly fewer (Figure 12B) and shorter (Figure 12A) hair bundles in v1f after 4dpf. These results show that V-ATPase plays a specific role in the development and survival of hair bundles in the IE through 4dpf. For the mutant groups, there were a significantly fewer number of hair cell bundles at 4dpf, allowing us to accept our hypothesis that V-ATPase is necessary for stereocilia proliferation and development in the IE. This corroborates with existing data that V-ATPase is needed for mechanotransduction, chiefly because it influences the integrity of stereocilia during embryonic development. Figure 11. ((Left column) Phalloidin staining stereocilia, to denote hair bundles(white) across the apical surface of HCs in the WT (top) and v1f (bottom) in IE.. (Center column) DAPI staining to show HC nuclei (white) in IE. (Right column) Merged image of hair bundles (pink) and hair cell nuclei (white). 4dpf, Figure 12. Difference in amount of hair bundles and length of hair bundles in IE at 4dpf. A) A significant decrease in IE hair bundle length was observed in the mutant line after three experiments. B) A significant decrease in the amount of IE hair bundles was observed in the mutant line after 3 experiments. 4dpf. Experiment 3In this set of experiments, we used phosphohistone 3B (PH3B) to act as a marker for mitotic (dividing) support cells in both the IE and NM at 2dpf and 4dpf. This experiment allowed us to see if the amount of support cells in the IE and NM regions was influencing HC growth and development. As mentioned earlier, support cells surround hair cell bundles and aid in their development and regeneration. Support cells can mature into hair cells, for example, if the pre-existing hair cells were damaged and needed to be replaced (Gompel et al., 2014). Thus, if they were actively dividing during the time periods observed (2dpf and 4dpf), we can surmise that there may have been an increased need for support cells, and potentially HCs at those times. This would suggest that there was a loss of HCs with regards to V-ATPase inhibition in the mutant embryos.That is, HCs developed, but died and needed to be replaced. Figure 13 depicts the qualitative differences in the WT embryos at the IE and NM. In the third column, PH3B was used to highlight mitotic cells; the results were such that only one PH3B+ cell (grey) is seen in an IE WT embryo, and none were seen in a NM WT embryo. Figure 14 depicts these differences in the v1f embryos at the IE and NM. In the third panel, no PH3B+ cells were observed for these embryos. Quantitative data from both the IE and NM (Figure 15) showed that the results were not significant for either cell line. Overall, this experiment showed that V-ATPase loss on HCs does not have any effect appreciable on support cell proliferation and differentiation. Figure 13: (Far left) acetylated tubulin staining (grey) on any microtubule based cell types within WT embryos of IE (top) and NM (bottom). (Center left) DAPI staining to denote HC nuclei (grey) in WT of IE (top) and NM (bottom). (Center right) PH3B+ cells in either region. One was observed in the IE (grey), and none in the NM. (Far right) Merged image of all three panels; microtubule-based cells (pink), HC nuclei (blue), and PH3B+ cells (green). Figure 14: (Far left) acetylated tubulin staining (grey) on any microtubule based cell types within WT embryos of IE (top) and NM (bottom). (Center left) DAPI staining to denote HC nuclei (grey) in WT of IE (top) and NM (bottom). (Center right) PH3B+ cells in either region. None were observed in the mutant lines in either region. (Far right) Merged image of all three panels; microtubule-based cells (pink), HC nuclei (blue), and PH3B+ cells (green). Figure 15: Graphical analysis of PH3B+ cells in the NM (left) and IE (right). No statistical significance was observed for any group; n=1. Discussion Experiment 1One major limitation of this experiment was that we were unable to obtain the HC quantification for the IE. A next step would be to complete this analysis to fully establish the results of this set of experiments. It is likely that the HCs in the IE will mimic the behavior of the HCs in the NM since the prevalence of PN was not significantly increased in the mutant IE embryos. If the results were the same as the NM, then V-ATPase presence is likely vital to HC survival in the IE, suggesting that the morphology of both regions is similar and conserved. If the results are the opposite, then it is possible that another variable is present in the IE at this time period to protect the HCs from death, even without V-ATPase functionality. To test for the effects of V-ATPase on HC proliferation, specifically to see if HCs had developed at earlier stages, or at all, this same experiment should be conducted at earlier times (i.e. 36hpf, 2dpf, and 3dpf). This would provide insight on the importance of V-ATPase before 4dpf. Experiment 2This experiment does not provide NM data unfortunately, and this is due to the anatomy of the NM and analysis. During microscopy, the NM is viewed top down, while the IE is viewed sagittally. A sagittal view allows us to observe all hair bundles and measure, but a top down view at the NM makes this impossible. The next step with this experiment would be to apply other methods of analysis that will allow an accurate view of NM stereocilia. This would provide further insight on how conserved the V-ATPase physiology is between the two regions. Experiment 3Due to time constraints, the data here was only following one trial. To establish validity of these results, they should be repeated. If there is still no significant relationship, then it can be confidently deduced that V-ATPase loss has no effect on support cell proliferation and differentiation. However, if V-ATPase loss significantly reduces HC proliferation, as seen in the first set of experiments, then there should be a likely mechanism that signals the support cells to increase their mitotic activity to replace the lost HCs. If this experiment continues to show no relationship, then it is possible that another variable is present, preventing any communication between HC death and support cell activity. A potential experiment could be to apply a pharmacological drug that increases the amount of support cell mitotic activity and see if this drug rescues the support cell mitotic activity in mutants. This experiment could also be performed slightly earlier than 4dpf to see if V-ATPase loss affects support cell mitotic activity during the initial development of HCs (At 3dpf, or 3.5dpf, for example). If this is true, then it would corroborate with the NM results from the first set of experiments, and suggest that V-ATPase is affecting support cell activity at an earlier time; another variable may be involved at 4dpf that is leading to the results presented here. General LimitationsThere are a few other limitations to address while acknowledging these results. The first is that structurally, humans lack the lateral line system that aquatic vertebrate’s species entail. Thus, the NM results are not directly generalizable to humans, but researching this area still allows us further insight into overall V-ATPase functionality in the context of mechanotransduction. Also, some results differed between the IE and NM, suggesting that potentially another variable is present at one of these locations which is influencing our results. Secondly, we had originally intended to provide results from several points in time: 2dpf, 3dpf, and 4dpf. This would allow us to confirm if hair cells, support cells, and/or ciliary groups were developing initially and then died later due to V-ATPase loss, or if they had never appeared at all during development. Due to time constraints and lost data, we were unable to provide this comprehensive view. Lastly, I had conducted a fourth, high-risk high-reward experiment solely on the NM hair cells using pharmacological drugs, Neomycin and Concanamycin A (a known V-ATPase inhibitor). Neomycin is an aminoglycoside that is commonly found in antibiotic creams to treat infections, but has also caused loss of HCs (He et al., 2017), interestingly. Furthermore, previous data from other experiments I completed showed that Concanamycin A had a beneficial effect on HCs. I tested whether V-ATPase inhibition (from Concanamycin A) could protect hair cells from damage imposed by Neomycin. Unfortunately, I was not able to obtain the results of this experiment due to diminished lab access from unforeseen global circumstances. A next step would be to analyze this data once lab access is allowed. If this experiment proved feasible in zebrafish, and Concanamycin A did protect the HCs from damage, then repeating this work in other animals may prove beneficial in working towards someday conducting tests clinically. Conclusion Our preliminary results suggest that genetic V-ATPase loss disrupts total hair cell bundle proliferation in the NM, negatively affects total hair bundle length and quantity in the IE, and does not significantly affect mitotic activity of the surrounding support cells in either region. Altogether, we can support the hypothesis that V-ATPase is needed for the efficacy of mechanotransduction in general, and it may specifically be the stereocilia and hair cells during early embryonic development that are most reliant on V-ATPase presence for survival. V-ATPase does not appear to greatly affect HC proliferation, as there are still HCs present in the NM and IE at 4dpf, but experiments investigating earlier times are necessary to corroborate these results. However, it does appear to affect stereocilia proliferation. Lastly, the results show that V-ATPase does not greatly affect differentiation of HCs, leaving survival as the likely factor that V-ATPase loss influences the most. Further experiments, including repetitions of some of the ones included here, should be conducted to fully discern the relationship of V-ATPase on survival. These data certainly contribute to our currently incomplete understanding of V-ATPase functionality during zebrafish embryonic development and provide a foundation for further research into the mechanisms of V-ATPase and chiefly, its role in HC survival. 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