Amack, Jeffrey, D.; Santra, Peu (2021-11)
      Vacuolar type H+-ATPase (V-ATPase) is a ubiquitously expressed enzyme complex that pumps protons across membranes. The proton-motive force generated by V-ATPase is used by cells to acidify intracellular compartments. Additionally, certain specialized tissue types have V-ATPase on plasma membranes where it secretes H+into the extracellular space. While V-ATPase activity is essential for several cellular functions, our understanding of cell-type specific functions for V-ATPase remains limited. Here, I focused on investigating V-ATPase functions in mechanosensory hair cells. Hair cells are functional units of mammalian auditory and vestibular systems. Consequently, hair cell loss leads to permanent deafness. Mutation in specific V-ATPase subunits causes sensorineural deafness in human, however, the mechanism is not well understood. I used zebrafish as model vertebrate to investigate how loss of V-ATPase function impacts hair cells. Using a combination of genetic mutations, pharmacological manipulations and live imaging of hair cells in vivo, I found that V-ATPase activity is critical for hair cell survival. Analysis of molecular markers and cellular morphologies indicates hair cells in V-ATPase mutants undergo a caspase-independent, necrosis-like death. V-ATPase mutant hair cells show a significant decrease in mitochondrial membrane potential (mPTP). On modulating mPTP pharmacologically, V-ATPase mutants show a modest but consistent improvement of hair cell survival. These results indicate mitochondrial dysfunction contributes to hair cell death in V-ATPase mutants. Next, I generated a novel cilia pH biosensor and found that hair cell kinocilia have a more basic pH than other primary cilia in zebrafish embryos. Interestingly, my collaborators and I discovered that V-ATPase subunits localize to hair cell kinocilia in zebrafish and mice, which suggests cell-type specific functions for V-ATPase in kinocilia. pH maintenance in kinocilia may be an essential function that contributes to proper kinocilia length and/or function. In conclusion, this work has uncovered a function for V-ATPase activity that is critical for hair cell survival, in part by maintaining mitochondrial health, and a function that mediates hair cell kinocilia form and function. The work presented in this thesis advances our understanding of V-ATPase functioning in hearing loss, more broadly elucidates new in vivo cell-type specific V-ATPase functions.
    • Expression and Function of Paxillin Genes in Zebrafish: A Role in Skeletal Muscle Development

      Turner, Chris; Amack, Jeffrey; Jacob, Andrew (2017)
      Paxillin is a key component of the Integrin adhesion complex, which regulates cellular signaling events in response to extracellular matrix interactions. Although the roles for Paxillin in cell migration have been extensively studied, less is understood about its role in vertebrate development. Depletion of Paxillin from mouse embryos results in early lethality due to impaired cardiovascular development and function, necessitating the development of alternative vertebrate genetic models for examining the role of Paxillin during embryogenesis. Zebrafish have emerged as an experimental vertebrate model amenable to genetic manipulation. The work compiled herein first characterizes the expression profiles for Paxillin genes in zebrafish, and then describes the embryonic phenotypes observed upon mutation of these genes. The identification of two Paxillin genes in zebrafish, pxnaand pxnb, provided new insight into the evolution of this gene family in the Teleost lineage. Both overlapping and unique expression profiles for these genes during zebrafish embryogenesis were uncovered. While both genes are expressed in developing skeletal muscle, pxnawas restricted to the notochord during earlier stages of embryogenesis and pxnbwas expressed in the developing heart. Targeted mutation of either gene alone did not impair embryonic development, suggesting partial functional redundancy between each gene during embryogenesis. Accordingly, combined mutations in pxnaand pxnbrevealed defects during the development ofseveral embryonic tissues. In particular, skeletal muscle morphogenesis iiiwas perturbed in these double mutant embryos. Further characterization revealed that Paxillin genes in zebrafish serve to regulate embryonic myotome shape and proper extracellular matrix composition during muscle development. The amount of Laminin was reduced, while the abundance of Fibronectin persisted, during myotome morphogenesis in Paxillin double mutant embryos. In addition, a role for cytoskeletal contractility in regulatingsubcellular localization of Paxillin in developing skeletal muscle was established. Defects in the development of the cardiovascular system were also apparent in Paxillin double mutant embryos, and future work will focus on characterizing these in further detail. Altogether, this work provides a new vertebrate model to use for understanding the role of Paxillin during embryonic development, and uncovers an unrecognized role for Paxillin in establishing the extracellular matrix of skeletal muscle.