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Date of Award
5-2025
Document Type
Restricted Thesis: Campus only access
Degree Name
Bachelor of Science
Department
Biochemistry & Molecular Biol.
First Advisor
Dr. B. Daniel Pierce
Second Advisor
Dr. Jonathan Dattelbaum
Abstract
Intracellular pH (pHi) dynamics are a critical regulator of cell behaviors such as directed cell migration, proliferation, and cell fate changes, including differentiation and lineage specification of adult and embryonic stem cells. However, we lack an understanding of how pHi dynamics regulate cell behaviors in vivo, largely due to a lack of amenable models. We aimed to address this gap by 1) establishing a new tool for quantifying pHidynamics in vivo and 2) investigating the role of pHi dynamics in epimorphic regeneration, a complex tissue process involving multiple pHi-regulated cell behaviors. We introduced the genetically-encoded ratiometric pHi biosensor mKate2-pHluorin into zebrafish, a model organism that retains the capacity to regenerate its tail fin from larval stages through adulthood. Using this zebrafish line, we identified previously unrecognized pHi dynamics during the early phase of larval tail fin regeneration. To determine the functional significance of these pHi changes, we pharmacologically and genetically inhibited the plasma membrane sodium-proton exchangers (NHEs), primary determinants of pHi changes in mammalian cells. We found that the NHE inhibitor 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) lowered pHi in amputated tail tissues, blocking the increases in pHi that normally occur at 0.5 and 6 hours post-amputation (hpa). Further, we found that EIPA and a second NHE inhibitor, BIX, impaired tail regeneration (area of regrowth reduced by 50% and 27%, respectively). To understand how NHE activity regulates zebrafish larval tail regeneration, we conducted bulk RNA-sequencing of amputated tails at 6 hpa. We observed that the genes downregulated by EIPA and BIX at 6 hpa were highly expressed in immune cells. Immune cells are known to be recruited by the production of reactive oxygen species (ROS) at 20 mpa. We therefore asked whether NHE activity regulates ROS signaling and found that EIPA decreased ROS production at 15-30 mpa. Our results reveal a previously unrecognized role of NHE activity in coordinating cell behaviors at the tissue-level in vivo. Furthermore, we identify NHEs as novel regulators of zebrafish larval tail regeneration, possibly acting through ROS-mediated immune cell recruitment. Understanding how pHi dynamics contribute to regeneration may offer new strategies for enhancing tissue repair in non-regenerative species, including humans.
Please note that this thesis is permanently restricted and not available.
Recommended Citation
Margaryan, Anush, "Sodium-Proton Exchanger Activity Is Required for Zebrafish Larval Tail Fin Regeneration" (2025). Honors Theses. 1841.
https://scholarship.richmond.edu/honors-theses/1841
