Molecular Regulation of Vertebrate Pigmentation and Stem Cell Quiescence in Larval Zebrafish

Vertebrate animals often recruit undifferentiated populations of precursors (adult stem cells; ASCs) for homeostasis and regeneration of adult tissues. A longstanding question in stem cell biology concerns how these long-lived ASCs switch between active, proliferating states and inactive, quiescent states throughout an organism’s lifetime. I use the melanocyte stem cell (MSC), which regenerates the melanocytes found in vertebrate hair and skin, to study ASC biology and dissect the molecular pathways that maintain ASC quiescence in larval zebrafish. An in vivo drug screen carried out in our lab identified a GABA-A receptor antagonist as an enhancer of melanocyte production in the larval zebrafish, suggesting GABA normally promotes MSC quiescence with pharmacological activation of GABA-A receptor function to inhibit melanocyte production and stem cell proliferation. GABA is a key neurotransmitter, and GABA receptors are well studied for their role in cell-to-cell communication, specifically in a neuronal context, but their role in stem cell biology is poorly characterized. A potential role for GABA signaling in the regulation of stem cell quiescence would represent a fundamental new molecular mechanism of vertebrate pigment biology and could inform our understanding of adult stem cell establishment, aging, and exhaustion in general. I found that pharmacological inhibition of the GABA-A receptor class, using four commercially available antagonists, enhanced melanocyte production in vivo. Conversely, I identified five drugs that activate GABA-A receptor signaling and that robustly inhibit melanocyte regeneration. In addition, I used CRISPR/CAS9 in zebrafish to generate two in-frame mutations in the GABA rho 1 gene (gabrr1), both of which have a robust melanocyte over-production phenotype. I cloned gabrr1 cDNA downstream of an inducible heat-shock promoter and generated a stable transgenic line to conditionally overexpress gabrr1 in larval zebrafish. Consistent with my model, I found that overexpression of gabrr1 inhibited melanocyte regeneration. In addition, my pharmacology experiments identified an additional molecular pathway, independent of GABA signaling, that appears to regulate melanocyte stem cell quiescence in parallel within larval zebrafish. In summary, I used pharmacological and genetic studies to investigate the genetic and molecular basis of adult stem cell quiescence in zebrafish, the results of which support a model of melanocyte stem cell biology wherein activation of GABA-A signaling (via gabrr1) and an independent pathway through TSPO signaling both maintain a quiescent state to prevent excessive pigmentation of the larval zebrafish