GABA-A receptor and mitochondrial TSPO signaling act in parallel to regulate melanocyte stem cell quiescence in larval zebrafish

Abstract

Tissue regeneration and homeostasis often require recruitment of undifferentiated precursors (adult stem cells; ASCs). While many ASCs continuously proliferate throughout the lifetime of an organism, others are recruited from a quiescent state to replenish their target tissue. A long-standing question in stem cell biology concerns how long-lived, non-dividing ASCs regulate the transition between quiescence and proliferation. We study the melanocyte stem cell (MSC) to investigate the molecular pathways that regulate ASC quiescence. Our prior work indicated that GABA-A receptor activation promotes MSC quiescence in larval zebrafish. Here, through pharmacological and genetic approaches we show that GABA-A acts through calcium signaling to maintain MSC quiescence. Unexpectedly, we identified translocator protein (TSPO), a mitochondrial membrane-associated protein that regulates mitochondrial function and metabolic homeostasis, as a parallel regulator of MSC quiescence. We found that both TSPO-specific ligands and induction of gluconeogenesis likely act in the same pathway to promote MSC activation and melanocyte production in larval zebrafish. In contrast, TSPO and gluconeogenesis appear to act in parallel to GABA-A receptor signaling to regulate MSC quiescence and vertebrate pigment patterning.

Keywords: GABA-A; melanocyte; stem cell; translocator protein; zebrafish.

Figure 1:. GABA-A antagonists and TSPO ligands increase melanocyte production in larval zebrafish.

(A) Cartoon of experimental timeline for PTU melanocyte differentiation assay. Experimental drugs and PTU are added to zebrafish embryos between 3 – 6 dpf. * indicates timepoint at which scoring is performed. (B) Schematic drawing of 6 dpf zebrafish larvae scored in PTU melanocyte differentiation assay. Region of interest (ROI) defined as dorsal area between otic vesicle and tail. White arrowheads indicate unpigmented melanocytes. (C–F) Representative 6 dpf larvae treated with vehicle control (C) or diazepam (40 µM; D), PK-11195 (100 µM; E), and Ro5-4864 (100 µM; F). (G) Quantification of the average number of melanin⁻, GFP⁺ dorsal melanocytes / ROI for each treatment group of 6 dpf larvae. White arrowheads indicate unpigmented melanocytes. Yellow box indicates representative region containing unpigmented melanocytes, displayed at higher magnification in bottom right insert. Data summarized in Table S2. Each experimental group was compared to vehicle control. *** indicate a statistical difference in our analysis (Tukey-HSD; p<0.001).

Figure 2:. GABA-A antagonists and TSPO ligands induced melanocyte production require MSCs.

(A) Cartoon of experimental timeline for AG1478 treatment. (B) Quantification of average melanin⁻, GFP⁺ dorsal melanocytes / ROI in each experimental group. Data summarized in Table S3. Each experimental group was compared to vehicle control. *** indicate a statistical difference in our analysis (Tukey-HSD; p<0.001).

Figure 3:. GABA inhibits GABA-A antagonist melanocyte production, but does not inhibit TSPO ligand induced melanocyte production.

(A) Cartoon of experimental timeline for GABA treatment. (B) Quantification of average melanin⁻, GFP⁺ dorsal melanocytes / ROI in each experimental group. Data summarized in Table S4. Each experimental group was compared to vehicle control. *** indicate a statistical difference in our analysis (Tukey-HSD; p<0.001).

Figure 4:. Additive drug effects between GABA-A antagonists and TSPO ligands suggest independent molecular pathways.

Quantification of average melanin⁻, GFP⁺ dorsal melanocytes / ROI in each experimental group. Data summarized in Table S5. *** indicate a statistical difference in our analysis (Tukey-HSD; p<0.001).

Figure 5:. Inhibition of Ca²⁺ signaling increases melanocyte production.

Quantification of average melanin⁻, GFP⁺ dorsal melanocytes / ROI in each group of 6 dpf larvae. Data summarized in Table S6. Each experimental group was compared to vehicle control. *** indicate a statistical difference in our analysis (Tukey-HSD; p<0.001).

Figure 6:. Pharmacological induction of gluconeogenesis pathways increases melanocyte production.

Quantification of average melanin⁻, GFP⁺ dorsal melanocytes / ROI in each group of 6 dpf larvae. Data summarized in Table S7. Each experimental group was compared to vehicle control. *** indicate a statistical difference in our analysis (Tukey-HSD; p<0.001).

Figure 7:. GABA pathway drugs are more sensitive to kita haploinsufficiency than TSPO pathway drugs.

Quantification of average melanin⁻, GFP⁺ dorsal melanocytes / ROI in each group of 6 dpf heterozygous kita^b5/+ larvae. Data summarized in Table S8. Each experimental group was compared to vehicle control. *** indicate a statistical difference in our analysis (Tukey-HSD; p<0.001).

Figure 8:. Inhibition of GABA / Ca²⁺ signaling and manipulation of gluconeogenesis pathways act in parallel pathways to regulate MSC quiescence.

Quantification of average melanin⁻, GFP⁺ dorsal melanocytes / ROI in each group of 6 dpf larvae. Data summarized in Table S9. Each experimental group was compared to vehicle control. *** indicate a statistical difference in our analysis (Tukey-HSD; p<0.01).