Maintenance of Melanocyte Stem Cell Quiescence by GABA-A Signaling in Larval Zebrafish

Abstract

In larval zebrafish, melanocyte stem cells (MSCs) are quiescent, but can be recruited to regenerate the larval pigment pattern following melanocyte ablation. Through pharmacological experiments, we found that inhibition of γ-aminobutyric acid (GABA)-A receptor function, specifically the GABA-A ρ subtype, induces excessive melanocyte production in larval zebrafish. Conversely, pharmacological activation of GABA-A inhibited melanocyte regeneration. We used clustered regularly interspaced short palindromic repeats/Cas9 to generate two mutant alleles of gabrr1, a subtype of GABA-A receptors. Both alleles exhibited robust melanocyte overproduction, while conditional overexpression of gabrr1 inhibited larval melanocyte regeneration. Our data suggest that gabrr1 signaling is necessary to maintain MSC quiescence and sufficient to reduce, but not eliminate, melanocyte regeneration in larval zebrafish.

Keywords: CRISPR; GABA; GABA-A receptors; inhibition; melanocyte; pigmentation; quiescence; zebrafish.

Figure 1

GABA-A antagonists increase melanocyte production in larval zebrafish. (A) Schematic of experimental timeline for PTU melanocyte differentiation assay. Drugs and PTU are added to zebrafish embryos between 3 and 6 dpf. (B–E) Images of representative 6 dpf larvae treated with vehicle control (B) or GABA-A antagonist CP-615003-27 (40 µM), (C) Picrotoxin (100 µM) (D), or TPMPA (100 µM); (E). (F) Quantification of the average number of melanin⁻, GFP⁺ dorsal melanocytes for each treatment group ± variation (vehicle control: 0.92 ± 1.15, N = 84; CP-615003-27: 4.27 ± 2.12, N = 81; Picrotoxin: 3.76 ± 1.38, N = 55; and TPMPA: 4.10 ± 2.14, N = 52). Following single-factor ANOVA, each experimental group was compared to vehicle control using Tukey’s HSD. *** P < 0.001 (Tukey’s HSD). dpf, days postfertilization; GABA, γ-aminobutyric acid; HSD, honestly significant difference; PTU, phenylthiourea.

Figure 2

GABA-A antagonist-induced melanocytes derive from MSCs. (A) Schematic of experimental timeline for drug treatment. (B) Quantification of the average melanin⁻, GFP⁺ dorsal melanocytes in each group ± variation (vehicle control: 1.76 ± 1.26, N = 39; vehicle control + AG1478: 0.32 ± 0.48, N = 28; TPMPA: 4.14 ± 1.43, N = 29; TPMPA + AG1478: 0.28 ± 0.54, N = 25; Picrotoxin: 5.2 ± 0.99, N = 30; and Picrotoxin + AG1478: 0.17 ± 0.38, N = 30). Following single-factor ANOVA, each experimental group was compared to vehicle control using Tukey’s HSD. *** P < 0.001 (Tukey’s HSD). dpf, days postfertilization; GABA, γ-aminobutyric acid; HSD, honestly significant difference; MSC, melanocyte stem cell; PTU, phenylthiourea.

Figure 3

Pharmacological activation of GABA-A signaling inhibits melanocyte regeneration. (A) Schematic of experimental timeline for drug treatment. Images of representative mitfa^vc7 7 dpf larvae treated with vehicle control (B), GABA (50 mM) (C), GABOB (100 µM) (D), L,838-417 (100 µM) (E), CI-966 HCL (20 µM) (F), and MK 0343 (100 µM) (G). (H) Quantification of the average number of dorsal melanocytes in each drug treatment group ± variation (vehicle control: 42.2 ± 9.38, N = 78; GABA: 21.2 ± 10.4, N = 42; GABOB: 26.8 ± 7.71, N = 41; L,838-417: 16 ± 11.2, N = 42; CI-966 HCL: 20.4 ± 7.81, N = 43; and MK 0343: 18.1 ± 9.55, N = 35). Following single-factor ANOVA, each experimental group was compared to vehicle control using Tukey’s HSD. *** P < 0.001 (Tukey’s HSD). dpf, days postfertilization; GABA, γ-aminobutyric acid; HSD, honestly significant difference.

Figure 4

gabrr1 mutations exhibit a dominant excess melanocyte phenotype during larval stages. (A) Partial sequence alignment of wild-type and CRISPR mutagenized gabrr1 genomic locus in zebrafish. (B) Partial peptide alignment of vertebrate gabrr1 homology reference protein (human: NP_002033; mouse: NP_032101; and zebrafish: NP_001020724) within the ligand-binding domain, with the predicted amino acid sequence of the two gabrr1 alleles generated in the study. (C) Quantification of the average number of melanin⁻, GFP⁺ dorsal melanocytes in each treatment group ± variation (wild-type: 2.04 ± 0.94, N = 51; gabrr1^j247/+: 9.56 ± 1.48, N = 39; gabrr1^j247/j247: 9.13 ± 1.41, N = 15; gabrr1^j248/+: 7.81 ± 1.17, N = 48; gabrr1^j248/j248: 10.2 ± 2.54, N = 13; and gabrr1^j247/j248: 9.72 ± 1.99, N = 29). Representative images of 6 dpf wild-type (D), gabrr1^j247/+ (E), gabrr1^j248/+ (F), and gabrr1^j247/j248 (G) larvae. Following single-factor ANOVA, each experimental group was compared to vehicle control using Tukey’s HSD. *** P < 0.001 (Tukey’s HSD). CRISPR, clustered regularly interspaced short palindromic repeats; dpf, days postfertilization; HSD, honestly significant difference.

Figure 5

Overexpression of gabrr1 inhibits melanocyte regeneration in larval zebrafish. (A) Schematic of experimental timeline. Arrows indicate timing of three 30-min 37° heat-shock treatments. (B) Quantification of average dorsal melanocytes in each treatment group ± variation. [mlpha + heat shock: 49.9 ± 6.01, N = 32; Tg(hsp70l:gabrr1)^j972: 51.1 ± 6.86, N = 38; and Tg(hsp70l:gabrr1)^j972+ heatshock: 32.3 ± 5.54, N = 46]. Images of representative mlpha + heat shock (C), Tg(hsp70l:gabrr1)^j972 (D), and Tg(hsp70l:gabrr1)^j972 + heatshock (E) larvae. Following single-factor ANOVA, each experimental group was compared to vehicle control using Tukey’s HSD. *** P < 0.001 (Tukey’s HSD). 4-HA, 4-hydroxyanisole; dpf, days postfertilization; HSD, honestly significant difference; NS, not significant.

Figure 6

gabrr1-mediated maintenance of MSC quiescence is sensitive to kita dosage. Quantification of the average number of melanin⁻, GFP⁺ dorsal melanocytes in gabrr1^j247/+ and kita^b5/+ ± variation (wild-type: 2.35 ± 1.03, N = 42; kita^b5/+: 0.88 ± 0.81, N = 56; gabrr1^j247/+: 9.55 ± 1.43, N = 40; and kita^b5/+; gabrr1^j247/+: 0.87 ± 0.87, S.E.M: 0.11, N = 60). Following single-factor ANOVA, each experimental group was compared to vehicle control using Tukey’s HSD. *** P < 0.001 (Tukey’s HSD). HSD, honestly significant difference; MSC, melanocyte stem cell;