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.

VERTEBRATE animals often rely on undifferentiated precursors to regulate the growth and homeostasis of specific tissues. These precursors, adult stem cells (ASCs), undergo long-term self-renewal throughout the lifetime of the organism to maintain the growth and regenerative potential of their target tissue. ASCs are found in many tissues including blood, muscle, skin, and the nervous system (Nishimura et al. 2002; Bertrand et al. 2007; Ma et al. 2009; Cheung and Rando 2013). While some ASCs continually proliferate to maintain their target tissue, others remain quiescent or dormant, and must be recruited to enter a proliferative state, often induced by depletion of differentiated cells in their respective tissues (Li and Bhatia 2011). Understanding the pathways that maintain ASC quiescence and that recruit quiescent ASCs to proliferate is critical to elucidate vertebrate tissue growth and homeostasis.

Zebrafish pigmentation, specifically melanocyte development, is an excellent model system to dissect the genetic and molecular basis of ASC quiescence, and recruitment. Both adult melanocytes and melanocytes that regenerate appear to derive from recruitable melanocyte stem cells (MSCs) (Johnson et al. 1995; Rawls and Johnson 2000). For example, genetic studies indicate that the embryonic melanocyte pattern develops from direct-developing melanocytes and is complete by 3 days postfertilization (dpf) (Hultman et al. 2009). Under normal conditions, few new melanocytes develop from 3 dpf until the onset of metamorphosis at ∼15 dpf (Hultman and Johnson 2010). However, when embryonic melanocytes are removed via laser or chemical treatment during this time, a rapid and complete regeneration of the melanocyte pattern occurs through the activation of cell division in melanocyte precursors, MSCs (Yang et al. 2004; Yang and Johnson 2006). MSCs normally lie dormant during larval zebrafish pigmentation, but can be recruited upon loss of differentiated melanocytes. The pathways that regulate MSC quiescence and recruitment are poorly understood.

Forward genetic studies have helped clarify the genetic regulatory hierarchy that controls melanocyte production and MSC proliferation in zebrafish. These studies highlight the importance of three genes in zebrafish pigmentation: the receptor tyrosine kinase erbb3b, the transcription factor mitfa, and the receptor tyrosine kinase kita. An adult zebrafish mutant for erbb3b, named picasso, exhibits defective melanocyte stripe formation, even though larval erbb3b mutants exhibit a wild-type pigment pattern (Budi et al. 2008). Critically, when picasso mutant zebrafish are challenged for melanocyte regeneration during larval stages, melanocyte regeneration is completely abrogated, suggesting that regenerating melanocytes require erbb3b function while early embryonic melanocytes do not (Hultman et al. 2009). This finding led to a model wherein a subset of migratory neural crest cells directly differentiate into embryonic melanocytes (direct-developing melanocytes) and other erbb3b-dependent neural crest cells establish undifferentiated melanocyte precursors, MSCs, that persist throughout zebrafish adult life and can be recruited to form new (stem-cell derived) melanocytes throughout the larval and adult stages (Dooley et al. 2013).

Additional insight into MSCs arose from experiments using a temperature-sensitive mutation of melanocyte-inducing transcription factor a (mitfa), which is required for all melanocyte development and survival across vertebrate biology (Lister et al. 1999; Levy et al. 2006). These studies revealed that mitfa function was required for the embryonic pigment pattern, but was not required for the survival of MSCs (Johnson et al. 2011). Therefore, while required for melanocyte survival, mitfa function is not required for the survival of MSCs that can regenerate larval melanocytes and produce the adult pigment pattern.

The receptor tyrosine kinase kita plays key roles during zebrafish pigment patterning. Removal of kita function, as seen in the sparse mutant, results in an ∼50% loss of larval melanocytes, but the adult melanocyte pattern is largely normal (Parichy et al. 1999). Thus, kita function is required for the development of direct developing melanocytes. However, kita does regulate MSC function. For example, kita function is required for melanocyte regeneration during larval stages and for melanocyte regeneration in the caudal fin at all stages (Rawls and Johnson 2001, 2003; O’Reilly-Pol and Johnson 2013).

GABA (γ-aminobutyric acid) is a major inhibitory neurotransmitter that transduces its signal by binding to and activating GABA receptors, such as the GABA-A receptor class (Bormann 2000). GABA-A receptors are voltage-gated chloride channels. When activated, they allow Cl⁻ ions to move down their electrochemical gradient into the cell, which hyperpolarizes the cell and inhibits action potential propagation along axons (Sigel and Steinmann 2012). Although GABA is best known to function as a neurotransmitter, prior studies indicated that GABA can inhibit the proliferation of murine embryonic stem cells and peripheral neural crest stem cells (Young and Bordey 2009; Teng et al. 2013). However, a role for GABA signaling in regulating vertebrate pigment patterning has not been shown.

Here, we show that pharmacological and genetic inhibition of GABA-A receptor function leads to excessive melanocyte production during larval zebrafish development, with the newly produced melanocytes likely arising from MSCs. Conversely, we show that pharmacological or genetic activation of GABA-A signaling inhibits melanocyte regeneration. Our work shows that GABA-mediated signaling promotes MSC quiescence during zebrafish development, and highlights the importance of membrane potential and bioelectric sensing in regulating pigment patterning in vertebrates.

Materials and Methods

Zebrafish stocks and husbandry

Adult fish were raised and maintained under a 14 h light:10 h dark light-to-dark cycle according to previously standardized protocols (Westerfield 2000). To facilitate melanocyte quantification, homozygous mlpha fish were used as wild-type and all experiments were performed in a homozygous mlpha genetic background unless otherwise indicated (Sheets et al. 2007). To perform our melanocyte differentiation assay, we used mlpha fish carrying Tg(fTyrp1:GFP)^j900 (Tryon and Johnson 2012). To genetically ablate melanocytes, we used mlpha fish homozygous for the temperature-sensitive mitfa^vc7 mutation (Johnson et al. 2011). The kita^b5 allele in a mlpha background was used in the study to test lineage specificity within MSCs (Parichy et al. 1999). We used the mlpha background to generate our two clustered regularly interspaced short palindromic repeat (CRISPR)-based mutations in gabrr1. Embryos of each genotype used in the present study were generated from in vitro fertilization.

Pharmacological reagents and drug screening

Our initial screen used a drug-repurposing panel (Pfizer) containing ∼500 unique compounds to identify melanocyte-promoting drugs. In this panel, each compound was supplied as a 30-mM stock solution in 96-well plates. We subsequently diluted each compound into 2-mM working solutions for further testing across multiple doses, generally ranging between 1 and 100 µM in 96-well plates. With this approach, we identified three compounds that increased melanocyte number: PF-04138835, a protein kinase B (AKT) inhibitor; PF-04269339-01, a 5-hydroxytryptamine_1A receptor partial agonist; and CP-615003-27, a GABA-A receptor antagonist. All other drugs used in the study were purchased from commercial vendors (Supplemental Material, Table S1). Each drug was handled according to the manufacturer’s guidelines, but in general each compound was dissolved in a solvent (DMSO or water) to a stock concentration of 20 mM. For drug experiments, 10–12 embryos were placed into 24-well plates with ∼2 ml egg water (60 mg/liter Instant Ocean: 0.06 parts per trillion (ppt)). The stock solution of each drug was then diluted and added to 2 ml egg water to a final vehicle concentration of 0.5% DMSO or water (Table S1). For our melanocyte differentiation assay, we used phenylthiourea (PTU) at a final concentration of 200 µM in egg water. Experiments were performed as parallel duplicates for each drug treatment.

Melanocyte counting

We focused our analysis of melanocyte development on the larval dorsal stripe. We quantified melanocytes along the dorsum, beginning with the melanocytes located immediately caudal to the otic vesicle and along the stripe to the posterior edge of the caudal fin. Each larvae was analyzed once as a biological replicant for the indicated experiment, and no further analyses on individual fish as technical replicants were used in this study. Larvae with severe morphological defects, such as cardiac edema, were excluded from analysis.

Generation of CRISPR/Cas9-mediated gabrr1 mutations

We used a previously described software tool to design guide RNAs (gRNAs) that target the gabrr1 locus [E-CRISP: http://www.e-crisp.org/E-CRISP/ (Heigwer et al. 2014)]. We chose a gRNA (Table S2) that targeted a highly conserved stretch of residues within the gabrr1 coding sequence (Wang et al. 1995). Briefly, we cloned our gabrr1 gRNA sequence into pT7-gRNA (plasmid # 46759; Addgene; Table S2). We used the mMessage mMachine T7 RNA synthesis kit (#AM1344; Thermo Fisher Scientific) to synthesize noncapped gRNA targeting gabrr1. We used the mMessage mMachine SP6 RNA synthesis kit (#AM1340; Thermo Fisher Scientific) to generate capped Cas9 mRNA from pCS2-nCas9n (Jao et al. 2013). Using a Olympus SZ40 microscope, we injected one-to-two-cell stage embryos with solution containing gabrr1 gRNA (75–100 ng/µl), Cas9 mRNA (300–400 ng/µl), and phenol red (1%). Injected embryos were reared to adulthood, and sperm samples were screened for Cas9-induced mutations. The genotyping assay was performed by amplifying a 500-bp amplicon flanking the gabrr1 target locus (Table S2), digesting with T7 endonuclease I (#M0302S; NEB) and Hpy166II (#R0616S; NEB), then analyzing with gel electrophoresis. T7-digested F₀ founders with putative gabrr1 lesions were outcrossed to mlpha and reared to adulthood. F₁ individuals were genotyped with Hpy166II and sequenced to identify mutation, outcrossed to Tg(fTyrp1:GFP)^j900, and analyzed for PTU melanocyte differentiation. F₁ individuals were intercrossed, and F₂ progeny were genotyped with Hpy166II and sequenced to identify homozygous gabrr1 mutant fish. Homozygous F₂ fish were outcrossed to Tg(fTyrp1:GFP)^j900 to generate clutches of F₃ heterozygotes, or intercrossed to generate clutches of homozygous embryos for analysis.

Generation of hsp70l:gabrr1 transgenic line

We generated a stable transgenic line to conditionally overexpress gabrr1 under the heat-shock promoter Tg(hsp70l:gabrr1)^j972. We used PCR-based methods to clone the full-length gabrr1 complementary DNA (cDNA) (Table S2) downstream of the hsp70l promoter (Pac1 site) in pT2-hsp70l (Halloran et al. 2000; Tryon and Johnson 2014) using an Infusion HD cloning kit (#638909; Takara Bio). We used the mMessage mMachine SP6 RNA synthesis kit to synthesize capped transposase mRNA from pCS-TP (Kawakami et al. 2004). To create a germline-integrated hsp70l:gabrr1, we injected embryos at the one- or two-cell stage with solution containing the pT2-hsp70l:gabrr1 plasmid (25–50 ng/µl), transposase mRNA (50–75 ng/µl), and phenol red (1%). Injected F₀ animals were screened at 1–2 dpf for the clonal marker xEf1α:GFP, indicating genomic integration of the construct. GFP⁺ embryos were reared to adulthood, outcrossed to mlpha, and the resulting progeny were screened for germline transmission of the Xenopus laevis EF1α:GFP clonal marker (Johnson and Krieg 1995). We established one stable hspl:gabrr1 transgenic line: Tg(hsp70l:gabrr1)^j972.

Heat-shock induction

Adult zebrafish carrying Tg(hsp70l:gabrr1)^j972 were outcrossed to mlpha strains to generate clutches of Tg(hsp70l:gabrr1)^j972/+; mlpha. From 1 to 3 dpf, embryos were then treated with the melanocyte prodrug 4-hydroxyanisole (4-HA) (M18655; Sigma [Sigma Chemical], St. Louis, MO; 10 mg/ml in DMSO) to ablate melanocytes. At 3 dpf, 4-HA was washed away, embryos were placed in 50-ml conical tubes, and heat shocked at 37° in a water bath for 30 min. The heat-shock treatment was repeated every 24 hr at 3, 4, and 5 dpf. At 6 dpf, the experiment was terminated and larvae were fixed in 3.7% formaldehyde for melanocyte quantification.

Microscopy and imaging

To screen for transgenic markers, embryos were anesthetized in tricaine mesylate and screened for GFP expression using an epifluorescence stereomicroscope (SMZ1500; Nikon, Garden City, NY). Images of representative larvae were taken with a Zeiss AxioCam MrC Digital Camera (Zeiss Carl Zeiss], Thornwood, NY; AxioVision imaging software). Images were then analyzed and processed using Fiji software (Schindelin et al. 2012).

Statistical analysis

In each experiment, we performed single-factor ANOVA (α: 0.01) to compare the means of each experimental group. We then performed Tukey’s honestly significant difference (HSD) post hoc tests to determine which groups were significantly different from their corresponding controls. All statistical tests and reported P-values were calculated in Microsoft Excel.

Data availability

Strains and plasmids are available upon request. The authors affirm that all data necessary for confirming the conclusions of the article are present within the article, figures, and tables. Supplemental material available at Figshare: https://doi.org/10.25386/genetics.9725753.

Results

GABA-A antagonists increase melanocyte production in larval zebrafish

We sought to explore the molecular regulation of MSC quiescence by searching for drugs that result in excess melanocyte development in the larval zebrafish. Previously, our laboratory found that the larval pigment pattern develops from direct-developing melanocytes and is largely complete by 3 dpf, but that melanocytes that develop after 3 dpf or those that regenerate the pigment pattern following melanocyte ablation develop from MSCs (Hultman et al. 2009; Hultman and Johnson 2010). We took advantage of this finding and designed a small-molecule screen to identify compounds that increase melanocyte output after 3 dpf. Our screen used larvae expressing the melanocyte marker fTyrp1:GFP^j900, and incubated them in a solution containing the screened compound and the melanin-inhibiting drug PTU. Newly generated melanocytes were uniquely identified based on the lack of melanin (mel−) and expression of GFP (GFP⁺): the mel⁻, GFP⁺ melanocytes. We focused on the dorsal larval stripe because we previously found that less than two new melanocytes develop within this region between 3 and 6 dpf (Hultman and Johnson 2010). This infrequent development of new melanocytes provided a low background that allowed us to screen for compounds that induced even a small increase in melanocyte production.

We screened > 500 compounds from a Pfizer repurposing panel, and identified a GABA-A receptor antagonist (CP-615003-27) that increased melanocyte production between 3 and 6 dpf. Consistent with previous findings from our laboratory, zebrafish treated with a vehicle control developed on average 1.05 mel⁻, GFP⁺ melanocytes in the dorsal larval stripe (Figure 1, B and F). Larvae treated with the GABA-A antagonist CP-615003-27 developed on average 4.0 newly formed mel⁻, GFP⁺ melanocytes in the same region (Figure 1, C and F), a significant increase over vehicle control-treated fish (Figure 1F). To confirm the effect of GABA-A inhibition on melanocyte production and development, we tested two other GABA-A antagonists. Zebrafish treated with the GABA-A antagonist Picrotoxin developed on average 3.7 mel⁻, GFP⁺ melanocytes (Figure 1, D and F), and zebrafish treated with the GABA-A ρ antagonist (1,2,5,6-Tetrahydropyridin-4-yl)methylphosphinic acid (TPMPA) developed on average 4.1 mel⁻, GFP⁺ melanocytes (Figure 1, E and F). Our finding that inhibition of GABA-A receptors through distinct GABA-A antagonists increases melanocyte production suggested that GABA-A signaling regulates MSC quiescence in larval 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.

GABA-A antagonist-induced melanocytes derive from MSCs

We next asked whether the newly formed melanocytes that develop following GABA-A antagonist treatment arise from an MSC or precursor lineage. Our previous work supported a model that melanocytes within the dorsal stripe primarily develop from undifferentiated MSCs or melanocyte precursors after 3 dpf, suggesting that GABA-A antagonists induce MSCs to produce new melanocytes. However, it remained formally possible that pharmacological inhibition of GABA-A signaling could activate aberrant melanocyte development from a nonstem cell source through an unknown mechanism. To distinguish between these mechanisms, we treated zebrafish embryos with either DMSO or the erbb3 inhibitor tyrphostin AG1478 (AG1478) from 8 to 48 hr postfertilization, washed out the drug, and then treated the larvae with solution containing PTU and a GABA-A antagonist from 3 to 6 dpf (Figure 2A). AG1478-mediated inhibition of erbb3 activity has been previously shown to inhibit melanocyte regeneration and metamorphic melanocyte development in zebrafish (Budi et al. 2008; Hultman et al. 2009). Early treatment with this small molecule is thought to block establishment of MSCs, removing the developmental source of new melanocytes (Dooley et al. 2013). Therefore, if new melanocytes arise from MSCs, we predicted that prior AG1478 treatment would inhibit the ability of GABA-A antagonists to induce melanocyte production.

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.

For this analysis, we focused on two representative GABA-A antagonists: TPMPA and Picrotoxin. After each drug treatment, individual larvae were scored for average mel⁻, GFP⁺ melanocytes in the dorsal stripe, which we interpreted as newly developed melanocytes in the presence of PTU. Zebrafish larvae treated with DMSO and vehicle control developed 1.76 mel⁻, GFP⁺ melanocytes, while larvae treated with AG1478 and vehicle control developed 0.32 mel⁻, GFP⁺ melanocytes (Figure 2B). This result suggested that the AG1478 treatment effectively blocked late (3–6 dpf) melanocyte production. Thus, our PTU assay could detect relatively small changes in melanocyte production, which allowed us to confidently test the combinatorial effects of AG1478 and GABA-A antagonists on melanocyte production. Larvae treated with DMSO and the GABA-A antagonist TPMPA developed 4.1 mel⁻, GFP⁺ melanocytes, but larvae treated with AG1478 and TPMPA developed only 0.28 mel⁻, GFP⁺ melanocytes. Similarly, larvae treated with DMSO and Picrotoxin developed 5.2 mel⁻, GFP⁺ melanocytes, but larvae treated with AG1478 and Picrotoxin developed only 0.17 mel⁻, GFP⁺ melanocytes (Figure 2B). We conclude that GABA-A antagonists induce melanocyte production from erbb3-dependent undifferentiated melanocyte precursors.

Pharmacological activation of GABA-A signaling inhibits melanocyte regeneration

Our data support the model that inhibition of GABA-A receptor signaling increases melanocyte production from undifferentiated precursors. This provided a clear prediction that activation of GABA-A signaling would inhibit melanocyte production. To test this hypothesis, we chose to treat larvae homozygous for the temperature-sensitive mitfa^vc7 allele with drugs that activate GABA-A receptor signaling. When raised at a restrictive temperature (32°), the temperature-sensitive nature of the mitfa^vc7 allele prevents melanoblast survival and mitfa^vc7 larvae develop no melanocytes. When shifted to a permissive temperature (25°), mitfa function is restored and mitfa^vc7 larvae exhibit near complete regeneration of the larval pigment pattern (Johnson et al. 2011). The homozygous mitfa^vc7 allele then provided us with temporal control of melanocyte development and regeneration to test the effects of GABA-A receptor activation.

To determine if GABA-A agonists inhibit melanocyte production, we reared mitfa^vc7 larvae to 3 dpf at 32°, and then downshifted to 25° in the presence of a GABA-A receptor drug (Figure 3A). As a measure of melanocyte regeneration following downshift, we scored larvae for the number of dorsal stripe melanocytes present as a developmental stage equivalent to 6 dpf when zebrafish are grown continuously at 28.5° (Kimmell et al. 1995). Mitfa^vc7 larvae treated with vehicle control regenerated 42.2 dorsal melanocytes (Figure 3, B and H). However, mitfa^vc7 larvae treated with the endogenous ligand GABA or the GABA-A ρ agonist γ-Amino-β-hydroxybutyric acid (GABOB) regenerated only 21.2 and 26.8 dorsal melanocytes, respectively (Figure 3, C, D, and H). The reduction of melanocyte regeneration following treatment of GABA-A agonists suggested that direct activation of GABA-A signaling partially inhibited melanocyte regeneration. To further challenge this idea, we treated mitfa^vc7 larvae with drugs that indirectly activated GABA-A receptor signaling and challenged for melanocyte regeneration. Larvae treated with the GABA-A partial agonists L,838,417 (Figure 3, E and H) and MK 0343 (Figure 3, G and H) regenerated, on average, 16 dorsal melanocytes and 18.1 dorsal melanocytes, respectively. Similarly, larvae treated with the GABA reuptake inhibitor CI-966, which increases synaptic concentrations of GABA (Ebert and Krnjevic 1990), regenerated only 20.4 dorsal melanocytes (Figure 3, F and H). The effects of these GABA-A-activating drugs were not restricted to the dorsal stripe, and appeared to reduce pigmentation across the ventral and lateral regions of the larvae as well (Figure S1). Our data suggest that pharmacological activation of GABA-A receptor signaling inhibits melanocyte production from MSCs.

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.

GABA-A ρ 1 is necessary for restriction of melanocyte production in larval zebrafish

To validate our pharmacology results, we sought to genetically remove GABA-A signaling and assess the impact on melanocyte production. Here, we focused on the GABA-A ρ receptor subtype, as the GABA-A ρ subtype-specific drug TPMPA yielded a robust increase in melanocyte production (Figure 1F). Fortunately, GABA-A ρ receptors are homopentameric, allowing us to target a single gene to disrupt receptor function (Martinez-Delgado et al. 2010). To target GABA-A ρ receptor function, we used a CRISPR-based strategy to target the GABA-A ρ 1 (gabrr1) gene. We specifically targeted a region in the ligand-binding domain that is critical for zinc inhibition to increase the likelihood of disrupting endogenous protein function (Wang et al. 1995). Using a PCR- and restriction enzyme-based method, we identified two putative gabrr1 alleles with altered DNA sequences at the targeted site (Figure 4A). Sequence analysis and protein alignments of both alleles revealed two gabrr1 in-frame mutations (Figure 4, A and B), both of which delete the conserved residues VHS from positions 146–148 of the polypeptide sequence, with one allele, gabrr1^j247, also substituting the lysine at position 149 to glutamic acid (Figure 4B).

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.

To determine if genetic reduction of gabrr1 function altered melanocyte development, we outcrossed carriers of each gabrr1 mutation to fTyrp1:GFP^j900, treated the F₁ progeny with PTU from 3 to 6 dpf, and quantified newly generated dorsal melanocytes. Both alleles demonstrated a robust dominant excess melanocyte phenotype. Zebrafish heterozygous for the gabrr1^j247 allele developed 9.56 mel⁻, GFP⁺ dorsal melanocytes (Figure 4, C and E), a fivefold increase over wild-type siblings (Figure 4, C and D). Zebrafish heterozygous for the gabrr1^j248 allele developed 7.81 mel⁻, GFP⁺ melanocytes (Figure 4, C and F), while trans-heterozygous gabrr1^j247/248 fish developed 9.72 mel⁻, GFP⁺ melanocytes on average. These results suggest that the two gabrr1 alleles function as dominant-negative alleles, although it remains possible that they are haploinsufficient for gabrr1 function. The gabrr1 mutant phenotypes mirror the excess melanocyte phenotype observed upon pharmacological inhibition of GABA receptor function (Figure 1). In our analysis, ventral pigmentation appeared unaffected in both gabrr1 alleles (Figure S2), suggesting spatial restriction of GABA-A signaling in early melanocyte patterning. In addition, we observe no gross change to adult melanocyte patterns in either heterozygous or homozygous adult mutant fish, suggesting the melanocyte pigment pattern recovers during the adult transition and that melanocyte patterning is most sensitive to gabrr1 signaling during larval development. We infer that gabrr1 function is necessary to inhibit excessive melanocyte production in the larval zebrafish, suggesting that GABA signaling through gabrr1 is a key regulatory pathway that normally maintains MSC quiescence in larval zebrafish.

GABA-A ρ 1 is sufficient to reduce, but not inhibit, melanocyte regeneration in larval zebrafish

Our observation that gabrr1 function is necessary to inhibit melanocyte production led us to test whether overexpression of gabrr1 was sufficient to inhibit melanocyte regeneration. To address this question, we cloned the gabrr1 cDNA under control of the heat-shock promoter element hsp70l within the Tol2 germline transformation vector and obtained a stable transgenic line: Tg(hsp70l:gabrr1)^j972 (Suster et al. 2009). We then treated Tg(hsp70l:gabrr1)^j972 and control larvae with the drug 4-HA from 1 to 3 dpf to ablate melanocytes, washed the drug out, induced heat shock at 37°, and then quantified melanocyte regeneration at 6 dpf (Figure 5A). Heat-shocked wild-type and nonheat-shocked Tg(hsp70l:gabrr1)^j972 larvae regenerated on average 49.9 and 51.1 melanocytes, respectively, whereas heat-shocked Tg(hsp70l:gabrr1)^j972 larvae regenerated on average 32.3 melanocytes, a roughly 40% reduction in melanocyte production (Figure 5, B and C). Thus, overexpression of gabrr1 can repress production of melanocytes during periods of regeneration. The overexpression of gabrr1 also appeared to inhibit melanocyte production both ventrally and laterally, but this effect was not as obvious as the effect of the dorsal stripe (Figure S3). Expression of gabrr1 then appears partially sufficient to inhibit melanocyte regeneration in larval zebrafish.

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.

Kita signaling and gabrr1 function within the same MSC lineage

We next determined whether kita function was required for GABAergic maintenance of MSC quiescence. Zebrafish heterozygous for the kita^b5 null allele regenerate only ∼50% of the larval pigment pattern, suggesting a reduction of the MSC pool consistent with the effects of kit haploinsufficiency observed in mammals (Geissler et al. 1988; O’Reilly-Pol and Johnson 2013). To test for possible interactions between kita and gabrr1, we asked whether kita haploinsufficiency inhibited the melanocyte overproduction phenotype observed in gabrr1 mutants. We generated control and kita^b5/+; gabrr1^j247/+ double-heterozygous larvae, reared them to 3 dpf, treated them with PTU, and then scored for excess melanocyte production at 6 dpf. As previously observed, wild-type larvae developed two excess melanocytes between 3 and 6 dpf, kita^b5/+ larvae developed 1.5 excess melanocytes, and gabrr1^j247/+ developed nine excess melanocytes on average (Figure 6). Of note, kita^b5/+; gabrr1^j247/+ larvae developed on average 1.5 excess melanocytes, indicating that the gabrr1 mutant melanocyte overproduction phenotype depends entirely on normal kita function, suggesting that GABA-A-mediated MSC quiescence is restricted within kita-dependent melanocyte lineages.

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;

Discussion

Our work provides evidence that GABAergic signaling promotes MSC quiescence in larval zebrafish. Both pharmacological and genetic studies indicate that reduction of GABA-A ρ signaling increases melanocyte production, whereas overexpression of GABA-A ρ signaling inhibits melanocyte production. Although both classical and recent studies have implicated membrane potential in pigmentation and stem cell proliferation, to our knowledge, our study is the first to uncover such a role for GABA-A receptor signaling in vertebrate pigment biology. Below, we propose a model for how GABA-A signaling regulates melanocyte development, discuss the nature of the GABA-A ρ mutants, and place our work in the context of old and new studies that highlight the importance of membrane potential, and cell excitability, in the regulation of stem cell proliferation and pigmentation.

Our work indicates that GABAergic signaling, directly or indirectly, maintains the MSC in a quiescent state. Prior studies suggest that differentiated larval melanocytes inhibit melanocyte differentiation by suppressing MSC proliferation. For example, chemical or laser ablation of differentiated larval melanocytes induces melanocyte regeneration by promoting the cell division of stem cell-like melanocyte progenitors. In this context, our work provides a conceptual model for how the presence of differentiated melanocytes promotes MSC quiescence, and how their absence triggers MSC proliferation and melanocyte production. Our pharmacological and genetic data support a model wherein melanocytes release the neurotransmitter GABA, which activates gabrr1 receptors on the MSC, maintaining the MSC in a quiescent state. Conversely, loss of melanocytes would trigger a reduction in GABA concentration and relieve gabrr1-mediated quiescence, triggering MSC proliferation and melanocyte production. Currently, this highly speculative model requires precise mapping of the cells that express GABA and gabbr1 in the melanocyte lineage to test its validity. Our data do not rule out the possibility that GABAergic signaling could act indirectly to regulate MSC quiescence, as melanocytes could release a non-GABA signal that triggers a GABA-to-GABA receptor relay in adjacent cells and tissues that ultimately promotes MSC quiescence. Clearly, additional work is required to address whether GABAergic signaling, directly or indirectly, controls MSC quiescence, but the presence of GABA synthesis enzymes (such as GAD67 mRNA) in human melanocytes hints that GABA signaling may be an evolutionarily conserved mechanism that regulates vertebrate pigmentation (Ito et al. 2007).

Dominant-negative nature of gabrr1 mutant alleles

Both gabrr1 alleles exhibit essentially identical melanocyte overproduction phenotypes when in the heterozygous, trans-heterozygous, or homozygous state, a phenotype similar to that observed upon pharmacological inhibition of GABA-A signaling. Both gabrr1 alleles remove a highly conserved triplet of amino acids in the ligand-binding domain of the receptor (Wang et al. 1995). Thus, each allele likely produces a nonfunctional subunit. Although it is formally possible that these mutant alleles are haploinsufficient, we favor the model they act in a dominant-negative manner since GABA-A ρ receptors are known to function as homopentamers. Thus, if the mutant form of the protein is expressed at roughly wild-type levels and can assemble into GABA-A ρ pentamers, only a tiny fraction of these pentamers would be composed of five wild-type subunits, providing a rational explanation for the dominant nature of the gabrr1 mutant alleles.

Do multiple extrinsic pathways regulate MSC quiescence?

When challenged for regeneration, zebrafish larvae produce hundreds of new melanocytes to repopulate the pigment pattern. These new melanocytes derive from a pool of established precursors: MSCs. Though usually quiescent, MSCs are capable of producing hundreds of new melanocytes throughout development. Using clonal analysis, we previously estimated that the developing zebrafish establishes between 150 and 200 MSCs before 2 dpf (Tryon et al. 2011). The MSC pool, though quiescent, then maintains their abundance in number and regenerative capability. Complete abrogation of the mechanisms that maintain MSC quiescence would then be expected to generate excess melanocytes proportional to the regenerative capabilities of all MSCs, i.e., generate hundreds of excess melanocytes. However, pharmacological or genetic inhibition of GABA-A signaling yields only 8–10 excess melanocytes on average (Figure 1 and Figure 4). Thus, the full regenerative capability of larval zebrafish likely involves the concerted actions of multiple pathways that converge on activation of MSC proliferation.

Our genetic studies with kita and gabrr1 suggest that GABA-A signaling may regulate a kita-dependent pool of MSCs (Tu and Johnson 2010). For example, our prior work indicated that haploinsufficiency for kita reduces the available MSC pool by ∼50% (O’Reilly-Pol and Johnson 2013), but haploinsufficiency for kita completely suppressed the overproduction of melanocytes caused by reduced gabrr1 function, suggesting that normal kita signaling is required for all GABA-sensitive MSCs, but that not all larval MSCs within are sensitive to either kita or GABA-A signaling. The requirement of kita signaling within the gabrr1-driven melanocyte lineage of zebrafish may then be indicative of regulatory pathways that suppress melanocyte production in a region-specific manner.

Which pathways function in parallel with gabrr1 to suppress MSC proliferation remain unclear. Recent work completed during the course of our study suggests that the endothelin receptor Aa (ednraa) acts in parallel to gabrr1 to maintain MSC quiescence. For example, loss of ednraa function leads to ectopic melanocyte production (via an MSC intermediate) specifically within the ventral trunk of larval zebrafish, whereas we find that genetic reduction of gabrr1 function increases MSC-derived melanocyte production in the dorsal stripe (Camargo-Sosa et al. 2019), even though pharmacological or genetic activation of gabrr1 signaling appears to reduce pigmentation in a larvae-wide manner. These studies support the idea that distinct genetic pathways maintain MSC quiescence in a region-specific manner throughout zebrafish development. Clearly, additional work is needed to determine whether other pathways act with gabbr1 and ednraa to promote MSC quiescence, but our work hints that GABA-A-mediated quiescence may be a hallmark of vertebrate pigment biology.

Bioelectric regulation of MSC quiescence and proliferation

GABA receptors function as ligand-gated channels that regulate membrane potential, suggesting that changes in membrane potential trigger the observed changes in melanocyte patterning, and MSC quiescence and proliferation. Inhibition of gabbr1 function, which should depolarize the cell, induced melanocyte production through an MSC intermediate. In this context, we note that prior work observed severe hyperpigmentation in X. laevis larvae due to melanocyte overproliferation and overproduction via pharmacological depolarization of glycine-gated chloride channels (Blackiston et al. 2011). In addition, the application of GABA and GABA-A agonists, which hyperpolarize cells by promoting Cl⁻ influx, inhibits proliferation of embryonic stem cells and peripheral neural crest stem cells in mice (Young and Bordey 2009; Teng et al. 2013). Moreover, in the mouse neocortex, neural progenitors become increasingly hyperpolarized as they produce their characteristic cell lineages (Vitali et al. 2018). Of note, artificial hyperpolarization of neural progenitor cells induced the premature production of late-stage cell types, revealing a functional link between changes in membrane potential and the temporal birth order of cells in the neocortex. Changes in the membrane potential of stem and progenitor cells can then alter cell division patterns and cellular behavior, supporting the idea that gabrr1-mediated regulation of membrane potential underlies its role in regulating early melanocyte patterning in zebrafish. Future work that can systematically assess the effects of membrane potential on the development of stem cells and their progeny, in a broad range of tissues, is required to reveal the extent to which this phenomenon occurs in vertebrate biology.