| INVESTIGATION

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

James R. Allen,1 James B. Skeath, and Stephen L. Johnson2

Department of Genetics, Washington University School of Medicine, St. Louis, Missouri 63110

ORCID ID: 0000-0003-1314-9122 (J.R.A.)

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

KEYWORDS GABA; melanocyte; GABA-A receptors; quiescence; zebrafish; pigmentation; inhibition; CRISPR

VERTEBRATE animals often rely on undifferentiated pre-cursors to regulate thegrowthandhomeostasis of specific

tissues. These precursors, adult stem cells (ASCs), undergolong-term self-renewal throughout the lifetime of the organ-ism tomaintain the growth and regenerative potential of theirtarget tissue. ASCs are found inmany tissues including blood,muscle, skin, and the nervous system (Nishimura et al. 2002;Bertrand et al. 2007; Ma et al. 2009; Cheung and Rando2013). While some ASCs continually proliferate to maintaintheir target tissue, others remain quiescent or dormant, andmust be recruited to enter a proliferative state, often inducedby depletion of differentiated cells in their respective tissues(Li and Bhatia 2011). Understanding the pathways thatmaintain ASC quiescence and that recruit quiescent ASCs toproliferate is critical to elucidate vertebrate tissue growth andhomeostasis.

Zebrafish pigmentation, specifically melanocyte develop-ment, is an excellent model system to dissect the genetic andmolecular basis of ASC quiescence, and recruitment. Bothadult melanocytes and melanocytes that regenerate appearto derive from recruitable melanocyte stem cells (MSCs)(Johnson et al. 1995; Rawls and Johnson 2000). For exam-ple, genetic studies indicate that the embryonic melanocytepattern develops from direct-developing melanocytes and iscomplete by 3 days postfertilization (dpf) (Hultman et al.2009). Under normal conditions, few new melanocytes de-velop from 3 dpf until the onset of metamorphosis at �15dpf (Hultman and Johnson 2010). However, when embry-onic melanocytes are removed via laser or chemical treat-ment during this time, a rapid and complete regenerationof the melanocyte pattern occurs through the activation ofcell division in melanocyte precursors, MSCs (Yang et al.2004; Yang and Johnson 2006). MSCs normally lie dor-mant during larval zebrafish pigmentation, but can berecruited upon loss of differentiated melanocytes. The path-ways that regulate MSC quiescence and recruitment arepoorly understood.

Forward genetic studies have helped clarify the geneticregulatory hierarchy that controlsmelanocyte production andMSC proliferation in zebrafish. These studies highlight theimportance of three genes in zebrafish pigmentation: thereceptor tyrosine kinase erbb3b, the transcription factor

Copyright © 2019 by the Genetics Society of Americadoi: https://doi.org/10.1534/genetics.119.302416Manuscript received June 10, 2019; accepted for publication August 12, 2019;published Early Online August 23, 2019.Supplemental material available at Figshare: https://doi.org/10.25386/genetics.9725753.This paper is dedicated to the late Stephen L. Johnson.1Corresponding author: Department of Genetics, Room 6315 Scott McKinleyResearch Bldg., 4523 Clayton Ave., Washington University School of Medicine,St. Louis, MO 63110. E-mail: jrallen@wustl.edu

2Deceased

Genetics, Vol. 213, 555–566 October 2019 555

mitfa, and the receptor tyrosine kinase kita. An adult zebra-fish mutant for erbb3b, named picasso, exhibits defectivemelanocyte stripe formation, even though larval erbb3b mu-tants exhibit a wild-type pigment pattern (Budi et al. 2008).Critically, when picasso mutant zebrafish are challenged formelanocyte regeneration during larval stages, melanocyteregeneration is completely abrogated, suggesting that regen-erating melanocytes require erbb3b function while earlyembryonic melanocytes do not (Hultman et al. 2009). Thisfinding led to a model wherein a subset of migratory neuralcrest cells directly differentiate into embryonic melano-cytes (direct-developing melanocytes) and other erbb3b-dependent neural crest cells establish undifferentiatedmelanocyte precursors, MSCs, that persist throughout zebra-fish adult life and can be recruited to form new (stem-cellderived) melanocytes throughout the larval and adult stages(Dooley et al. 2013).

Additional insight into MSCs arose from experimentsusinga temperature-sensitivemutationofmelanocyte-inducingtranscription factor a (mitfa), which is required for all melano-cyte development and survival across vertebrate biology (Listeret al. 1999; Levy et al. 2006). These studies revealed thatmitfafunctionwas required for the embryonic pigment pattern, butwas 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 thatcan regenerate larval melanocytes and produce the adultpigment pattern.

The receptor tyrosine kinase kita plays key roles duringzebrafish pigment patterning. Removal of kita function, asseen in the sparse mutant, results in an �50% loss of larvalmelanocytes, but the adult melanocyte pattern is largely nor-mal (Parichy et al. 1999). Thus, kita function is required forthe development of direct developing melanocytes. However,kita does regulate MSC function. For example, kita functionis required for melanocyte regeneration during larval stagesand for melanocyte regeneration in the caudal fin at allstages (Rawls and Johnson 2001, 2003; O’Reilly-Pol andJohnson 2013).

GABA (g-aminobutyric acid) is a major inhibitory neuro-transmitter that transduces its signal by binding to and acti-vating GABA receptors, such as the GABA-A receptor class(Bormann 2000). GABA-A receptors are voltage-gated chlo-ride channels. When activated, they allow Cl2 ions to movedown their electrochemical gradient into the cell, which hy-perpolarizes the cell and inhibits action potential propagationalong axons (Sigel and Steinmann 2012). Although GABA isbest known to function as a neurotransmitter, prior studiesindicated that GABA can inhibit the proliferation of murineembryonic stem cells and peripheral neural crest stem cells(Young and Bordey 2009; Teng et al. 2013). However, a rolefor GABA signaling in regulating vertebrate pigment pattern-ing has not been shown.

Here, we show that pharmacological and genetic inhibi-tion of GABA-A receptor function leads to excessive melano-cyte production during larval zebrafish development, with

the newly produced melanocytes likely arising from MSCs.Conversely, we show that pharmacological or genetic activa-tion of GABA-A signaling inhibits melanocyte regeneration.Our work shows that GABA-mediated signaling promotesMSC quiescence during zebrafish development, and high-lights the importance of membrane potential and bioelectricsensing in regulating pigment patterning in vertebrates.

Materials and Methods

Zebrafish stocks and husbandry

Adult fish were raised andmaintained under a 14 h light:10 hdark light-to-dark cycle according to previously standardizedprotocols (Westerfield 2000). To facilitate melanocyte quan-tification, homozygousmlpha fish were used as wild-type andall experiments were performed in a homozygous mlpha ge-netic background unless otherwise indicated (Sheets et al.2007). To perform our melanocyte differentiation assay, weused mlpha fish carrying Tg(fTyrp1:GFP)j900 (Tryon andJohnson 2012). To genetically ablate melanocytes, we usedmlpha fish homozygous for the temperature-sensitivemitfavc7

mutation (Johnson et al. 2011). The kitab5 allele in a mlphabackground was used in the study to test lineage specificitywithin MSCs (Parichy et al. 1999). We used the mlpha back-ground to generate our two clustered regularly interspacedshort palindromic repeat (CRISPR)-based mutations in gabrr1.Embryos of each genotype used in the present study weregenerated 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 suppliedas a 30-mM stock solution in 96-well plates. We subsequentlydiluted each compound into 2-mM working solutions for fur-ther testing across multiple doses, generally ranging be-tween 1 and 100 mM in 96-well plates. With this approach,we identified three compounds that increased melanocytenumber: PF-04138835, a protein kinase B (AKT) inhibitor;PF-04269339-01, a 5-hydroxytryptamine1A receptor partialagonist; and CP-615003-27, a GABA-A receptor antagonist.All other drugs used in the study were purchased from com-mercial vendors (Supplemental Material, Table S1). Eachdrug was handled according to the manufacturer’s guide-lines, but in general each compound was dissolved in a sol-vent (DMSO or water) to a stock concentration of 20mM. Fordrug experiments, 10–12 embryos were placed into 24-wellplates with�2ml egg water (60mg/liter Instant Ocean: 0.06parts per trillion (ppt)). The stock solution of each drug wasthen diluted and added to 2 ml egg water to a final vehicleconcentration of 0.5% DMSO or water (Table S1). For ourmelanocyte differentiation assay, we used phenylthiourea(PTU) at a final concentration of 200 mM in egg water. Ex-periments were performed as parallel duplicates for eachdrug treatment.

556 J. R. Allen, J. B. Skeath, and S. L. Johnson

Melanocyte counting

We focused our analysis of melanocyte development on thelarval dorsal stripe. We quantified melanocytes along thedorsum, beginningwith themelanocytes located immediatelycaudal to the otic vesicle and along the stripe to the posterioredge of the caudal fin. Each larvae was analyzed once as abiological replicant for the indicated experiment, and nofurther analyses on individualfish as technical replicantswereused 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 guideRNAs (gRNAs) that target the gabrr1 locus [E-CRISP: http://www.e-crisp.org/E-CRISP/ (Heigwer et al. 2014)]. We chosea gRNA (Table S2) that targeted a highly conserved stretchof residues within the gabrr1 coding sequence (Wang et al.1995). Briefly, we cloned our gabrr1 gRNA sequence intopT7-gRNA (plasmid # 46759; Addgene; Table S2). We usedthe mMessage mMachine T7 RNA synthesis kit (#AM1344;Thermo Fisher Scientific) to synthesize noncapped gRNA tar-geting gabrr1. We used the mMessage mMachine SP6 RNAsynthesis kit (#AM1340; Thermo Fisher Scientific) to gener-ate 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/ml), Cas9 mRNA (300–400 ng/ml), and phenolred (1%). Injected embryos were reared to adulthood, andsperm samples were screened for Cas9-induced mutations.The genotyping assay was performed by amplifying a 500-bpamplicon flanking the gabrr1 target locus (Table S2), digest-ing with T7 endonuclease I (#M0302S; NEB) and Hpy166II(#R0616S; NEB), then analyzing with gel electrophoresis.T7-digested F0 founders with putative gabrr1 lesions wereoutcrossed to mlpha and reared to adulthood. F1 individualswere genotyped with Hpy166II and sequenced to identifymutation, outcrossed to Tg(fTyrp1:GFP)j900, and analyzedfor PTUmelanocyte differentiation. F1 individuals were inter-crossed, and F2 progeny were genotyped with Hpy166II andsequenced to identify homozygous gabrr1 mutant fish. Ho-mozygous F2 fish were outcrossed to Tg(fTyrp1:GFP)j900 togenerate clutches of F3 heterozygotes, or intercrossed to gen-erate clutches of homozygous embryos for analysis.

Generation of hsp70l:gabrr1 transgenic line

We generated a stable transgenic line to conditionallyoverexpress gabrr1 under the heat-shock promoter Tg(hsp70l:gabrr1)j972. We used PCR-based methods to clonethe full-length gabrr1 complementary DNA (cDNA) (TableS2) downstream of the hsp70l promoter (Pac1 site) in pT2-hsp70l (Halloran et al. 2000; Tryon and Johnson 2014) usingan Infusion HD cloning kit (#638909; Takara Bio). We usedthemMessagemMachine SP6 RNA synthesis kit to synthesizecapped 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 solutioncontaining the pT2-hsp70l:gabrr1 plasmid (25–50 ng/ml),transposase mRNA (50–75 ng/ml), and phenol red (1%). In-jected F0 animals were screened at 1–2 dpf for the clonalmarker xEf1a:GFP, indicating genomic integration of the con-struct. GFP+ embryos were reared to adulthood, outcrossed tomlpha, and the resulting progeny were screened for germlinetransmission of the Xenopus laevis EF1a: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 out-crossed to mlpha strains to generate clutches of Tg(hsp70l:gabrr1)j972/+; mlpha. From 1 to 3 dpf, embryos were thentreated with the melanocyte prodrug 4-hydroxyanisole (4-HA) (M18655; Sigma [Sigma Chemical], St. Louis, MO; 10mg/ml in DMSO) to ablate melanocytes. At 3 dpf, 4-HA waswashed 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 werefixed in 3.7% formaldehyde for melanocyte quantification.

Microscopy and imaging

To screen for transgenic markers, embryos were anesthe-tized in tricaine mesylate and screened for GFP expressionusing an epifluorescence stereomicroscope (SMZ1500; Nikon,Garden City, NY). Images of representative larvae were takenwith a Zeiss AxioCam MrC Digital Camera (Zeiss Carl Zeiss],Thornwood, NY; AxioVision imaging software). Images werethen analyzed and processed using Fiji software (Schindelinet al. 2012).

Statistical analysis

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

Data availability

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

Results

GABA-A antagonists increase melanocyte production inlarval zebrafish

We sought to explore the molecular regulation of MSC qui-escencebysearching fordrugs that result inexcessmelanocytedevelopment in the larval zebrafish. Previously, our laboratory

GABA-A Inhibits Zebrafish Pigmentation 557

found that the larval pigment pattern develops from direct-developing melanocytes and is largely complete by 3 dpf, butthat melanocytes that develop after 3 dpf or those that re-generate the pigment pattern following melanocyte ablationdevelop from MSCs (Hultman et al. 2009; Hultman andJohnson 2010). We took advantage of this finding anddesigned a small-molecule screen to identify compounds thatincrease melanocyte output after 3 dpf. Our screen used lar-vae expressing the melanocyte marker fTyrp1:GFPj900, andincubated them in a solution containing the screened com-pound and the melanin-inhibiting drug PTU. Newly gener-ated melanocytes were uniquely identified based on the lackof melanin (mel2) and expression of GFP (GFP+): the mel2,GFP+ melanocytes. We focused on the dorsal larval stripebecause we previously found that less than two new melano-cytes develop within this region between 3 and 6 dpf(Hultman and Johnson 2010). This infrequent developmentof newmelanocytes provided a low background that allowedus to screen for compounds that induced even a small in-crease in melanocyte production.

We screened. 500 compounds from a Pfizer repurposingpanel, and identified a GABA-A receptor antagonist (CP-615003-27) that increased melanocyte production between3 and 6 dpf. Consistent with previous findings from our lab-oratory, zebrafish treated with a vehicle control developed onaverage 1.05 mel2, GFP+ melanocytes in the dorsal larvalstripe (Figure 1, B and F). Larvae treated with the GABA-Aantagonist CP-615003-27 developed on average 4.0 newlyformed mel2, GFP+ melanocytes in the same region (Figure1, C and F), a significant increase over vehicle control-treatedfish (Figure 1F). To confirm the effect of GABA-A inhibitionon melanocyte production and development, we tested twoother GABA-A antagonists. Zebrafish treated with theGABA-A antagonist Picrotoxin developed on average 3.7mel2, GFP+ melanocytes (Figure 1, D and F), and zebrafishtreated with the GABA-A r antagonist (1,2,5,6-Tetrahydropyridin-4-yl)methylphosphinic acid (TPMPA) developed onaverage 4.1mel2, GFP+melanocytes (Figure 1, E and F). Ourfinding that inhibition of GABA-A receptors through distinctGABA-A antagonists increases melanocyte production sug-gested that GABA-A signaling regulates MSC quiescence inlarval zebrafish.

GABA-A antagonist-induced melanocytes derivefrom MSCs

We next asked whether the newly formed melanocytes thatdevelop following GABA-A antagonist treatment arise froman MSC or precursor lineage. Our previous work supporteda model that melanocytes within the dorsal stripe primarilydevelop from undifferentiated MSCs or melanocyte precur-sors after 3 dpf, suggesting that GABA-A antagonists induceMSCs to produce new melanocytes. However, it remainedformally possible that pharmacological inhibition of GABA-Asignaling could activate aberrant melanocyte developmentfrom a nonstem cell source through an unknown mecha-nism. To distinguish between these mechanisms, we treated

zebrafish embryos with either DMSO or the erbb3 inhibitortyrphostin AG1478 (AG1478) from 8 to 48 hr postfertiliza-tion, washed out the drug, and then treated the larvae withsolution containing PTU and a GABA-A antagonist from 3 to6 dpf (Figure 2A). AG1478-mediated inhibition of erbb3 ac-tivity has been previously shown to inhibit melanocyte re-generation and metamorphic melanocyte development inzebrafish (Budi et al. 2008; Hultman et al. 2009). Early treat-ment with this small molecule is thought to block establish-ment of MSCs, removing the developmental source of newmelanocytes (Dooley et al. 2013). Therefore, if new melano-cytes arise fromMSCs, we predicted that prior AG1478 treat-ment would inhibit the ability of GABA-A antagonists toinduce melanocyte production.

For this analysis,we focusedon tworepresentativeGABA-Aantagonists: TPMPA and Picrotoxin. After each drug treat-ment, individual larvae were scored for average mel2, GFP+

melanocytes in the dorsal stripe, which we interpreted asnewly developed melanocytes in the presence of PTU. Zebra-fish larvae treated with DMSO and vehicle control developed1.76 mel2, GFP+ melanocytes, while larvae treated withAG1478 and vehicle control developed 0.32 mel2, GFP+ me-lanocytes (Figure 2B). This result suggested that the AG1478treatment effectively blocked late (3–6 dpf) melanocyte pro-duction. Thus, our PTU assay could detect relatively smallchanges in melanocyte production, which allowed us to con-fidently test the combinatorial effects of AG1478 andGABA-Aantagonists on melanocyte production. Larvae treated withDMSO and the GABA-A antagonist TPMPA developed 4.1mel2, GFP+ melanocytes, but larvae treated with AG1478and TPMPA developed only 0.28 mel2, GFP+ melanocytes.Similarly, larvae treated with DMSO and Picrotoxin devel-oped 5.2 mel2, GFP+ melanocytes, but larvae treated withAG1478 and Picrotoxin developed only 0.17 mel2, GFP+

melanocytes (Figure 2B). We conclude that GABA-A antago-nists induce melanocyte production from erbb3-dependentundifferentiated melanocyte precursors.

Pharmacological activation of GABA-A signaling inhibitsmelanocyte regeneration

Our data support the model that inhibition of GABA-A re-ceptor signaling increases melanocyte production from un-differentiatedprecursors. Thisprovidedaclearprediction thatactivation of GABA-A signaling would inhibit melanocyteproduction. To test this hypothesis, we chose to treat larvaehomozygous for the temperature-sensitivemitfavc7 allelewithdrugs that activate GABA-A receptor signaling. When raisedat a restrictive temperature (32�), the temperature-sensitivenature of the mitfavc7 allele prevents melanoblast survivaland mitfavc7 larvae develop no melanocytes. When shiftedto a permissive temperature (25�), mitfa function is restoredandmitfavc7 larvae exhibit near complete regeneration of thelarval pigment pattern (Johnson et al. 2011). The homozy-gousmitfavc7 allele then provided us with temporal control ofmelanocyte development and regeneration to test the effectsof GABA-A receptor activation.

558 J. R. Allen, J. B. Skeath, and S. L. Johnson

To determine if GABA-A agonists inhibit melanocyteproduction, we rearedmitfavc7 larvae to 3 dpf at 32�, and thendownshifted to 25� in the presence of a GABA-A receptor drug(Figure 3A). As a measure of melanocyte regeneration fol-lowing downshift, we scored larvae for the number of dorsalstripe melanocytes present as a developmental stage equiva-lent to 6 dpf when zebrafish are grown continuously at 28.5�(Kimmell et al. 1995). Mitfavc7 larvae treated with vehiclecontrol regenerated 42.2 dorsal melanocytes (Figure 3, Band H). However, mitfavc7 larvae treated with the endog-enous ligand GABA or the GABA-A r agonist g-Amino-b-hydroxybutyric acid (GABOB) regenerated only 21.2and 26.8 dorsal melanocytes, respectively (Figure 3, C, D,and H). The reduction of melanocyte regeneration followingtreatment of GABA-A agonists suggested that direct activa-tion of GABA-A signaling partially inhibited melanocyte

regeneration. To further challenge this idea, we treatedmitfavc7 larvae with drugs that indirectly activated GABA-Areceptor signaling and challenged for melanocyte regenera-tion. Larvae treated with the GABA-A partial agonistsL,838,417 (Figure 3, E and H) and MK 0343 (Figure 3, Gand H) regenerated, on average, 16 dorsal melanocytesand 18.1 dorsal melanocytes, respectively. Similarly, larvaetreated with the GABA reuptake inhibitor CI-966, which in-creases synaptic concentrations of GABA (Ebert and Krnjevic1990), regenerated only 20.4 dorsal melanocytes (Figure 3,F and H). The effects of these GABA-A-activating drugswere not restricted to the dorsal stripe, and appeared to re-duce pigmentation across the ventral and lateral regionsof the larvae as well (Figure S1). Our data suggest that phar-macological activation of GABA-A receptor signaling inhibitsmelanocyte production from MSCs.

Figure 1 GABA-A antagonists in-crease melanocyte production inlarval zebrafish. (A) Schematic ofexperimental timeline for PTU mela-nocyte differentiation assay. Drugsand PTU are added to zebrafish em-bryos between 3 and 6 dpf. (B–E)Images of representative 6 dpf lar-vae treated with vehicle control (B)or GABA-A antagonist CP-615003-27 (40 mM), (C) Picrotoxin (100 mM)(D), or TPMPA (100 mM); (E). (F)Quantification of the average num-ber of melanin2, GFP+ dorsal mela-nocytes for each treatment group 6variation (vehicle control: 0.926 1.15,N = 84; CP-615003-27: 4.276 2.12,N = 81; Picrotoxin: 3.76 6 1.38, N =55; and TPMPA: 4.10 6 2.14, N =52). Following single-factor ANOVA,each experimental group was com-pared to vehicle control using Tukey’sHSD. *** P , 0.001 (Tukey’s HSD).dpf, days postfertilization; GABA,g-aminobutyric acid; HSD, hon-estly significant difference; PTU,phenylthiourea.

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GABA-A r 1 is necessary for restriction of melanocyteproduction in larval zebrafish

To validate our pharmacology results, we sought to geneti-cally remove GABA-A signaling and assess the impact onmelanocyte production. Here, we focused on the GABA-A r

receptor subtype, as the GABA-A r subtype-specific drugTPMPA yielded a robust increase in melanocyte production(Figure 1F). Fortunately, GABA-A r receptors are homopen-tameric, allowing us to target a single gene to disrupt recep-tor function (Martinez-Delgado et al. 2010). To targetGABA-A r receptor function, we used a CRISPR-based strat-egy to target the GABA-A r 1 (gabrr1) gene. We specificallytargeted a region in the ligand-binding domain that is criticalfor zinc inhibition to increase the likelihood of disruptingendogenous protein function (Wang et al. 1995). Using aPCR- and restriction enzyme-based method, we identifiedtwo putative gabrr1 alleles with altered DNA sequences atthe targeted site (Figure 4A). Sequence analysis and proteinalignments of both alleles revealed two gabrr1 in-frame mu-tations (Figure 4, A and B), both of which delete the con-served residues VHS from positions 146–148 of thepolypeptide sequence, with one allele, gabrr1j247, alsosubstituting the lysine at position 149 to glutamic acid (Fig-ure 4B).

To determine if genetic reduction of gabrr1 function al-tered melanocyte development, we outcrossed carriers ofeach gabrr1 mutation to fTyrp1:GFPj900, treated the F1 prog-eny with PTU from 3 to 6 dpf, and quantified newly gener-ated dorsal melanocytes. Both alleles demonstrated a robustdominant excess melanocyte phenotype. Zebrafish heterozy-gous for the gabrr1j247 allele developed 9.56 mel2, GFP+

dorsal melanocytes (Figure 4, C and E), a fivefold increaseover wild-type siblings (Figure 4, C and D). Zebrafish hetero-zygous for the gabrr1j248 allele developed 7.81 mel2, GFP+

melanocytes (Figure 4, C and F), while trans-heterozygousgabrr1j247/248 fish developed 9.72 mel2, GFP+ melanocyteson average. These results suggest that the two gabrr1 allelesfunction as dominant-negative alleles, although it remainspossible that they are haploinsufficient for gabrr1 function.The gabrr1mutant phenotypes mirror the excess melanocytephenotype observed upon pharmacological inhibition ofGABA receptor function (Figure 1). In our analysis, ventralpigmentation appeared unaffected in both gabrr1 alleles(Figure S2), suggesting spatial restriction of GABA-A signal-ing in early melanocyte patterning. In addition, we observeno gross change to adult melanocyte patterns in eitherheterozygous or homozygous adult mutant fish, suggestingthe melanocyte pigment pattern recovers during the adult

Figure 2 GABA-A antagonist-inducedmelanocytes derive from MSCs. (A)Schematic of experimental timeline fordrug treatment. (B) Quantification ofthe average melanin2, GFP+ dorsal me-lanocytes in each group 6 variation (ve-hicle control: 1.76 6 1.26, N = 39;vehicle control + AG1478: 0.32 60.48, N = 28; TPMPA: 4.14 6 1.43,N = 29; TPMPA + AG1478: 0.286 0.54,N = 25; Picrotoxin: 5.2 6 0.99, N = 30;and Picrotoxin + AG1478: 0.17 6 0.38,N = 30). Following single-factor ANOVA,each experimental group was comparedto vehicle control using Tukey’s HSD. ***P , 0.001 (Tukey’s HSD). dpf, days po-stfertilization; GABA, g-aminobutyricacid; HSD, honestly significant dif-ference; MSC, melanocyte stem cell;PTU, phenylthiourea.

560 J. R. Allen, J. B. Skeath, and S. L. Johnson

transition and that melanocyte patterning is most sensitive togabrr1 signaling during larval development. We infer thatgabrr1 function is necessary to inhibit excessive melanocyteproduction in the larval zebrafish, suggesting that GABA sig-naling through gabrr1 is a key regulatory pathway that nor-mally maintains MSC quiescence in larval zebrafish.

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

Our observation that gabrr1 function is necessary to inhibitmelanocyte production led us to test whether overexpressionof 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 theTol2 germline transformation vector and obtained a stabletransgenic line: Tg(hsp70l:gabrr1)j972 (Suster et al. 2009).We then treated Tg(hsp70l:gabrr1)j972 and control larvaewith the drug 4-HA from 1 to 3 dpf to ablate melanocytes,washed the drug out, induced heat shock at 37�, andthen quantified melanocyte regeneration at 6 dpf(Figure 5A). Heat-shocked wild-type and nonheat-shockedTg(hsp70l:gabrr1)j972 larvae regenerated on average 49.9and 51.1 melanocytes, respectively, whereas heat-shockedTg(hsp70l:gabrr1)j972 larvae regenerated on average 32.3melanocytes, a roughly 40% reduction in melanocyte pro-duction (Figure 5, B and C). Thus, overexpression of

Figure 3 Pharmacological acti-vation of GABA-A signaling in-hibits melanocyte regeneration. (A)Schematic of experimental time-line for drug treatment. Imagesof representative mitfavc7 7 dpflarvae treated with vehicle control(B), GABA (50 mM) (C), GABOB(100 mM) (D), L,838-417 (100 mM)(E), CI-966 HCL (20 mM) (F), andMK0343 (100 mM) (G). (H) Quantifi-cation of the average number ofdorsal melanocytes in each drugtreatment group 6 variation (ve-hicle control: 42.2 6 9.38, N =78; GABA: 21.2 6 10.4, N = 42;GABOB: 26.8 6 7.71, N = 41;L,838-417: 16 6 11.2, N = 42;CI-966 HCL: 20.4 6 7.81, N =43; and MK 0343: 18.1 6 9.55,N = 35). Following single-factorANOVA, each experimental groupwas compared to vehicle controlusing Tukey’s HSD. *** P, 0.001(Tukey’s HSD). dpf, days postferti-lization; GABA, g-aminobutyricacid; HSD, honestly significantdifference.

GABA-A Inhibits Zebrafish Pigmentation 561

gabrr1 can repress production of melanocytes during pe-riods of regeneration. The overexpression of gabrr1 alsoappeared to inhibit melanocyte production both ventrallyand laterally, but this effect was not as obvious as the effectof the dorsal stripe (Figure S3). Expression of gabrr1 then ap-pears partially sufficient to inhibit melanocyte regeneration inlarval zebrafish.

Kita signaling and gabrr1 function within the sameMSC lineage

We next determined whether kita function was required forGABAergic maintenance of MSC quiescence. Zebrafish het-erozygous for the kitab5 null allele regenerate only �50% ofthe larval pigment pattern, suggesting a reduction of theMSCpool consistent with the effects of kit haploinsufficiency ob-served in mammals (Geissler et al. 1988; O’Reilly-Pol and

Johnson 2013). To test for possible interactions betweenkita and gabrr1, we asked whether kita haploinsufficiencyinhibited the melanocyte overproduction phenotypeobserved in gabrr1 mutants. We generated control andkitab5/+; gabrr1j247/+ double-heterozygous larvae, rearedthem to 3 dpf, treated them with PTU, and then scored forexcess melanocyte production at 6 dpf. As previously ob-served, wild-type larvae developed two excess melanocytesbetween 3 and 6 dpf, kitab5/+ larvae developed 1.5 excessmelanocytes, and gabrr1j247/+ developed nine excess mela-nocytes on average (Figure 6). Of note, kitab5/+; gabrr1j247/+

larvae developed on average 1.5 excess melanocytes, indicat-ing that the gabrr1 mutant melanocyte overproduction phe-notype depends entirely on normal kita function, suggestingthat GABA-A-mediated MSC quiescence is restricted withinkita-dependent melanocyte lineages.

Figure 4 gabrr1 mutations exhibit adominant excess melanocyte phenotypeduring larval stages. (A) Partial sequencealignment of wild-type and CRISPRmutagenized gabrr1 genomic locus inzebrafish. (B) Partial peptide alignmentof vertebrate gabrr1 homology refer-ence protein (human: NP_002033;mouse: NP_032101; and zebrafish:NP_001020724) within the ligand-binding domain, with the predictedamino acid sequence of the twogabrr1 alleles generated in the study.(C) Quantification of the average num-ber of melanin2, GFP+ dorsal melano-cytes in each treatment group 6variation (wild-type: 2.04 6 0.94, N =51; gabrr1j247/+: 9.56 6 1.48, N = 39;gabrr1j247/j247: 9.13 6 1.41, N = 15;gabrr1j248/+: 7.81 6 1.17, N = 48;gabrr1j248/j248: 10.2 6 2.54, N = 13;and gabrr1j247/j248: 9.72 6 1.99, N =29). Representative images of 6 dpfwild-type (D), gabrr1j247/+ (E), gabrr1j248/+

(F), and gabrr1j247/j248 (G) larvae. Follow-ing single-factor ANOVA, each experi-mental group was compared to vehiclecontrol using Tukey’s HSD. *** P ,0.001 (Tukey’s HSD). CRISPR, clusteredregularly interspaced short palindromicrepeats; dpf, days postfertilization; HSD,honestly significant difference.

562 J. R. Allen, J. B. Skeath, and S. L. Johnson

Discussion

Ourworkprovides evidence thatGABAergic signalingpromotesMSC quiescence in larval zebrafish. Both pharmacological andgenetic studies indicate that reduction of GABA-A r signalingincreases melanocyte production, whereas overexpression ofGABA-A r signaling inhibits melanocyte production. Althoughboth classical and recent studies have implicated membranepotential in pigmentation and stem cell proliferation, to ourknowledge, our study is the first to uncover such a role forGABA-A receptor signaling in vertebrate pigment biology. Be-low, we propose a model for how GABA-A signaling regulatesmelanocyte development, discuss the nature of the GABA-A r

mutants, and place our work in the context of old and newstudies that highlight the importance of membrane potential,and cell excitability, in the regulation of stem cell proliferationand pigmentation.

Our work indicates that GABAergic signaling, directly orindirectly,maintains theMSC inaquiescent state. Prior studiessuggest that differentiated larval melanocytes inhibit mela-nocyte differentiation by suppressing MSC proliferation. Forexample, chemical or laser ablation of differentiated larval

melanocytes induces melanocyte regeneration by promotingthe cell division of stem cell-like melanocyte progenitors. Inthis context, our work provides a conceptual model for howthe presence of differentiated melanocytes promotes MSCquiescence, and how their absence triggers MSC proliferationandmelanocyteproduction.Ourpharmacological andgeneticdata support a model wherein melanocytes release the neu-rotransmitter GABA, which activates gabrr1 receptors on theMSC, maintaining the MSC in a quiescent state. Conversely,loss of melanocytes would trigger a reduction in GABA con-centration and relieve gabrr1-mediated quiescence, trigger-ing MSC proliferation and melanocyte production. Currently,this highly speculative model requires precise mapping of thecells that express GABA and gabbr1 in themelanocyte lineageto test its validity. Our data do not rule out the possibility thatGABAergic signaling could act indirectly to regulate MSCquiescence, as melanocytes could release a non-GABA signalthat triggers a GABA-to-GABA receptor relay in adjacent cellsand tissues that ultimately promotes MSC quiescence.Clearly, additional work is required to address whetherGABAergic signaling, directly or indirectly, controls MSC

Figure 5 Overexpression of gabrr1 inhibits melanocyte regeneration in larval zebrafish. (A) Schematic of experimental timeline. Arrows indicate timingof three 30-min 37� heat-shock treatments. (B) Quantification of average dorsal melanocytes in each treatment group6 variation. [mlpha + heat shock:49.96 6.01, N = 32; Tg(hsp70l:gabrr1)j972: 51.16 6.86, N = 38; and Tg(hsp70l:gabrr1)j972+ heatshock: 32.36 5.54, N = 46]. Images of representativemlpha + heat shock (C), Tg(hsp70l:gabrr1)j972 (D), and Tg(hsp70l:gabrr1)j972 + heatshock (E) larvae. Following single-factor ANOVA, each experimentalgroup 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.

GABA-A Inhibits Zebrafish Pigmentation 563

quiescence, but the presence of GABA synthesis enzymes(such as GAD67 mRNA) in human melanocytes hints thatGABA signaling may be an evolutionarily conserved mecha-nism that regulates vertebrate pigmentation (Ito et al. 2007).

Dominant-negative nature of gabrr1 mutant alleles

Both gabrr1 alleles exhibit essentially identical melanocyteoverproduction phenotypes when in the heterozygous, trans-heterozygous, or homozygous state, a phenotype similar tothat observed upon pharmacological inhibition of GABA-Asignaling. Both gabrr1 alleles remove a highly conserved trip-let of amino acids in the ligand-binding domain of the recep-tor (Wang et al. 1995). Thus, each allele likely produces anonfunctional subunit. Although it is formally possible thatthese mutant alleles are haploinsufficient, we favor themodel they act in a dominant-negative manner since GABA-Ar 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 r pentamers, onlya tiny fraction of these pentamers would be composed of fivewild-type subunits, providing a rational explanation for thedominant nature of the gabrr1 mutant alleles.

Do multiple extrinsic pathways regulateMSC quiescence?

When challenged for regeneration, zebrafish larvae producehundreds of new melanocytes to repopulate the pigmentpattern. These new melanocytes derive from a pool of estab-lished precursors: MSCs. Though usually quiescent, MSCs arecapable of producing hundreds of new melanocytes through-out development. Using clonal analysis, we previously esti-mated that the developing zebrafish establishes between150 and 200 MSCs before 2 dpf (Tryon et al. 2011). The

MSC pool, though quiescent, then maintains their abundancein number and regenerative capability. Complete abrogationof themechanisms thatmaintainMSC quiescence would thenbe expected to generate excess melanocytes proportional tothe regenerative capabilities of all MSCs, i.e., generate hun-dreds of excess melanocytes. However, pharmacological orgenetic inhibition of GABA-A signaling yields only 8–10 ex-cess melanocytes on average (Figure 1 and Figure 4). Thus,the full regenerative capability of larval zebrafish likely in-volves the concerted actions of multiple pathways that con-verge on activation of MSC proliferation.

Our genetic studies with kita and gabrr1 suggest thatGABA-A signaling may regulate a kita-dependent pool ofMSCs (Tu and Johnson 2010). For example, our prior workindicated that haploinsufficiency for kita reduces the avail-able MSC pool by �50% (O’Reilly-Pol and Johnson 2013),but haploinsufficiency for kita completely suppressed theoverproduction of melanocytes caused by reduced gabrr1function, suggesting that normal kita signaling is requiredfor all GABA-sensitive MSCs, but that not all larval MSCswithin are sensitive to either kita or GABA-A signaling. Therequirement of kita signaling within the gabrr1-driven mela-nocyte lineage of zebrafish may then be indicative of regula-tory pathways that suppress melanocyte production in aregion-specific manner.

Which pathways function in parallel with gabrr1 to sup-press MSC proliferation remain unclear. Recent work com-pleted during the course of our study suggests that theendothelin receptor Aa (ednraa) acts in parallel to gabrr1to maintain MSC quiescence. For example, loss of ednraafunction leads to ectopic melanocyte production (via anMSC intermediate) specifically within the ventral trunk oflarval zebrafish, whereas we find that genetic reduction of

Figure 6 gabrr1-mediated mainte-nance of MSC quiescence is sensitiveto kita dosage. Quantification of the av-erage number of melanin2, GFP+ dorsalmelanocytes in gabrr1j247/+ and kitab5/+

6 variation (wild-type: 2.356 1.03, N =42; kitab5/+: 0.88 6 0.81, N = 56;gabrr1j247/+: 9.55 6 1.43, N = 40; andkitab5/+; gabrr1j247/+: 0.87 6 0.87,S.E.M: 0.11, N = 60). Following single-factor ANOVA, each experimental groupwas compared to vehicle control usingTukey’s HSD. *** P , 0.001 (Tukey’sHSD). HSD, honestly significant differ-ence; MSC, melanocyte stem cell;

564 J. R. Allen, J. B. Skeath, and S. L. Johnson

gabrr1 function increases MSC-derived melanocyte produc-tion in the dorsal stripe (Camargo-Sosa et al. 2019), eventhough pharmacological or genetic activation of gabrr1 sig-naling appears to reduce pigmentation in a larvae-wide man-ner. These studies support the idea that distinct geneticpathways maintain MSC quiescence in a region-specific man-ner throughout zebrafish development. Clearly, additionalwork is needed to determine whether other pathways actwith gabbr1 and ednraa to promote MSC quiescence, butour work hints that GABA-A-mediated quiescence may be ahallmark of vertebrate pigment biology.

Bioelectric regulation of MSC quiescence andproliferation

GABA receptors function as ligand-gated channels that regu-late membrane potential, suggesting that changes in mem-brane potential trigger the observed changes in melanocytepatterning, and MSC quiescence and proliferation. Inhibitionof gabbr1 function, which should depolarize the cell, inducedmelanocyte production through anMSC intermediate. In thiscontext, we note that prior work observed severe hyperpig-mentation in X. laevis larvae due to melanocyte overprolifer-ation and overproduction via pharmacological depolarizationof glycine-gated chloride channels (Blackiston et al. 2011). Inaddition, the application of GABA and GABA-A agonists,which hyperpolarize cells by promoting Cl2 influx, inhibitsproliferation of embryonic stem cells and peripheral neuralcrest stem cells in mice (Young and Bordey 2009; Teng et al.2013). Moreover, in the mouse neocortex, neural progenitorsbecome increasingly hyperpolarized as they produce theircharacteristic cell lineages (Vitali et al. 2018). Of note, arti-ficial hyperpolarization of neural progenitor cells induced thepremature production of late-stage cell types, revealing afunctional link between changes in membrane potentialand the temporal birth order of cells in the neocortex.Changes in the membrane potential of stem and progenitorcells can then alter cell division patterns and cellular behav-ior, supporting the idea that gabrr1-mediated regulation ofmembrane potential underlies its role in regulating early me-lanocyte patterning in zebrafish. Future work that can sys-tematically assess the effects of membrane potential on thedevelopment of stem cells and their progeny, in a broad rangeof tissues, is required to reveal the extent to which this phe-nomenon occurs in vertebrate biology.

Acknowledgments

We thank Brian Stephens and Sinan Li for fish husbandryduring the majority of the study; the Washington UniversitySchool of Medicine Genetics Department and WashingtonUniversity Zebrafish Facility for providing critical support incompleting this research; Rob Tryon and Ryan McAdow, forassistance and guidance in generating mutant and trans-genic lines; and Michael Nonet, Cristina Strong, CharlesKaufman, and Douglas Chalker for critical comments on themanuscript. J.R.A. was a Howard Hughes Medical Institute

Gilliam Fellow during the course of this study. This workwas funded by National Institutes of Health (NIH) grantRO1 GM-056988 to S.L.J. J.B.S. was supported by NIH grantRO1 NS-036570. The authors declare no competing finan-cial interests.

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Communicating editor: B. Draper

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