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. Author manuscript; available in PMC: 2021 May 1.
Published in final edited form as: Pigment Cell Melanoma Res. 2019 Nov 11;33(3):416–425. doi: 10.1111/pcmr.12836

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

James R Allen 1,*, James B Skeath 1, Stephen L Johnson 1,†
PMCID: PMC7176537  NIHMSID: NIHMS1056262  PMID: 31642595

Abstract

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

Keywords: Melanocyte, zebrafish, TSPO, GABA-A, stem cell

Introduction:

Many adult tissues depend on undifferentiated precursors, adult stem cells (ASCs), to maintain tissue homeostasis and to regenerate damaged tissue. ASCs are found in many tissues, such as blood (Bertrand et al., 2007) and skin (Nishimura et al., 2002), and are often capable of long-term self-renewal to maintain their regenerative properties. In some tissues, ASCs continuously proliferate so that progenitors cells are available for high tissue turnover. In other tissues, ASCs remain quiescent until recruited to proliferate (L. Li & Bhatia, 2011). Depletion of cells within the target tissue often triggers ASC recruitment. Currently, the pathways that regulate the transition between ASC quiescence and recruitment are unclear.

Vertebrate pigmentation is an excellent system to investigate the molecular basis of ASC quiescence and recruitment. In zebrafish, melanocytes comprise most of the pigment pattern of adult and larval fish and regenerate from undifferentiated precursors (Johnson, Africa, Walker, & Weston, 1995; Rawls & Johnson, 2000), which we term melanocyte stem cells (MSCs). In zebrafish, MSCs mostly contribute to the adult pigment pattern, rather than larval pigmentation. The initial embryonic pigment pattern develops from direct-developing neural crest cells fated towards melanocyte differentiation and is complete by 3 days post fertilization (dpf) (Hultman & Johnson, 2010). Under normal development, few new melanocytes develop within larval zebrafish between 3 dpf and the onset of metamorphosis at 15 dpf. Ablation of embryonic melanocytes, however, triggers complete regeneration of the larval pigment pattern through recruitment and proliferation of quiescent MSCs (Yang & Johnson, 2006). Thus, MSCs are maintained in a quiescent state during larval development, but the pathways that normally maintain MSC quiescence remain unclear. Genetic studies indicate that adult and regenerating, but not direct-developing, melanocytes require erbb3b signaling, suggesting a critical role for this receptor tyrosine kinase within MSCs and demonstrating that genetically distinct melanocyte populations form the zebrafish pigment pattern (Budi, Patterson, & Parichy, 2008; Dooley, Mongera, Walderich, & Nusslein-Volhard, 2013; Hultman et al., 2009).

Previously, we reported pharmacological and genetic evidence that gabrr1 signaling regulates MSC quiescence in larval zebrafish (Allen, Skeath, & Johnson, 2019). GABA-A receptors signal through multiple pathways, although increased intracellular calcium is a major effector of GABA-A signaling (Aguayo, Espinoza, Kunos, & Satin, 1998). Increased intracellular calcium activates calcineurin, a calcium-dependent protein phosphatase. Calcineurin then activates calmodulin, an ubiquitously expressed calcium-binding messenger protein that mediates calcium signal transduction in all eukaryotic cells (Chin & Means, 2000). Although calcium sensing often acts downstream of GABA-A receptors, whether calcium signaling maintains MSC quiescence is unknown.

Translocator protein (TSPO) is a mitochondrial protein that controls mitochondrial function. Recent work has explored TSPO as a regulator of metabolic homeostasis (F. Li, Liu, Garavito, & Ferguson-Miller, 2015). TSPO ligands were identified as glucose-lowering agents in a screen for modulators of hepatic gluconeogenesis in larval zebrafish (Gut et al., 2013). Most animals, including zebrafish, initiate gluconeogenesis to increase available glucose reserves when yolk-derived carbohydrates are depleted early in development. Application of TSPO ligands to zebrafish larvae reduced whole-body glucose and increased pck1 expression in the liver, a known fasting response gene and inducer of gluconeogenesis (Hatting, Tavares, Sharabi, Rines, & Puigserver, 2018). Rather than directly activating gluconeogenesis, TSPO was proposed to trigger a metabolic fasting response that delayed replenishment of carbohydrates from compensatory gluconeogenic activity. This metabolic fasting also activated transcriptional changes beyond gluconeogenesis genes and modulated other fasting adaptation pathways, such as β-oxidation and cellular respiration, which drive ATP production in the mitochondria during low-energy states (Houten, Violante, Ventura, & Wanders, 2016). A role for TSPO in regulating pigment patterning has not been uncovered, although changes in mitochondrial function and metabolic regulation have been noted to disrupt pigmentation in vertebrates (D’Agati et al., 2017).

Here, we show that GABA-A receptor signaling acts through calcineurin and calcium signaling to maintain MSC quiescence. We also uncover a link between metabolic regulation and pigment patterning, as TSPO-ligands and induction of gluconeogenesis appear to act in the same pathway to drive excess melanocyte production. To regulate melanocyte production, TSPO and gluconeogenesis appear to act in parallel to GABA-A signaling, suggesting multiple pathways converge to regulate MSC quiescence.

Materials and Methods:

Zebrafish stocks and husbandry:

Adult fish were reared at 14L:10D light-to-dark cycle. All experiments were performed with mlphaj120 larvae heterozygous for the transgene Tg(fTyrp1:GFP)j900, which expresses GFP in differentiated melanocytes. Clutches of mlphaj120; Tg(fTyrp1:GFP)j900/+ embryos were generated via in vitro fertilization and maintained at 25°C during the course of each experiment. All experiments were performed in accordance with animal protocols approved by the Animal Studies Committee of Washington University.

Pharmacological reagents and drug screening:

Drugs used in this study were purchased from commercial vendors (Table S1). A concentrated stock solution, 20mM if possible, was created for each compound in DMSO or water and stored at −80°C. For each condition, 10–12 embryos were placed into 24-well plates with ~2 ml Egg water (60 mg/L Instant Ocean: 0.06 ppt) containing 200 µM phenylthiocarbamide (PTU), which blocks melanin synthesis. The stock solution was diluted and added to 2 ml Egg water to the indicated concentration in a final vehicle of 0.5% DMSO or water. AG1478 was maintained as a 20mM stock (DMSO), and used at 3 µM from 8 – 48 hours post fertilization (hpf). Experiments were performed with parallel duplicates for each drug treatment.

Melanocyte quantification:

We quantified melanocyte production within the larval dorsal stripe. We analyzed PTU-treated mlpha j120; Tg(fTyrp1:GFP)j900/+, where all melanocytes express GFP, but newly formed melanocytes lack pigment. We quantified mel−, GFP+ melanocytes along the dorsum from immediately caudal to the otic vesicle to the posterior edge of the caudal fin (Figure 1B). Following drug treatments, larvae with severe morphological defects were excluded from analysis.

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

Figure 1:

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

Statistical Analysis:

We used single factor ANOVA (α: 0.001 unless otherwise noted) to compare the means of each experimental group. We then performed Tukey’s HSD post hoc tests to determine significant differences between treatment groups within each experiment. All statistical tests and reported p-values were calculated using Microsoft Excel.

Results:

GABA-A antagonists and TSPO ligands increase melanocyte production

We previously found that disruption of GABA-A receptor function triggers new melanocyte production in larval zebrafish, likely by activating the normally quiescent MSC (Allen et al., 2019). To extend our pharmacological interrogation of GABA-A receptor signaling and larval melanocyte regeneration, we conducted a systematic analysis of the effects of GABA receptor antagonists, agonists, re-uptake inhibitors, indirect agonists, and positive allosteric modulators on melanocyte production (Olsen, 2014). We also obtained drugs that manipulate the structurally distinct metabotropic GABA-B receptor (Table S1) (Bormann, 2000). To assess the effect of each compound on melanocyte regeneration, we generated embryos expressing the melanocyte marker Tg(fTyrp1:GFP)j900, added the melanin-inhibiting drug PTU at 3 dpf, and quantified new melanocytes as those lacking melanin pigment and expressing GFP (mel−, GFP+ melanocytes). In this design, new melanocytes form but lack pigment due to PTU treatment, thus allowing identification of melanocytes that form in the dorsal stripe (Figure 1B) within a specific temporal window (Figure 1A). In total, we tested the effects of 32 compounds on larval melanocyte production. Larval zebrafish treated with vehicle control had on average 1.2 new melanocytes in the dorsal stripe (Figure 1C; 1G). Validating our prior results, we found that GABA-A antagonists produced between 3-to-4 new melanocytes (Figure 1G). Most GABA-A receptor agonists and compounds expected to increase GABA-A receptor function, such as barbiturates, or GABA concentration, such as GABA uptake inhibitors, did not increase melanocyte production. Larvae treated with diazepam (10 µM), a benzodiazepine expected to increase GABA-A receptor function, however, increased melanocyte production (4.1±1.9 new melanocytes; Table S1). This was unexpected, as we had previously shown that reduction of GABA-A function increased melanocyte production and over-expression of a GABA-A rho receptor inhibited melanocyte regeneration (Allen et al., 2019).

The diazepam-induced melanocyte production presented two possibilities: 1) inhibition and enhancement of GABA-A receptor activity were equally sufficient to increase melanocyte production; 2) Diazepam treatment activated a non-GABA pathway to increase melanocyte production. As our prior study indicated that GABA-A receptor inhibition produced new melanocytes, we tested whether diazepam acts through a non-GABA pathway to induce melanocyte production. Diazepam also binds the mitochondrial associated translocator protein (TSPO; also called the peripheral benzodiazepine receptor) (Papadopoulos et al., 2006). Several synthetic TSPO ligands have been identified that are structurally distinct from diazepam and do not bind GABA-A receptors, e.g., PK-11195 and Ro5–4864 (Rupprecht et al., 2010). When we treated zebrafish with TSPO ligands PK-11195, Ro5–4864, or FGIN-143, we observed a clear increase in new melanocytes for all three drugs (Figure 1G). In addition, treatment of zebrafish with a 4-fold higher dose of diazepam (40 µM) doubled the number of new melanocytes (Figure 1D; 1F). Our results indicated that diazepam yielded a stronger effect than any of the synthetic TSPO ligands we tested in our assay. This was surprising as diazepam is expected to bind TSPO and activate GABA-A receptors, two molecular pathways which this and our prior study suggest should exhibit opposing effects on larval pigmentation. Our interpretation is that our diazepam likely binds and activates TSPO with greater affinity than the synthetic TSPO ligands at the concentrations used in our assay. Therefore, we infer that pharmacological modulation of TSPO function increases melanocyte production and that the effect of diazepam on melanocyte number is mediated through TSPO, but not GABA-A receptor function.

GABA-A antagonists and TSPO ligands require MSCs to increase melanocyte production

We next tested whether TSPO-ligand induced melanocytes originated from MSCs. Our previous studies suggested that melanocytes after 3 dpf develop from MSCs (Hultman & Johnson, 2010). However, our drug treatments could activate melanocyte development from a non-stem cell source, such as delayed melanoblasts. To test this possibility, we treated larval zebrafish with the small molecule AG1478 between 8 to 48 hpf. AG1478 inhibits all ErbB signaling, including erbb3b signaling, which blocks establishment of the MSC compartment. We then treated the larvae with PTU, to block melanin synthesis, and either the TSPO ligands Ro5–4864 and PK-11195 or the GABA-A antagonist TPMPA and quantified new melanocyte production (Figure 2A). Larvae treated with vehicle control and DMSO or AG1478 developed 1.9 or 0.4 new melanocytes respectively (Figure 2B). Larvae treated with Ro5–4864 and PK-11195 produced 6.4 and 6.3 new melanocytes in the presence of DMSO, and 0.7 or 0.3 new melanocytes with AG1478. Confirming our prior work (Allen et al., 2019), larvae treated with TPMPA and DMSO developed 5.2 new melanocytes, and 0.4 new melanocytes with AG1478. We infer that GABA-A antagonists and TSPO ligands trigger MSCs to produce melanocytes.

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

Figure 2:

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

GABA competes with GABA-A antagonists, but not TSPO ligands

We next tested whether GABA-A antagonists and TSPO ligands interact with distinct molecular targets. We reasoned that high concentrations of the endogenous ligand of each respective pathway would competitively inhibit melanocyte production. While some endogenous TSPO ligands, such as cholesterol and protoporphyrin IX are known, their exact mode of action on TSPO is unclear and their effects across biological systems is nonuniform (Veenman & Gavish, 2012). Therefore, we tested the competitive effect of GABA on both GABA-A antagonists and synthetic TSPO-ligands. We treated larvae with GABA-A antagonists or TSPO ligands in the presence or absence of 50 mM GABA (Figure 3A). Control larvae treated with vehicle or GABA developed 1.6 and 1.5 new melanocytes on average per larvae, respectively (Figure 3B). Larvae treated with the GABA-A antagonists TPMPA or Picrotoxin exhibited 4.4 and 4.7 new melanocytes respectively in the absence of GABA, but only 1.4 and 1.3 new melanocytes respectively in the presence of GABA. In contrast, larvae treated with the TSPO ligands Ro5–4864 or PK-11195 exhibited 6.1 and 5.8 new melanocytes respectively in the absence of GABA, and 6.3 and 6.0 new melanocytes in the presence of GABA. We infer that GABA out competes GABA-A antagonists for binding to the GABA-A receptor, but has no measurable effect on new melanocyte production triggered by TSPO ligands, indicating GABA-A antagonists and TSPO ligands target distinct molecules in one pathway or act in parallel to increase melanocyte production.

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

Figure 3:

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

GABA-A antagonists and TSPO ligands act additively to regulate melanocyte production

If GABA-A antagonists and TSPO ligand act in parallel to drive melanocyte production, co-application of both drugs should exhibit an additive effect on melanocyte production. We performed a series of dose responses to determine the ideal concentrations to yield the near saturated biological effects of each compound on melanocyte differentiation (Figure S1–4). Larvae treated with the GABA-A antagonists TPMPA or Picrotoxin produced 4.9 and 5.5 new melanocytes, respectively, and larvae treated with both TPMPA and Picrotoxin produced 6.4 new melanocytes, suggesting saturation of the GABA-A mediated melanocyte production phenotype. Larvae treated with the TSPO ligands Ro5–4864 or PK-11195 produced 5.1 and 6.7 new melanocytes respectively, and larvae treated with both Ro5–4864 and PK-11195 produced 6.6 new melanocytes, suggesting saturation of the effect of TSPO ligands on melanocyte production. Larvae treated with TPMPA and Ro5–4864 produced 9.7 new melanocytes, a significantly greater effect than that observed with either individual treatment (Figure 4). We infer that GABA-A antagonists and TSPO ligands produce an additive effect on melanocyte development, suggesting these compounds act in parallel to increase melanocyte production.

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

Figure 4:

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

Inhibition of calcium mobilization and calcium sensors increases melanocyte production

As GABA-A receptors often transduce their signal by increasing intracellular calcium (Bormann, 2000), we tested whether inhibition of calcium receptors or cellular calcium sensing via calcineurin inhibition increased melanocyte production. We used the calcium entry and voltage-gated calcium receptor blocker SKF-96365 and Calcineurin inhibitors Cyclosporin-A and FK506 to determine if inhibition of calcium signaling increased melanocyte production. Larvae treated with SKF-96365 produced approximately 4.2 new melanocytes compared to 1.7 new melanocytes with vehicle control, a response similar to the GABA-A antagonist TPMPA (Figure 5). Larvae treated with Cyclosporin-A or FK506 produced 10.4 and 11.3 new melanocytes respectively. We conclude that pharmacological inhibition of calcium signaling increases melanocyte output, suggesting GABA acts via calcium transduction to maintain MSC quiescence.

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

Figure 5:

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

Pharmacological activation of gluconeogenesis increases melanocyte production

TSPO ligands have been shown to promote gluconeogenesis in larval zebrafish (Gut et al., 2013); our data show they also increase melanocyte production. Gluconeogenesis is a metabolic pathway that generates glucose from non-dietary sources to maintain optimal blood glucose levels (Hers & Hue, 1983). Therefore, we tested whether gluconeogenesis drives increased melanocyte production by obtaining compounds that induce or inhibit gluconeogenesis. First, we tested the anti-diabetic drug metformin, shown to suppress gluconeogenesis and lower blood sugar. Larvae treated with vehicle control or metformin produced 1.6 and 1.9 new melanocytes respectively, indicating suppression of gluconeogenesis has no detectable effect on pigmentation. Next, we tested the β-adrenergic agonist isoprenaline, shown to activate gluconeogenesis and raise blood sugar (Gut et al., 2013; Jones & Ritchie, 1978). Larvae treated with isoprenaline produced 7.5 new melanocytes (Figure 6), a significant increase over vehicle control, indicating activation of gluconeogenesis drives melanocyte production.

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

Figure 6:

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

GABA-A antagonists and TSPO ligands are differentially sensitive to kita dosage

We next sought genetic evidence to determine if GABA antagonists and TSPO ligands act through similar or distinct MSC pools. The kit receptor tyrosine kinase plays several roles within MSCs in zebrafish (Hultman, Bahary, Zon, & Johnson, 2007; Parichy, Rawls, Pratt, Whitfield, & Johnson, 1999; Rawls & Johnson, 2000, 2001). While its exact function remains unclear, melanocyte survival, migration, and regeneration all depend on kita function. Larval melanocytes are completely lost in a null kita allele, kitab5, indicating kita function is necessary for melanocyte survival (Parichy et al., 1999). Zebrafish heterozygous for kitab5 exhibit normal larval pigmentation, but when challenged, melanocyte regeneration is reduced by ~50% (O’Reilly-Pol & Johnson, 2013), indicating MSCs require full kita signaling to completely regenerate the larval pigment pattern. MSC lineages within larval zebrafish can then be broadly separated into kita-sensitive or kita-insensitive classes.

We tested whether the effects of GABA-A antagonists or TSPO ligands on melanocyte production were sensitive to kita dosage. We generated clutches of kitab5/+ heterozygotes and treated with each compound and PTU at 3 dpf. kitab5/+ larvae treated with vehicle control had 0.3 excess melanocytes, indicating a low background of melanocyte production in kita heterozygotes compared to wild-type (Figure 1). kitab5/+ larvae treated with the GABA-A antagonists TPMPA, the calcium receptor antagonist SKF-96365, or the calcineurin inhibitors Cyclosporin-A and FK506 exhibited 0.6, 2.4, 3.9, and 4.3 excess melanocytes respectively (Figure 7), a three-to-eight fold decrease in melanocyte production relative to wildtype. This indicates that the excess melanocytes produced by these drugs are sensitive to kita dosage. In contrast, kitab5/+ larvae treated with TSPO ligands Ro5–4864 and PK-11195, or the gluconeogenesis drug isoprenaline exhibited 4.4, 3.4, and 6.7 excess melanocytes respectively (Figure 7), a 20–30% decrease in melanocyte production relative to the effect of each drug treatment in wildtype. Thus, TSPO ligands and gluconeogenesis drugs are less sensitive to kita dosage than GABA-A antagonists or calcium signaling inhibitors, suggesting GABA signaling and TSPO ligands/gluconeogenesis exert distinct effects on kita-sensitive or kita-insensitive pools of MSCs.

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

Figure 7:

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Quantification of average melanin−, GFP+ dorsal melanocytes / ROI in each group of 6 dpf heterozygous kitab5/+ larvae. Data summarized in Table S8. Each experimental group was compared to vehicle control. *** indicate a statistical difference in our analysis (Tukey-HSD; p<0.001).

GABA and calcium signaling act in parallel to TSPO and gluconeogenesis to regulate MSC quiescence

Our results suggest GABA-A receptors and calcium signaling act in parallel to TSPO and gluconeogenesis to maintain MSC quiescence. If this model is correct, TSPO ligands, but not GABA-A antagonists, should enhance the melanocyte phenotype of calcium receptor and sensing inhibitors. Similarly, GABA-A antagonists and Calcineurin inhibitors, but not synthetic TSPO ligands, should enhance the melanocyte phenotype of the gluconeogenesis drug isoprenaline. We performed a series of dose responses to determine the ideal concentrations to yield the near saturated biological effects of each compound in our assays (Figure S5–9). As shown in Figure 8 and Table S9, the TSPO ligand Ro5–4864, but not the GABA-A antagonist TPMPA, enhances the increased melanocyte phenotype observed with calcium inhibitors. In addition, the GABA-A antagonist TPMPA, but not the TSPO ligand Ro5–4864, enhances the increased melanocyte phenotype observed with isoprenaline and is not inhibited by Metformin. Interestingly, co-treatment with the TSPO ligand Ro5–4864 and metformin reduced melanocyte production compared to Ro5–4864 (p<0.01; *), suggesting that gluconeogenesis pathways act downstream of TSPO signaling. However, we also found that co-treatment of TPMPA and metformin increased melanocyte production relative to TPMPA alone (p<0.01; *), suggesting that metformin potentiates the effect of the GABA-A antagonist. This was an unexpected result that hints at potential crosstalk between GABA and TSPO downstream. Overall, however, our data support the model that GABA-A and calcium signaling act in parallel to TSPO function and gluconeogenesis to maintain MSC quiescence in larval zebrafish.

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

Figure 8:

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

Discussion:

We find that GABA-A receptor signaling acts through calcium signaling to maintain MSC dormancy. We also show that the mitochondrial protein TSPO and gluconeogenesis act in parallel to GABA-A signaling to maintain MSC dormancy. Below, we propose a model wherein multiple molecular pathways maintain MSC quiescence in larval zebrafish, briefly discuss the mechanisms through which each newly identified pathway could act to regulate MSC activity, and speculate on why starvation might induce pigmentation.

Independent pathways maintain MSC quiescence

Our findings suggest that zebrafish pigmentation depends on the integration of distinct inputs that converge on MSCs. Here, MSCs likely interpret both local and systemic cues. We speculate that GABAergic signaling functions as a local cue, providing a link between differentiated melanocytes and their MSC precursors. Several studies indicate that melanocytes may synthesize and release GABA (Ganesan et al., 2008; Ito, Tanaka, Nishibe, Hasegawa, & Ueno, 2007). GABA-A signaling from melanocytes to MSCs may act as a negative feedback loop, repressing MSC proliferation in the presence of melanocytes and releasing MSC from this repression when melanocytes are depleted. Although we favor the model that melanocytes signal directly to MSCs via GABA/GABA receptor signaling, our data do not exclude that GABA/GABA receptor signaling act indirectly to regulate MSC activity. For example, MSCs associate with the peripheral nervous system, and GABA and GABA receptors are abundant in neurons and glia. Thus, it is possible that a GABA/GABA receptor relay acts in neurons, glia, and/or other associated cell types to help transduce a signal from melanocytes to MSCs. In contrast to GABA, which we expect acts locally to control MSC quiescence, we speculate that the mitochondrial protein TSPO and gluconeogenesis act as systemic cues to link nutritional status to MSC activation or quiescence and pigmentation.

Our work suggests GABA-A signaling and the nutritional state act in parallel to regulate MSC quiescence, with our kita dosage experiments suggesting GABA-A signaling and the nutritional state exert distinct effects on kita-sensitive and insensitive pools of MSCs. Even as our data suggests these molecular pathways function independently, our data also hints that early pigmentation might involve crosstalk between these pathways. For example, our finding that metformin, which itself had little effect on pigmentation, demonstrates opposing effects on melanocyte production when combined with GABA or TSPO pathway compounds, suggests that melanocyte development must integrate both local and systemic signals during pigmentation. In addition to local and systemic cues, MSCs also appear to respond to spatial cues, as recent work indicates the ednraa RTK pathway regulates MSC quiescence specifically in the ventral trunk of larval zebrafish (Camargo-Sosa et al., 2019). In the future, it will be interesting to determine how GABA and TSPO function integrate with ednraa activity to regulate MSC quiescence and activation.

Conserved from bacteria to humans, TSPO is a nuclear-encoded outer-mitochondrial membrane protein. TSPO has been implicated in many biological processes, e.g., neuroinflammation, cell fate, oxidation stress response, cholesterol transport. Despite its clear biological importance, TSPO’s exact molecular function and mechanisms of action remain unclear (Bonsack & Sukumari-Ramesh, 2018). Thus, the precise physiological effects of TSPO and the effects its ligands have on it are unknown. The ligands we used bind the protein with great affinity (Rupprecht et al., 2010), but whether they inhibit or activate TSPO activity remains unknown. TSPO pharmacology then identifies a biological process - mitochondrial function - that appears to act in the same pathway as gluconeogenesis to maintain MSC quiescence. Future work is required to discern the function of TSPO in the mitochondria, the impact of TSPO ligands on this activity, and how TSPO function integrates with metabolism to control melanocyte production.

Why does starvation promote excess pigmentation?

Our work indicates that induction of gluconeogenesis and starvation-like states induces excess pigmentation during zebrafish development. Why would starvation increase pigmentation? Currently, we do not know the answer to this question, but an intriguing, yet purely speculative, model is that increased melanocyte production in times of low food availability provides a selective advantage to zebrafish in terms of enhanced camouflage or other property, e.g. thermoregulation, that promotes survival. Testing this, and other, models, will help clarify whether the link between starvation and pigmentation observed in this paper represents an evolutionary adaptive response or not, and if so, the exact mechanism through which it provides a selective advantage.

Supplementary Material

Supp TableS1-9
Supp figS1-9
Supp legends

Significance:

Adult stem cells (ASCs) underlie the regenerative potential of many tissues and organs in vertebrates, and dysregulation of ASC function can lead to cancer. Understanding how ASCs alternate between periods of quiescence and proliferation to regenerate lost tissue or maintain tissue homeostasis is poorly understood. Our work indicates the mitochondrial translocator protein acts in the same pathway as gluconeogenesis to promote ASC quiescence in the melanocyte lineage. This new pathway appears to link nutritional or metabolic status to melanocyte pigmentation in zebrafish, with the potential for this link to be an evolutionarily adaptive response.

Acknowledgements:

We thank the Washington University Genetics Department and Zebrafish Facility for critical support in this research. We thank Dr. Rob Tryon and Jonathan Spalding for critical input on experimental design. We thank Dr. Joe Henry Steinbach for generously sharing samples of pentobarbital, phenobarbital, diazepam, and allopregnanolone. We thank Dr. Charles Kaufman for critical review of the manuscript. J.R.A was funded by a HHMI Gilliam Fellowship. This work was funded by NIH RO1-GM056988 to S.L.J. J.B.S was supported by NIH RO1-NS036570.

Footnotes

Competing Interests:

The authors declare no competing interests.

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Supp TableS1-9
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