Defects in ErbB-Dependent Establishment of Adult Melanocyte Stem Cells Reveal Independent Origins for Embryonic and Regeneration Melanocytes

Adult stem cells are responsible for maintaining and repairing tissues during the life of an organism. Tissue repair in humans, however, is limited compared to the regenerative capabilities of other vertebrates, such as the zebrafish (Danio rerio). An understanding of stem cell mechanisms, such as how they are established, their self-renewal properties, and their recruitment to produce new cells is therefore important for the application of regenerative medicine. We use larval melanocyte regeneration following treatment with the melanocytotoxic drug MoTP to investigate these mechanisms in Melanocyte Stem Cell (MSC) regulation. In this paper, we show that the receptor tyrosine kinase, erbb3b, is required for establishing the adult MSC responsible for regenerating the larval melanocyte population. Both the erbb3b mutant and wild-type fish treated with the ErbB inhibitor, AG1478, develop normal embryonic melanocytes but fail to regenerate melanocytes after MoTP-induced melanocyte ablation. By administering AG1478 at different time points, we show that ErbB signaling is only required for regeneration prior to MoTP treatment and before 48 hours of development, consistent with a role in establishing MSCs. We then show that overexpression of kitla, the Kit ligand, in transgenic larvae leads to recruitment of MSCs, resulting in overproliferation of melanocytes. Furthermore, kitla overexpression can rescue AG1478-blocked regeneration, suggesting that ErbB signaling is required to promote the progression and specification of the MSC from a pre–MSC state. This study provides evidence that ErbB signaling is required for the establishment of adult MSCs during embryonic development. That this requirement is not shared with the embryonic melanocytes suggests that embryonic melanocytes develop directly, without proceeding through the ErbB-dependent MSC. Moreover, the shared requirement of larval melanocyte regeneration and metamorphic melanocytes that develops at the larval-to-adult transition suggests that these post-embryonic melanocytes develop from the same adult MSC population. Lastly, that kitla overexpression can recruit the MSC to develop excess melanocytes raises the possibility that Kit signaling may be involved in MSC recruitment during regeneration.     

Clonal analyses reveal roles of organ founding stem cells, melanocyte stem cells and melanoblasts in establishment, growth and regeneration of the adult zebrafish fin

In vertebrates, the adult form emerges from the embryo by mobilization of precursors or adult stem cells. What different cell types these precursors give rise to, how many precursors establish the tissue or organ, and how they divide to establish and maintain the adult form remain largely unknown. We use the pigment pattern of the adult zebrafish fin, with a variety of clonal and lineage analyses, to address these issues. Early embryonic labeling with lineage-marker-bearing transposons shows that all classes of fin melanocytes (ontogenetic, regeneration and kit-independent melanocytes) and xanthophores arise from the same melanocyte-producing founding stem cells (mFSCs), whereas iridophores arise from distinct precursors. Additionally, these experiments show that, on average, six and nine mFSCs colonize the caudal and anal fin primordia, and daughters of different mFSCs always intercalate to form the adult pattern. Labeled clones are arrayed along the proximal-distal axis of the fin, and melanocyte time-of-differentiation lineage assays show that although most of the pigment pattern growth is at the distal edge of the fin, significant growth also occurs proximally. This suggests that leading edge melanocyte stem cells (MSCs) divide both asymmetrically to generate new melanocytes, and symmetrically to expand the MSCs and leave quiescent MSCs in their wake. Clonal labeling in adult stages confirms this and reveals different contributions of MSCs and transient melanoblasts during growth. These analyses build a comprehensive picture for how MSCs are established and grow to form the pigment stripes of the adult zebrafish fins.     

Melanocyte regeneration reveals mechanisms of adult stem cell regulation

Utilization of adult stem cells in regenerative therapies may require a thorough understanding of the mechanisms that establish, recruit and renew the stem cell, promote the differentiation of its daughters, or how the stem cell is repressed by its target tissue. Regeneration of melanocytes in the regenerating zebrafish caudal fin, or following larval melanocyte-specific ablation, or recruitment of new melanocytes during pigment pattern metamorphosis each provides evidence for melanocyte stem cells (MSCs) that support the melanocyte pigment pattern. We discuss the mechanisms of MSC regulation provided from analysis of normal or mutant regeneration in each of these systems, including the implications drawn from evidence that regeneration does not simply recapitulate ontogenetic development. These results suggest that analysis of melanocyte regeneration in zebrafish will provide a fine scale dissection of mechanisms establishing or regulating adult stem cells.     

Differential contribution of direct-developing and stem cell-derived melanocytes to the zebrafish larval pigment pattern

The extent of adult stem cell involvement in embryonic growth is often unclear, as reliable markers or assays for whether a cell is derived from an adult stem cell, such as the melanocyte stem cell (MSC), are typically not available. We have previously shown that two lineages of melanocytes can contribute to the larval zebrafish pigment pattern. The embryo first develops an ontogenetic pattern that is largely composed of ErbB-independent, direct-developing melanocytes. This population can be replaced during regeneration by an ErbB-dependent MSC-derived population following melanocyte ablation. In this study, we developed a melanocyte differentiation assay used together with drugs that ablate the MSC to investigate whether MSC-derived melanocytes contribute to the ontogenetic pattern. We found that essentially all melanocytes that develop before 3 dpf arise from the ErbB-independent, direct-developing population. Similarly, late-developing (after 3 dpf) melanocytes of the head are also ErbB independent. In contrast, the melanocytes that develop after 3 days postfertilization in the lateral and dorsal stripe are sensitive to ErbB inhibitor, indicating that they are derived from the MSC. We show that melanocyte regeneration mutants kit^j1e99 and skiv2l2^j24e1 that are grossly normal for the overall ontogenetic pattern also lack the MSC-derived contribution to the lateral stripe. This result suggests that the underlying regeneration defect of these mutations is a defect in MSC regulation. We suggest that the regulative functions of the MSC may serve quality control roles during larval development, in addition to its established roles in larval regeneration and growth and homeostasis in the adult.     

Requirements for the kit receptor tyrosine kinase during regeneration of zebrafish fin melanocytes

Embryonic neural crest-derived melanocytes and their precursors express the kit receptor tyrosine kinase and require its function for their migration and survival. However, mutations in kit also cause deficits in melanocytes that make up adult pigment patterns, including melanocytes that re-establish the zebrafish fin stripes during regeneration. As adult melanocytes in mice and zebrafish are generated and maintained by stem cell populations that are presumably established during embryonic development, it has been proposed that adult phenotypes in kit mutants result from embryonic requirements for kit. We have used a temperature-sensitive zebrafish kit mutation to show that kit is required during adult fin regeneration to promote melanocyte differentiation, rather than during embryonic stages to establish their stem cell precursors. We also demonstrate a transient role for kit in promoting the survival of newly differentiated regeneration melanocytes.     

Small molecule-induced ablation and subsequent regeneration of larval zebrafish melanocytes

We developed a method to efficiently ablate a single cell type, the zebrafish melanocyte, and study the mechanisms of its regeneration. We found that a small molecule, (2-morpholinobutyl)-4-thiophenol (MoTP), specifically ablates zebrafish larval melanocytes or melanoblasts, and that this melanocytotoxicity is dependent on tyrosinase activity, which presumably converts MoTP to cytotoxic quinone species. Following melanocyte ablation by MoTP treatment, we demonstrate by BrdU incorporation experiments that regenerated melanocytes are derived from the division of otherwise quiescent melanocyte precursors or stem cells. We further show that larval melanocyte regeneration requires the kit receptor tyrosine kinase. Our results suggest that a small number of melanocyte precursors or stem cells unevenly distributed in larvae are drawn upon to reconstitute the larval melanocyte population following melanocyte ablation by MoTP.     

Zebrafish kit mutation reveals primary and secondary regulation of melanocyte development during fin stripe regeneration

Fin regeneration in adult zebrafish is accompanied by re-establishment of the pigment stripes. To understand the mechanisms underlying fin stripe regeneration and regulation of normal melanocyte stripe morphology, we investigated the origins of melanocytes in the regenerating fin and their requirement for the kit receptor tyrosine kinase. Using pre-existing melanin as a lineage tracer, we show that most fin regeneration melanocytes develop from undifferentiated precursors, rather than from differentiated melanocytes. Mutational analysis reveals two distinct classes of regeneration melanocytes. First, an early regeneration class develops dependent on kit function. In the absence of kit function and kit-dependent melanocytes, a second class of melanocytes develops at later stages of regeneration. This late kit-independent class of regeneration melanocytes has little or no role in wild-type fin stripe development, thus revealing a secondary mode for regulation of fin stripes. Expression of melanocyte markers in regenerating kit mutant fins suggests that kit normally acts after mitf and before dct to promote development of the primary kit-dependent melanocytes. kit-dependent and kit-independent melanocytes are also present during fin stripe ontogeny in patterns similar to those observed during regeneration.     

Fate restriction in the growing and regenerating zebrafish fin

We use transposon-based clonal analysis to identify the lineage classes that make the adult zebrafish caudal fin. We identify nine distinct lineage classes, including epidermis, melanocyte/xanthophore, iridophore, intraray glia, lateral line, osteoblast, dermal fibroblast, vascular endothelium, and resident blood. These lineage classes argue for distinct progenitors, or organ founding stem cells (FSCs), for each lineage, which retain fate restriction throughout growth of the fin. Thus, distinct FSCs exist for the four neuroectoderm lineages, and dermal fibroblasts are not progenitors for fin ray osteoblasts; however, artery and vein cells derive from a shared lineage in the fin. Transdifferentiation of cells or lineages in the regeneration blastema is often postulated. However, our studies of single progenitors or FSCs reveal no transfating or transdifferentiation between these lineages in the regenerating fin. This result shows that, the same as in growth, lineages retain fate restriction when passed through the regeneration blastema.     

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

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

Quantitative control of end products in the melanocyte lineage of the ascidian embryo.

Terminal amounts of tyrosinase (EC 1.10.3.1) activity and melanin pigment in the giant melanocytes of cleavage-arrestedCiona intestinalis (L.) embryos are regulated independently of cell size and number of nuclei in the cells. Embryos were cleavage-arrested in cytochalasin B at a time before the last two divisions of the melanocyte lineage took place. The resulting two giant melanocytes, one from each of the two bilateral melanocyte lineages, developed tyrosinase and melanin. The cells were about three times larger in volume than the normal larval melanocytes and each contained four nuclei instead of just one. Quantitative measurements of melanin synthesized and tyrosinase activity in embryos with the giant melanocytes revealed amounts identical to those found in normal embryos. This specification of exact quantities differs markedly from the situation in mammalian melanocytes where cell volume and gene dosage influence the extent of melanotic differentiation. Quantitative control of differentiation in ascidian melanocytes appears to be mediated by a cytoplasmic determinant segregated through the melanocyte lineage and inherited by one daughter at each division of the lineages.     

Mutational analysis of endothelin receptor b1 (rose) during neural crest and pigment pattern development in the zebrafish Danio rerio

Pigment patterns of fishes are a tractable system for studying the genetic and cellular bases for postembryonic phenotypes. In the zebrafish Danio rerio, neural crest-derived pigment cells generate different pigment patterns during different phases of the life cycle. Whereas early larvae exhibit simple stripes of melanocytes and silver iridophores in a background of yellow xanthophores, this pigment pattern is transformed at metamorphosis into that of the adult, comprising a series of dark melanocyte and iridophore stripes, alternating with light stripes of iridophores and xanthophores. Although several genes have been identified in D. rerio that contribute to the development of both early larval and adult pigment patterns, comparatively little is known about genes that are essential for pattern formation during just one or the other life cycle phase. In this study, we identify the gene responsible for the rose mutant phenotype in D. rerio. rose mutants have wild-type early larval pigment patterns, but fail to develop normal numbers of melanocytes and iridophores during pigment pattern metamorphosis and exhibit a disrupted pattern of these cells. We show that rose corresponds to endothelin receptor b1 (ednrb1), an orthologue of amniote Ednrb genes that have long been studied for their roles in neural crest and pigment cell development. Furthermore, we demonstrate that D. rerio ednrb1 is expressed both during pigment pattern metamorphosis and during embryogenesis, and cells of melanocyte, iridophore, and xanthophore lineages all express this gene. These analyses suggest a phylogenetic conservation of roles for Ednrb signaling in the development of amniote and teleost pigment cell precursors. As murine Ednrb is essential for the development of all neural crest derived melanocytes, and D. rerio ednrb1 is required only by a subset of adult melanocytes and iridophores, these analyses also reveal variation among vertebrates in the cellular requirements for Ednrb signaling, and suggest alternative models for the cellular and genetic bases of pigment pattern metamorphosis in D. rerio.     

An orthologue of the kit-related gene fms is required for development of neural crest-derived xanthophores and a subpopulation of adult melanocytes in the zebrafish, Danio rerio

Developmental mechanisms underlying traits expressed in larval and adult vertebrates remain largely unknown. Pigment patterns of fishes provide an opportunity to identify genes and cell behaviors required for postembryonic morphogenesis and differentiation. In the zebrafish, Danio rerio, pigment patterns reflect the spatial arrangements of three classes of neural crest-derived pigment cells: black melanocytes, yellow xanthophores and silver iridophores. We show that the D. rerio pigment pattern mutant panther ablates xanthophores in embryos and adults and has defects in the development of the adult pattern of melanocyte stripes. We find that panther corresponds to an orthologue of the c-fms gene, which encodes a type III receptor tyrosine kinase and is the closest known homologue of the previously identified pigment pattern gene, kit. In mouse, fms is essential for the development of macrophage and osteoclast lineages and has not been implicated in neural crest or pigment cell development. In contrast, our analyses demonstrate that fms is expressed and required by D. rerio xanthophore precursors and that fms promotes the normal patterning of melanocyte death and migration during adult stripe formation. Finally, we show that fms is required for the appearance of a late developing, kit-independent subpopulation of adult melanocytes. These findings reveal an unexpected role for fms in pigment pattern development and demonstrate that parallel neural crest-derived pigment cell populations depend on the activities of two essentially paralogous genes, kit and fms.     

Neuroepithelial cells supply an initial transient wave of MSC differentiation

Mesenchymal stem cells (MSCs) are defined as cells that undergo sustained in vitro growth and are able to give rise to multiple mesenchymal lineages. Although MSCs are already used in regenerative medicine little is known about their in vivo behavior and developmental derivation. Here, we show that the earliest wave of MSC in the embryonic trunk is generated from Sox1+ neuroepithelium but not from mesoderm. Using lineage marking by direct gfp knock-in and Cre-recombinase mediated lineage tracing, we provide evidence that Sox1+ neuroepithelium gives rise to MSCs in part through a neural crest intermediate stage. This pathway can be distinguished from the pathway through which Sox1+ cells give rise to oligodendrocytes by expression of PDGFRbeta and A2B5. MSC recruitment from this pathway, however, is transient and is replaced by MSCs from unknown sources. We conclude that MSC can be defined as a definite in vivo entity recruited from multiple developmental origins.     

Zebrafish adult pigment stem cells are multipotent and form pigment cells by a progressive fate restriction process

Skin pigment pattern formation is a paradigmatic example of pattern formation. In zebrafish, the adult body stripes are generated by coordinated rearrangement of three distinct pigment cell‐types, black melanocytes, shiny iridophores and yellow xanthophores. A stem cell origin of melanocytes and iridophores has been proposed although the potency of those stem cells has remained unclear. Xanthophores, however, seemed to originate predominantly from proliferation of embryonic xanthophores. Now, data from Singh et al. shows that all three cell‐types derive from shared stem cells, and that these cells generate peripheral neural cell‐types too. Furthermore, clonal compositions are best explained by a progressive fate restriction model generating the individual cell‐types. The numbers of adult pigment stem cells associated with the dorsal root ganglia remain low, but progenitor numbers increase significantly during larval development up to metamorphosis, likely via production of partially restricted progenitors on the spinal nerves.     

Mutations in gfpt1 and skiv2l2 cause distinct stage-specific defects in larval melanocyte regeneration in zebrafish

The establishment of a single cell type regeneration paradigm in the zebrafish provides an opportunity to investigate the genetic mechanisms specific to regeneration processes. We previously demonstrated that regeneration melanocytes arise from cell division of the otherwise quiescent melanocyte precursors following larval melanocyte ablation with a small molecule, MoTP. The ease of ablating melanocytes by MoTP allows us to conduct a forward genetic screen for mechanisms specific to regeneration from such precursors or stem cells. Here, we reported the identification of two mutants, eartha^j23e1 and julie^j24e1 from a melanocyte ablation screen. Both mutants develop normal larval melanocytes, but upon melanocyte ablation, each mutation results in a distinct stage-specific defect in melanocyte regeneration. Positional cloning reveals that the eartha^j23e1 mutation is a nonsense mutation in gfpt1 (glutamine:fructose-6-phosphate aminotransferase 1), the rate-limiting enzyme in glucosamine-6-phosphate biosynthesis. Our analyses reveal that a mutation in gfpt1 specifically affects melanocyte differentiation (marked by melanin production) at a late stage during regeneration and that gfpt1 acts cell autonomously in melanocytes to promote ontogenetic melanocyte darkening. We identified that the julie^j24e1 mutation is a splice-site mutation in skiv2l2 (superkiller viralicidic activity 2-like 2), a predicted DEAD-box RNA helicase. Our in situ analysis reveals that the mutation in skiv2l2 causes defects in cell proliferation, suggesting that skiv2l2 plays a role in regulating melanoblast proliferation during early stages of melanocyte regeneration. This finding is consistent with previously described role for cell division during larval melanocyte regeneration. The analyses of these mutants reveal their stage-specific roles in melanocyte regeneration. Interestingly, these mutants identify regeneration-specific functions not only in early stages of the regeneration process, but also in late stages of differentiation of the regenerating melanocyte. We suggest that mechanisms of regeneration identified in this mutant screen may reveal fundamental differences between the mechanisms that establish differentiated cells during embryogenesis, and those involved in larval or adult growth.     

Zebrafish sparse corresponds to an orthologue of c-kit and is required for the morphogenesis of a subpopulation of melanocytes, but is not essential for hematopoiesis or primordial germ cell development.

The relative roles of the Kit receptor in promoting the migration and survival of amniote melanocytes are unresolved. We show that, in the zebrafish, Danio rerio, the pigment pattern mutation sparse corresponds to an orthologue of c-kit. This finding allows us to further elucidate morphogenetic roles for this c-kit-related gene in melanocyte morphogenesis. Our analyses of zebrafish melanocyte development demonstrate that the c-kit orthologue identified in this study is required both for normal migration and for survival of embryonic melanocytes. We also find that, in contrast to mouse, the zebrafish c-kit gene that we have identified is not essential for hematopoiesis or primordial germ cell development. These unexpected differences may reflect evolutionary divergence in c-kit functions following gene duplication events in teleosts.     

pyewacket, a new zebrafish fin pigment pattern mutant

Many mutants that disrupt zebrafish embryonic pigment pattern have been isolated, and subsequent cloning of the mutated genes causing these phenotypes has contributed to our understanding of pigment cell development. However, few mutants have been identified that specifically affect development of the adult pigment pattern. Through a mutant screen for adult pigment pattern phenotypes, we identified pyewacket (pye), a novel zebrafish mutant in which development of the adult caudal fin pigment pattern is aberrant. Specifically, pye mutants have fin melanocyte pigment pattern defects and fewer xanthophores than wild-type fins. We mapped pye to an interval where a single gene, the zebrafish ortholog of the human gene DHRSX, is present. pye will be an informative mutant for understanding how xanthophores and melanocytes interact to form the pigment pattern of the adult zebrafish fin.     

A requirement for <Emphasis Type="Italic">kit</Emphasis> in embryonic zebrafish melanocyte differentiation is revealed by melanoblast delay

Exploring differences in gene requirements between species can allow us to delineate basic developmental mechanisms, provide insight into patterns of evolution, and explain heterochronic differences in developmental processes. One example of differences in gene requirements between zebrafish and mammals is the requirement of the kit receptor tyrosine kinase in melanocyte development. kit is required for migration, survival and differentiation of all neural crest-derived melanocytes in mammals. In contrast, zebrafish kit is not required for differentiation of embryonic melanocytes during normal development. When melanoblast development in zebrafish embryos is delayed by injecting morpholinos targeted to the mitfa gene, we show that these delayed melanoblasts fail to differentiate in kit mutants. Thus, we show that there is a kit requirement for melanocyte differentiation in zebrafish when melanoblast development is delayed. Furthermore, we show that kit is not involved in maintaining melanocyte precursors through the developmental delay, but instead is required for differentiation of melanocytes after the block on their development is removed. Finally, we suggest there is a heterochronic shift in the onset of melanocyte differentiation between fish and mouse, and developmental delay of melanoblast development in zebrafish removes this heterochronic difference.Edited by D. Tautz     

Functionally distinct melanocyte populations revealed by reconstitution of hair follicles in mice

Hair follicle reconstitution analysis was used to test the contribution of melanocytes or their precursors to regenerated hair follicles. In this study, we first confirmed the process of chimeric hair follicle regeneration by both hair keratinocytes and follicular melanocytes. Then, as first suggested from the differential growth requirements of epidermal skin melanocytes and non‐cutaneous or dermal melanocytes, we confirmed the inability of the latter to be involved as follicular melanocytes to regenerate hair follicles during the hair reconstitution assay. This clear functional discrimination between non‐cutaneous or dermal melanocytes and epidermal melanocytes suggests the presence of two different melanocyte cell lineages, a finding that might be important in the pathogenesis of melanocyte‐related diseases and melanomas.