Melanophore sublineage-specific requirement for zebrafish touchtone during neural crest development

The specification, differentiation and maintenance of diverse cell types are of central importance to the development of multicellular organisms. The neural crest of vertebrate animals gives rise to many derivatives, including pigment cells, peripheral neurons, glia and elements of the craniofacial skeleton. The development of neural crest-derived pigment cells has been studied extensively to elucidate mechanisms involved in cell fate specification, differentiation, migration and survival. This analysis has been advanced considerably by the availability of large numbers of mouse and, more recently, zebrafish mutants with defects in pigment cell development. We have identified the zebrafish mutant touchtone (tct), which is characterized by the selective absence of most neural crest-derived melanophores. We find that although wild-type numbers of melanophore precursors are generated in the first day of development and migrate normally in tct mutants, most differentiated melanophores subsequently fail to appear. We demonstrate that the failure in melanophore differentiation in tct mutant embryos is due at least in part to the death of melanoblasts and that tct function is required cell autonomously by melanoblasts. The tct locus is located on chromosome 18 in a genomic region apparently devoid of genes known to be involved in melanophore development. Thus, zebrafish tct may represent a novel as well as selective regulator of melanoblast development within the neural crest lineage. Further, our results suggest that, like other neural crest-derived sublineages, melanogenic precursors constitute a heterogeneous population with respect to genetic requirements for development.     

Zebrafish endzone regulates neural crest-derived chromatophore differentiation and morphology

The development of neural crest-derived pigment cells has been studied extensively as a model for cellular differentiation, disease and environmental adaptation. Neural crest-derived chromatophores in the zebrafish (Danio rerio) consist of three types: melanophores, xanthophores and iridiphores. We have identified the zebrafish mutant endzone (enz), that was isolated in a screen for mutants with neural crest development phenotypes, based on an abnormal melanophore pattern. We have found that although wild-type numbers of chromatophore precursors are generated in the first day of development and migrate normally in enz mutants, the numbers of all three chromatophore cell types that ultimately develop are reduced. Further, differentiated melanophores and xanthophores subsequently lose dendricity, and iridiphores are reduced in size. We demonstrate that enz function is required cell autonomously by melanophores and that the enz locus is located on chromosome 7. In addition, zebrafish enz appears to selectively regulate chromatophore development within the neural crest lineage since all other major derivatives develop normally. Our results suggest that enz is required relatively late in the development of all three embryonic chromatophore types and is normally necessary for terminal differentiation and the maintenance of cell size and morphology. Thus, although developmental regulation of different chromatophore sublineages in zebrafish is in part genetically distinct, enz provides an example of a common regulator of neural crest-derived chromatophore differentiation and morphology.     

oca2 regulation of chromatophore differentiation and number is cell type specific in zebrafish

We characterized a zebrafish mutant that displays defects in melanin synthesis and in the differentiation of melanophores and iridophores of the skin and retinal pigment epithelium. Positional cloning and candidate gene sequencing link this mutation to a 410‐kb region on chromosome 6, containing the oculocutaneous albinism 2 (oca2) gene. Quantification of oca2 mutant melanophores shows a reduction in the number of differentiated melanophores compared with wildtype siblings. Consistent with the analysis of mouse Oca2‐deficient melanocytes, zebrafish mutant melanophores have immature melanosomes which are partially rescued following treatment with vacuolar‐type ATPase inhibitor/cytoplasmic pH modifier, bafilomycin A1. Melanophore‐specific gene expression is detected at the correct time and in anticipated locations. While oca2 zebrafish display unpigmented gaps on the head region of mutants 3 days post‐fertilization, melanoblast quantification indicates that oca2 mutants have the correct number of melanoblasts, suggesting a differentiation defect explains the reduced melanophore number. Unlike melanophores, which are reduced in number in oca2 mutants, differentiated iridophores are present at significantly higher numbers. These data suggest distinct mechanisms for oca2 in establishing differentiated chromatophore number in developing zebrafish.     

Sdf1a patterns zebrafish melanophores and links the somite and melanophore pattern defects in choker mutants

Pigment pattern formation in zebrafish presents a tractable model system for studying the morphogenesis of neural crest derivatives. Embryos mutant for choker manifest a unique pigment pattern phenotype that combines a loss of lateral stripe melanophores with an ectopic melanophore ;collar' at the head-trunk border. We find that defects in neural crest migration are largely restricted to the lateral migration pathway, affecting both xanthophores (lost) and melanophores (gained) in choker mutants. Double mutant and timelapse analyses demonstrate that these defects are likely to be driven independently, the collar being formed by invasion of melanophores from the dorsal and ventral stripes. Using tissue transplantation, we show that melanophore patterning depends upon the underlying somitic cells, the myotomal derivatives of which--both slow--and fast-twitch muscle fibres--are themselves significantly disorganised in the region of the ectopic collar. In addition, we uncover an aberrant pattern of expression of the gene encoding the chemokine Sdf1a in choker mutant homozygotes that correlates with each aspect of the melanophore pattern defect. Using morpholino knock-down and ectopic expression experiments, we provide evidence to suggest that Sdf1a drives melanophore invasion in the choker mutant collar and normally plays an essential role in patterning the lateral stripe. We thus identify Sdf1 as a key molecule in pigment pattern formation, adding to the growing inventory of its roles in embryonic development.     

Zebrafish colourless encodes sox10 and specifies non-ectomesenchymal neural crest fates.

Waardenburg-Shah syndrome combines the reduced enteric nervous system characteristic of Hirschsprung's disease with reduced pigment cell number, although the cell biological basis of the disease is unclear. We have analysed a zebrafish Waardenburg-Shah syndrome model. We show that the colourless gene encodes a sox10 homologue, identify sox10 lesions in mutant alleles and rescue the mutant phenotype by ectopic sox10 expression. Using iontophoretic labelling of neural crest cells, we demonstrate that colourless mutant neural crest cells form ectomesenchymal fates. By contrast, neural crest cells which in wild types form non-ectomesenchymal fates generally fail to migrate and do not overtly differentiate. These cells die by apoptosis between 35 and 45 hours post fertilisation. We provide evidence that melanophore defects in colourless mutants can be largely explained by disruption of nacre/mitf expression. We propose that all defects of affected crest derivatives are consistent with a primary role for colourless/sox10 in specification of non-ectomesenchymal crest derivatives. This suggests a novel mechanism for the aetiology of Waardenburg-Shah syndrome in which affected neural crest derivatives fail to be generated from the neural crest.     

Genetic and experimental studies on a new pigment mutant in Xenopus laevis.

White lethal (wl) is a recessive mutation affecting the differentiation of the three types of chromatophores in Xenopus laevis and eventually leading to the death of the mutants around stage 50. Melanophores appear at st. 33 but differentiate abnormally, remaining pale grey, and do not proliferate after st. 41. The rare xanthophores present contain only a few differentiated pterinosomes, and the iridophores consist of noniridescent white dots. When the albino gene (ap) is combined with wl, melanophores do not differentiate. Reciprocal heterotopic and orthotopic trunk neural crest grafts have shown that the defect is intrinsic to the neural crest cells but is not due, in the case of melanophores, to a tyrosinase deficiency as revealed by the dopa reaction. The mode of action of the gene, the abnormal pattern, and lethality are discussed.     

Embryonic requirements for ErbB signaling in neural crest development and adult pigment pattern formation

Vertebrate pigment cells are derived from neural crest cells and are a useful system for studying neural crest-derived traits during post-embryonic development. In zebrafish, neural crest-derived melanophores differentiate during embryogenesis to produce stripes in the early larva. Dramatic changes to the pigment pattern occur subsequently during the larva-to-adult transformation, or metamorphosis. At this time, embryonic melanophores are replaced by newly differentiating metamorphic melanophores that form the adult stripes. Mutants with normal embryonic/early larval pigment patterns but defective adult patterns identify factors required uniquely to establish, maintain or recruit the latent precursors to metamorphic melanophores. We show that one such mutant, picasso, lacks most metamorphic melanophores and results from mutations in the ErbB gene erbb3b, which encodes an EGFR-like receptor tyrosine kinase. To identify critical periods for ErbB activities, we treated fish with pharmacological ErbB inhibitors and also knocked down erbb3b by morpholino injection. These analyses reveal an embryonic critical period for ErbB signaling in promoting later pigment pattern metamorphosis, despite the normal patterning of embryonic/early larval melanophores. We further demonstrate a peak requirement during neural crest migration that correlates with early defects in neural crest pathfinding and peripheral ganglion formation. Finally, we show that erbb3b activities are both autonomous and non-autonomous to the metamorphic melanophore lineage. These data identify a very early, embryonic, requirement for erbb3b in the development of much later metamorphic melanophores, and suggest complex modes by which ErbB signals promote adult pigment pattern development.     

Genetic analysis of melanophore development in zebrafish embryos

Vertebrate pigment cells are derived from neural crest, a tissue that also forms most of the peripheral nervous system and a variety of ectomesenchymal cell types. Formation of pigment cells from multipotential neural crest cells involves a number of common developmental processes. Pigment cells must be specified; their migration, proliferation, and survival must be controlled and they must differentiate to the final pigment cell type. We previously reported a large set of embryonic mutations that affect pigment cell development from neural crest (R. N. Kelsh et al., 1996, Development 123, 369-389). Based on distinctions in pigment cell appearance between mutants, we proposed hypotheses as to the process of pigment cell development affected by each mutation. Here we describe the cloning and expression of an early zebrafish melanoblast marker, dopachrome tautomerase. We used this marker to test predictions about melanoblast number and pattern in mutant embryos, including embryos homozygous for mutations in the colourless, sparse, touchdown, sunbleached, punkt, blurred, fade out, weiss, sandy, and albino genes. We showed that in homozygous mutants for all loci except colourless and sparse, melanoblast number and pattern are normal. colourless mutants have a pronounced decrease in melanoblast cell number from the earliest stages and also show poor melanoblast differentiation and migration. Although sparse mutants show normal numbers of melanoblasts initially, their number is reduced later. Furthermore, their distribution indicates a defect in melanoblast dispersal. These observations permit us to refine our model of the genetic control of melanophore development in zebrafish embryos.     

The Fugu tyrp1 promoter directs specific GFP expression in zebrafish: tools to study the RPE and the neural crest‐derived melanophores

In vertebrates, pigment cells account for a small percentage of the total cell population and they intermingle with other cell types. This makes it difficult to isolate them for analyzes of their functions in the context of development. To alleviate such difficulty, we generated two stable transgenic zebrafish lines (pt101 and pt102) that express green fluorescent protein (GFP) in melanophores under the control of the 1 kb Fugu tyrp1 promoter. In pt101, GFP is expressed in both retinal pigment epithelium (RPE) cells and the neural crest‐derived melanophores (NCDM), whereas in pt102, GFP is predominately expressed in the NCDM. Our results indicate that the Fugu tyrp1 promoter can direct transgene expression in a cell‐type‐specific manner in zebrafish. In addition, our findings provide evidence supporting differential regulations of melanin‐synthesizing genes in RPE cells and the NCDM in zebrafish. Utilizing the varying GFP expression levels in these fish, we have isolated melanophores via flow cytometry and revealed the capability of sorting the NCDM from RPE cells as well. Thus, these transgenic lines are useful tools to study melanophores in zebrafish.     

Delta signaling mediates segregation of neural crest and spinal sensory neurons from zebrafish lateral neural plate

We examined the role of Delta signaling in specification of two derivatives in zebrafish neural plate: Rohon-Beard spinal sensory neurons and neural crest. deltaA-expressing Rohon-Beard neurons are intermingled with premigratory neural crest cells in the trunk lateral neural plate. Embryos homozygous for a point mutation in deltaA, or with experimentally reduced delta signalling, have supernumerary Rohon-Beard neurons, reduced trunk-level expression of neural crest markers and lack trunk neural crest derivatives. Fin mesenchyme, a putative trunk neural crest derivative, is present in deltaA mutants, suggesting it segregates from other neural crest derivatives as early as the neural plate stage. Cranial neural crest derivatives are also present in deltaA mutants, revealing a genetic difference in regulation of trunk and cranial neural crest development.     

alyron, an Insertional Mutation Affecting Early Neural Crest Development in Zebrafish

alyronz12 (aln) is a recessive lethal mutation that affects early stages of neural crest development in the zebrafish. alyron appears to be an insertional mutation as the mutation was generated following microinjection of plasmid DNA into one-cell embryos and the stably integrated transgenic sequences are closely linked to the mutation. The insertion site harbors multiple copies of the plasmid sequence that have experienced complex rearrangements. Host-insert junction fragments have been molecularly cloned and host sequences adjacent to the transgene have been used to map the mutation to the distal arm of linkage group 15. alyron function is required cell-autonomously in the neural crest lineage. alyron mutants have a severe but not complete deficit of premigratory neural crest as judged by reduced expression of several markers associated with early stages of neural crest development. Lack of premigratory neural crest is likely to account for the two most conspicuous characteristics of alyron mutants: the absence of body pigmentation and the inability to affect blood circulation. The neural crest phenotype of alyron mutants resembles that observed in mouse mutants that lack Pax-3 or both Wnt-1 and Wnt-3a function, and expression of the zebrafish homologues of these genes is greatly reduced in the dorsal neural keels of alyron mutants. In contrast, ventral neural keel identity appears unaffected. Given our findings that the mutation is unlinked to pax or wnt genes that have been described in the zebrafish, we propose that alyron is a novel gene function required for the specification and/or proliferative expansion of neural crest progenitors.     

Pigment pattern evolution by differential deployment of neural crest and post-embryonic melanophore lineages in Danio fishes

Latent precursors or stem cells of neural crest origin are present in a variety of post-embryonic tissues. Although these cells are of biomedical interest for roles in human health and disease, their potential evolutionary significance has been underappreciated. As a first step towards elucidating the contributions of such cells to the evolution of vertebrate form, we investigated the relative roles of neural crest cells and post-embryonic latent precursors during the evolutionary diversification of adult pigment patterns in Danio fishes. These pigment patterns result from the numbers and arrangements of embryonic melanophores that are derived from embryonic neural crest cells, as well as from post-embryonic metamorphic melanophores that are derived from latent precursors of presumptive neural crest origin. In the zebrafish D. rerio, a pattern of melanophore stripes arises during the larval-to-adult transformation by the recruitment of metamorphic melanophores from latent precursors. Using a comparative approach in the context of new phylogenetic data, we show that adult pigment patterns in five additional species also arise from metamorphic melanophores, identifying this as an ancestral mode of adult pigment pattern development. By contrast, superficially similar adult stripes of D. nigrofasciatus (a sister species to D. rerio) arise by the reorganization of melanophores that differentiated at embryonic stages, with a diminished contribution from metamorphic melanophores. Genetic mosaic and molecular marker analyses reveal evolutionary changes that are extrinsic to D. nigrofasciatus melanophore lineages, including a dramatic reduction of metamorphic melanophore precursors. Finally, interspecific complementation tests identify a candidate genetic pathway for contributing to the evolutionary reduction in metamorphic melanophores and the increased contribution of early larval melanophores to D. nigrofasciatus adult pigment pattern development. These results demonstrate an important role for latent precursors in the diversification of pigment patterns across danios. More generally, differences in the deployment of post-embryonic neural crest-derived stem cells or their specified progeny may contribute substantially to the evolutionary diversification of adult form in vertebrates, particularly in species that undergo a metamorphosis.     

Tetraspanin 3c requirement for pigment cell interactions and boundary formation in zebrafish adult pigment stripes

Skin pigment pattern formation in zebrafish requires pigment‐cell autonomous interactions between melanophores and xanthophores, yet the molecular bases for these interactions remain largely unknown. Here, we examined the dali mutant that exhibits stripes in which melanophores are intermingled abnormally with xanthophores. By in vitro cell culture, we found that melanophores of dali mutants have a defect in motility and that interactions between melanophores and xanthophores are defective as well. Positional cloning and rescue identified dali as tetraspanin 3c (tspan3c), encoding a transmembrane scaffolding protein expressed by melanophores and xanthophores. We further showed that dali mutant Tspan3c expressed in HeLa cell exhibits a defect in N‐glycosylation and is retained inappropriately in the endoplasmic reticulum. Our results are the first to identify roles for a tetraspanin superfamily protein in skin pigment pattern formation and suggest new mechanisms for the establishment and maintenance of zebrafish stripe boundaries.