Duplicate mitf genes in zebrafish: complementary expression and conservation of melanogenic potential.

Mutations in the zebrafish nacre/mitfa gene, expressed in all embryonic melanogenic cells, perturb only neural crest melanocytes, suggesting redundancy of mitfa with another gene in the zebrafish retinal pigment epithelium (RPE). Here, we describe a second zebrafish mitf gene, mitfb, which may fulfill this role. The proteins encoded by the two zebrafish mitf genes appear homologous to distinct isoforms generated by alternately spliced mRNAs of the single mammalian Mitf gene, suggesting specialization of the two zebrafish genes following a duplication event. Consistent with this hypothesis, expression of mitfa and mitfb is partially overlapping. mitfb is coexpressed with mitfa in the RPE at an appropriate time to compensate for loss of mitfa function in the nacre mutant but is not expressed in neural crest melanoblasts. Additionally, mitfb is expressed in the epiphysis and olfactory bulb where mitfa is not, and where Mitf expression has not previously been reported in other species. mitfb, but not a zebrafish ortholog of the closely related gene tfe3, can rescue neural crest melanophore development in nacre/mitfa mutant embryos when expressed via the mitfa promoter. These data suggest that mitfa and mitfb together may recapitulate the expression and functions of a single ancestral Mitf gene, and that mitfb may serve additional novel functions.     

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.     

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.     

Phox2b function in the enteric nervous system is conserved in zebrafish and is sox10-dependent

Zebrafish lacking functional sox10 have defects in non-ectomesenchymal neural crest derivatives including the enteric nervous system (ENS) and as such provide an animal model for human Waardenburg Syndrome IV. Here, we characterize zebrafish phox2b as a functionally conserved marker of the developing ENS. We show that morpholino-mediated knockdown of Phox2b generates fish modeling Hirschsprung disease. Using markers, including phox2b, we investigate the ontogeny of the sox10 ENS phenotype. As previously shown for melanophore development, ENS progenitor fate specification fails in these mutant fish. However, in addition, we trace back the sox10 mutant ENS defect to an even earlier time point, finding that most neural crest cells fail to migrate ventrally to the gut primordium.     

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.     

A direct role for Sox10 in specification of neural crest-derived sensory neurons

sox10 is necessary for development of neural and pigment cell derivatives of the neural crest (NC). However, whereas a direct role for Sox10 activity has been established in pigment and glial lineages, this is more controversial in NC-derived sensory neurons of the dorsal root ganglia (DRGs). We proposed that sox10 functioned in specification of sensory neurons, whereas others suggested that sensory neuronal defects were merely secondary to absence of glia. Here we provide evidence that in zebrafish, early DRG sensory neuron survival is independent of differentiated glia. Critically, we demonstrate that Sox10 is expressed transiently in the sensory neuron lineage, and specifies sensory neuron precursors by regulating the proneural gene neurogenin1. Consistent with this, we have isolated a novel sox10 mutant that lacks glia and yet displays a neurogenic DRG phenotype. In conjunction with previous findings, these data establish the generality of our model of Sox10 function in NC fate specification.     

Visualization of Craniofacial Development in the sox10: kaede Transgenic Zebrafish Line Using Time-lapse Confocal Microscopy

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, convergence and extension of facial prominences, and maturation of the craniofacial skeleton. To study the contribution of the cranial neural crest to specific regions of the zebrafish palate a sox10: kaede transgenic zebrafish line was generated. Sox10 provides lineage restriction of the kaede reporter protein to the neural crest, thereby making the cell labeling a more precise process than traditional dye or reporter mRNA injection. Kaede is a photo-convertible protein that turns from green to red after photo activation and makes it possible to follow cells precisely. The sox10: kaede transgenic line was used to perform lineage analysis to delineate CNC cell populations that give rise to maxillary versus mandibular elements and illustrate homology of facial prominences to amniotes. This protocol describes the steps to generate a live time-lapse video of a sox10: kaede zebrafish embryo. Development of the ethmoid plate will serve as a practical example. This protocol can be applied to making a time-lapse confocal recording of any kaede or similar photoconvertible reporter protein in transgenic zebrafish. Furthermore, it can be used to capture not only normal, but also abnormal development of craniofacial structures in the zebrafish mutants.