colgate/hdac1 Repression of foxd3 expression is required to permit mitfa-dependent melanogenesis

Neural crest-derived pigment cell development has been used extensively to study cell fate specification, migration, proliferation, survival and differentiation. Many of the genes and regulatory mechanisms required for pigment cell development are conserved across vertebrates. The zebrafish mutant colgate (col)/histone deacetylase1 (hdac1) has reduced numbers, delayed differentiation and decreased migration of neural crest-derived melanophores and their precursors. In hdac1^col mutants normal numbers of premigratory neural crest cells are induced. Later, while there is only a slight reduction in the number of neural crest cells in hdac1^col mutants, there is a severe reduction in the number of mitfa-positive melanoblasts suggesting that hdac1 is required for melanoblast specification. Concomitantly, there is a significant increase in and prolonged expression of foxd3 in neural crest cells in hdac1^col mutants. We found that partially reducing Foxd3 expression in hdac1^col mutants rescues mitfa expression and the melanophore defects in hdac1^col mutants. Furthermore, we demonstrate the ability of Foxd3 to physically interact at the mitfa promoter. Because mitfa is required for melanoblast specification and development, our results suggest that hdac1 is normally required to suppress neural crest foxd3 expression thus de-repressing mitfa resulting in melanogenesis by a subset of neural crest-derived cells.     

Zebrafish msxB, msxC and msxE function together to refine the neural-nonneural border and regulate cranial placodes and neural crest development

The zebrafish muscle segment homeobox genes msxB, msxC and msxE are expressed in partially overlapping domains in the neural crest and preplacodal ectoderm. We examined the roles of these msx genes in early development. Disrupting individual msx genes causes modest variable defects, whereas disrupting all three produces a reproducible severe phenotype, suggesting functional redundancy. Neural crest differentiation is blocked at an early stage. Preplacodal development begins normally, but placodes arising from the msx expression domain later show elevated apoptosis and are reduced in size. Cell proliferation is normal in these tissues. Unexpectedly, Msx-deficient embryos become ventralized by late gastrulation whereas misexpression of msxB dorsalizes the embryo. These effects appear to involve Distal-less (Dlx) protein activity, as loss of dlx3b and dlx4b suppresses ventralization in Msx-depleted embryos. At the same time, Msx-depletion restores normal preplacodal gene expression to dlx3b-dlx4b mutants. These data suggest that mutual antagonism between Msx and Dlx proteins achieves a balance of function required for normal preplacodal differentiation and placement of the neural-nonneural border.     

Sequential actions of Pax3 and Pax7 drive xanthophore development in zebrafish neural crest

The Pax3/7 gene family has a fundamental and conserved role during neural crest formation. In people, PAX3 mutation causes Waardenburg syndrome, and murine Pax3 is essential for pigment formation. However, it is unclear exactly how Pax3 functions within the neural crest. Here we show that pax3 is expressed before other pax3/7 members, including duplicated pax3b, pax7 and pax7b genes, early in zebrafish neural crest development. Knockdown of Pax3 protein by antisense morpholino oligonucleotides results in defective fate specification of xanthophores, with complete ablation in the trunk. Other pigment lineages are specified and differentiate. As a consequence of xanthophore loss, expression of pax7, a marker of the xanthophore lineage, is reduced in neural crest. Morpholino knockdown of Pax7 protein shows that Pax7 itself is dispensable for xanthophore fate specification, although yellow pigmentation is reduced. Loss of xanthophores after reduction of Pax3 correlates with a delay in melanoblast differentiation followed by significant increase in melanophores, suggestive of a Pax3-driven fate switch within a chromatophore precursor or stem cell. Analysis of other neural crest derivatives reveals that, in the absence of Pax3, the enteric nervous system is ablated from its inception. Therefore, Pax3 in zebrafish is required for specification of two specific lineages of neural crest, xanthophores and enteric neurons.     

Live image profiling of neural crest lineages in zebrafish transgenic lines

Zebrafish transgenic lines are important experimental tools for lineage tracing and imaging studies. It is crucial to precisely characterize the cell lineages labeled in transgenic lines to understand their limitations and thus properly interpret the data obtained from their use; only then can we confidently select a line appropriate for our particular research objectives. Here we profiled the cell lineages labeled in the closely related neural crest transgenic lines Tg(foxd3:GFP), Tg(sox10:eGFP) and Tg(sox10:mRFP). These fish were crossed to generate embryos, in which foxd3 and sox10 transgenic neural crest labeling could be directly compared at the cellular level using live confocal imaging. We have identified key differences in the cell lineages labeled in each line during early neural crest development and demonstrated that the most anterior cranial neural crest cells initially migrating out of neural tube at the level of forebrain and anterior midbrain express sox10:eGFP and sox10:mRFP, but not foxd3:GFP. This differential profile was robustly maintained in the different-tiating progeny of the neural crest lineages until 3.5dpf. Our data will enable researchers to make an informed choice in selecting transgenic lines for future neural crest research.     

Zic2 is required for neural crest formation and hindbrain patterning during mouse development

The Zic genes are the vertebrate homologues of the Drosophila pair rule gene odd-paired. It has been proposed that Zic genes play several roles during neural development including mediolateral segmentation of the neural plate, neural crest induction, and inhibition of neurogenesis. Initially during mouse neural development Zic2 is expressed throughout the neural plate while later on expression in the neurectoderm becomes restricted to the lateral region of the neural plate. A hypomorphic allele of Zic2 has demonstrated that in the mouse Zic2 is required for the timing of neurulation. We have isolated a new allele of Zic2 that behaves as a loss of function allele. Analysis of this mutant reveals two further functions for Zic2 during early neural development. Mutation of Zic2 results in a delay of neural crest production and a decrease in the number of neural crest cells that are produced. These defects are independent of mediolateral segmentation of the neurectoderm and of dorsal neurectoderm proliferation, both of which occur normally in the mutant embryos. Additionally Zic2 is required during hindbrain patterning for the normal development of rhombomeres 3 and 5. This work provides the first genetic evidence that the Zic genes are involved in neural crest production and the first demonstration that Zic2 functions during hindbrain patterning.     

Eif3ba regulates cranial neural crest development by modulating p53 in zebrafish

Congenital diseases caused by abnormal development of the cranial neural crest usually present craniofacial malformations and heart defects while the precise mechanism is not fully understood. Here, we show that the zebrafish eif3ba mutant caused by pseudo-typed retrovirus insertion exhibited a similar phenotype due to the hypogenesis of cranial neural crest cells (NCCs). The derivatives of cranial NCCs, including the NCC-derived cell population of pharyngeal arches, craniofacial cartilage, pigment cells and the myocardium derived from cardiac NCCs, were affected in this mutant. The expression of several neural crest marker genes, including crestin, dlx2a and nrp2b, was specifically reduced in the cranial regions of the eif3ba mutant. Through fluorescence-tracing of the cranial NCC migration marker nrp2b, we observed reduced intensity of NCC-derived cells in the heart. In addition, p53 was markedly up-regulated in the eif3ba mutant embryos, which correlated with pronounced apoptosis in the cranial area as shown by TUNEL staining. These findings suggest a novel function of eif3ba during embryonic development and a novel level of regulation in the process of cranial NCC development, in addition to providing a potential animal model to mimic congenital diseases due to cranial NCC defects. Furthermore, we report the identification of a novel transgenic fish line Et(gata2a:EGFP)^pku418 to trace the migration of cranial NCCs (including cardiac NCCs); this may serve as an invaluable tool for investigating the development and dynamics of cranial NCCs during zebrafish embryogenesis.     

Fgf20b is required for the ectomesenchymal fate establishment of cranial neural crest cells in zebrafish

In cranial skeletal development, the establishment of the ectomesenchymal lineage within the cranial neural crest is of great significance. Fgfs are polypeptide growth factors with diverse functions in development and metabolism. Fgf20b knockdown zebrafish embryos showed dysplastic neurocranial and pharyngeal cartilages. Ectomesenchymal cells from cranial neural crest cells were significantly decreased in Fgf20b knockdown embryos, but cranial neural crest cells with a non-ectomesnchymal fate were increased. However, the proliferation and apoptosis of cranial neural crest cells were essentially unchanged. Fgfr1 knockdown embryos also showed dysplastic neurocranial and pharyngeal cartilages. The present findings indicate that Fgf20b is required for ectomesenchymal fate establishment via the activation of Fgfr1 in zebrafish.     

Dicer is required for survival of differentiating neural crest cells

The neural crest (NC) lineage gives rise to a wide array of cell types ranging from neurons and glia of the peripheral nervous system to skeletal elements of the head. The mechanisms regulating NC differentiation into such a large number of cell types remain largely unknown. MicroRNAs (miRNAs) play key roles in regulating developmental events suggesting they may also play a role during NC differentiation. To determine what roles miRNAs play in differentiation of NC-derived tissues, we deleted the miRNA processing gene Dicer in NC cells using the Wnt1-Cre deleter line. We show that deletion of Dicer soon after NC cells have formed does not affect their migration and colonization of their targets in the embryo. However, the post-migratory NC is dependent on Dicer for survival. In the head, loss of Dicer leads to a loss of NC-derived craniofacial bones while in the trunk, cells of the enteric, sensory and sympathetic nervous systems are lost during development. We found that loss of Dicer does not prevent the initial differentiation of NC but as development progresses, NC derivatives are lost due to apoptotic cell death. When Dicer is deleted, both Caspase-dependent and -independent apoptotic pathways are activated in the sensory ganglia but only the Caspase-dependent apoptotic program was activated in the sympathetic nervous system showing that the specific endogenous apoptotic programs are turned on by loss of Dicer. Our results show that Dicer and miRNAs, are required for survival of NC-derived tissues by preventing apoptosis during differentiation.