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.     

Cardiac neural crest contributes to cardiomyogenesis in zebrafish

In birds and mammals, cardiac neural crest is essential for heart development and contributes to conotruncal cushion formation and outflow tract septation. The zebrafish prototypical heart lacks outflow tract septation, raising the question of whether cardiac neural crest exists in zebrafish. Here, results from three distinct lineage-labeling approaches identify zebrafish cardiac neural crest cells and indicate that these cells have the ability to generate MF20-positive muscle cells in the myocardium of the major chambers during development. Fate-mapping demonstrates that cardiac neural crest cells originate both from neural tube regions analogous to those found in birds, as well as from a novel region rostral to the otic vesicle. In contrast to other vertebrates, cardiac neural crest invades the myocardium in all segments of the heart, including outflow tract, atrium, atrioventricular junction, and ventricle in zebrafish. Three distinct groups of premigratory neural crest along the rostrocaudal axis have different propensities to contribute to different segments in the heart and are correspondingly marked by unique combinations of gene expression patterns. Zebrafish will serve as a model for understanding interactions between cardiac neural crest and cardiovascular development.     

Xenopus Teashirt1 regulates posterior identity in brain and cranial neural crest

We have isolated two related Xenopus homologues of the homeotic zinc finger protein Teashirt1 (Tsh1), XTsh1a and XTsh1b. While Drosophila teashirt specifies trunk identity in the fly, the developmental relevance of vertebrate Tsh homologues is unknown. XTsh1a/b are expressed in prospective trunk CNS throughout early neurula stages and later in the migrating cranial neural crest (CNC) of the third arch. In postmigratory CNC, XTsh1a/b is uniformly activated in the posterior arches. Gain- and loss-of-function experiments reveal that reduction or increase of XTsh1 levels selectively inhibits specification of the hindbrain and mid/hindbrain boundary in Xenopus embryos. In addition, both overexpression and depletion of XTsh1 interfere with the determination of CNC segment identity. In transplantation assays, ectopic XTsh1a inhibits the routing of posterior, but not of mandibular CNC streams. The loss of function phenotype could be rescued with low amounts either of XTsh1a or murine Tsh3. Our results demonstrate that proper expression of XTsh1 is essential for segmentally restricted gene expression in the posterior brain and CNC and suggest for the first time that teashirt genes act as positional factors also in vertebrate development.     

Semaphorin signaling guides cranial neural crest cell migration in zebrafish

Cranial neural crest cells (NCCs) migrate into the pharyngeal arches in three primary streams separated by two cranial neural crest (NC)-free zones. Multiple tissues have been implicated in the guidance of cranial NCC migration; however, the signals provided by these tissues have remained elusive. We investigate the function of semaphorins (semas) and their receptors, neuropilins (nrps), in cranial NCC migration in zebrafish. We find that genes of the sema3F and sema3G class are expressed in the cranial NC-free zones, while nrp2a and nrp2b are expressed in the migrating NCCs. sema3F/3G expression is expanded homogeneously in the head periphery through which the cranial NCCs migrate in lzr/pbx4 mutants, in which the cranial NC streams are fused. Antisense morpholino knockdown of Sema3F/3G or Nrp2 suppresses the abnormal cranial NC phenotype of lzr/pbx4 mutants, demonstrating that aberrant Sema3F/3G-Nrp2 signaling is responsible for this phenotype and suggesting that repulsive Sema3F/3G-Npn2 signaling normally contributes to the guidance of migrating cranial NCCs. Furthermore, global over-expression of sema3Gb phenocopies the aberrant cranial NC phenotype of lzr/pbx4 mutants when endogenous Sema3 ligands are knocked down, consistent with a model in which the patterned expression of Sema3 ligands in the head periphery coordinates the migration of Nrp-expressing cranial NCCs.     

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.     

Requirement for frzb and fzd7a in cranial neural crest convergence and extension mechanisms during zebrafish palate and jaw morphogenesis

Regulation of convergence and extension by wnt-frizzled signaling is a common theme in embryogenesis. This study examines the functional requirements of frzb and fzd7a in convergence and extension mechanisms during craniofacial development. Using a morpholino knockdown approach, we found that frzb and fzd7a are dispensable for directed migration of the bilateral trabeculae, but necessary for the convergence and extension of the palatal elements, where the extension process is mediated by chondrocyte proliferation, morphologic change and intercalation. In contrast, frzb and fzd7a are required for convergence of the mandibular prominences, where knockdown of either frzb or fzd7a resulted in complete loss of lower jaw structures. Further, we found that bapx1 was specifically downregulated in the wnt9a/frzb/fzd7a morphants, while general neural crest markers were unaffected. In addition, expression of wnt9a and frzb was also absent in the edn−/− mutant. Notably, over-expression of bapx1 was sufficient to partially rescue mandibular elements in the wnt9a/frzb/fzd7a morphants, demonstrating genetic epistasis of bapx1 acting downstream of edn1 and wnt9a/frzb/fzd7a in lower jaw development. This study underscores the important role of wnt-frizzled signaling in convergence and extension in palate and craniofacial morphogenesis, distinct regulation of upper vs. lower jaw structures, and integration of wnt-frizzled with endothelin signaling to coordinate shaping of the facial form.     

The development and distribution of the cranial neural crest in the rat embryo

Summary The head region of rat embryos was investigated by scanning electron microscopy after removal of the surface ectoderm with adhesive tape. Observations were made in embryos from 6-somite to 11-somite stages of development, in order to determine: (1) the sequence of emigration of neural crest cells from the different regions of the future brain; (2) the appearance of crest cells before, during, and after their conversion from an epithelial to a mesenchymal form; (3) the migration pathways.Emigration occurs first from the midbrain, and next from the rostral hindbrain; crest cells from these two regions migrate into the first visceral arch. Subsequently cells emigrate from the caudal hindbrain, but not in a rostrocaudal sequence. At the time of crest cell emigration, the neural fold morphology varies from a slightly convex, widely open plate (midbrain) to a closed tube (caudal hindbrain). Thus the timing of emigration is related neither to age (as reflected in rostrocaudal levels) nor to morphology of the neural epithelium.     

Neph3 associates with regulation of glomerular and neural development in zebrafish

Neph3 (filtrin) is a membrane protein expressed in the glomerular epithelial cells (podocytes), but its role in the glomerulus is still largely unknown. To characterize the function of Neph3 in the glomerulus, we employed the zebrafish as a model system. Here we show that the expression of neph3 in pronephros starts before the onset of nephrin and podocin expression, peaks when the nephron primordium differentiates into glomerulus and tubulus, and is then downregulated upon glomerular maturation. By histology, we found that neph3 is specifically expressed in pronephric podocytes at 36 hpf. Furthermore, disruption of neph3 expression by antisense morpholino oligonucleotides results in distorted body curvature and transient pericardial edema, the latter likely reflecting perturbation of glomerular osmoregulatory function. Histological analysis of neph3 morphants reveals altered glomerular morphology and dilated pronephric tubules. The phenotype of neph3 morphants, curved body and pericardial edema, is rescued by wild-type zebrafish neph3 mRNA. In addition to glomerulus, neph3 is highly expressed in the developing brain and specific regions of mature midbrain and hindbrain. In line with this, neph3 morphants show aberrant brain morphology. Collectively, the expression of neph3 in glomerulus and brain together with the morphant phenotype imply that neph3 is a pleiotropic gene active during distinct stages of tissue differentiation and associates directly in the regulation of both glomerular and neural development.     

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.     

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.     

meis1 regulates the development of endothelial cells in zebrafish

The differentiation of endothelial cells is tightly connected with the formation of blood vessels during vertebrate development. The signaling pathways mediated by vascular endothelial growth factor (vegf) are required for these processes. Here we show that a proto-oncogene, meis1, plays important roles in the vascular development in zebrafish. Knockdown of meis1 by anti-sense meis1 morpholino (meis1 MO) led to the impairment of intersegmental vessel (ISV) formation. In meis1 morphants, the expression of an artery marker was reduced in dorsal aorta (DA), and the expression of vein markers was expanded in DA and posterior cardinal vein (PCV), suggesting the defects on artery development. Furthermore, the expression of vegf receptor, flk1, was significantly decreased in these embryos. Interestingly, flk1 MO-injected embryos exhibited similar defects as meis1 morphants. Thus, these results implicate that meis1 is a novel regulator involved in endothelial cell development, presumably affecting the vegf signaling pathway.     

Conservation and diversity in the cis-regulatory networks that integrate information controlling expression of Hoxa2 in hindbrain and cranial neural crest cells in vertebrates

The Hoxa2 and Hoxb2 genes are members of paralogy group II and display segmental patterns of expression in the developing vertebrate hindbrain and cranial neural crest cells. Functional analyses have demonstrated that these genes play critical roles in regulating morphogenetic pathways that direct the regional identity and anteroposterior character of hindbrain rhombomeres and neural crest-derived structures. Transgenic regulatory studies have also begun to characterize enhancers and cis-elements for those mouse and chicken genes that direct restricted patterns of expression in the hindbrain and neural crest. In light of the conserved role of Hoxa2 in neural crest patterning in vertebrates and the similarities between paralogs, it is important to understand the extent to which common regulatory networks and elements have been preserved between species and between paralogs. To investigate this problem, we have cloned and sequenced the intergenic region between Hoxa2 and Hoxa3 in the chick HoxA complex and used it for making comparative analyses with the respective human, mouse, and horn shark regions. We have also used transgenic assays in mouse and chick embryos to test the functional activity of Hoxa2 enhancers in heterologous species. Our analysis reveals that three of the critical individual components of the Hoxa2 enhancer region from mouse necessary for hindbrain expression (Krox20, BoxA, and TCT motifs) have been partially conserved. However, their number and organization are highly varied for the same gene in different species and between paralogs within a species. Other essential mouse elements appear to have diverged or are absent in chick and shark. We find the mouse r3/r5 enhancer fails to work in chick embryos and the chick enhancer works poorly in mice. This implies that new motifs have been recruited or utilized to mediate restricted activity of the enhancer in other species. With respect to neural crest regulation, cis-components are embedded among the hindbrain control elements and are highly diverged between species. Hence, there has been no widespread conservation of sequence identity over the entire enhancer domain from shark to humans, despite the common function of these genes in head patterning. This provides insight into how apparently equivalent regulatory regions from the same gene in different species have evolved different components to potentiate their activity in combination with a selection of core components.