Zic1 and Zic4 regulate zebrafish roof plate specification and hindbrain ventricle morphogenesis

During development, the lumen of the neural tube develops into a system of brain cavities or ventricles, which play important roles in normal CNS function. We have established that the formation of the hindbrain (4th) ventricle in zebrafish is dependent upon the pleiotropic functions of the genes implicated in human Dandy Walker Malformation, Zic1 and Zic4. Using morpholino knockdown we show that zebrafish Zic1 and Zic4 are required for normal morphogenesis of the 4th ventricle. In Zic1 and/or Zic4 morphants the ventricle does not open properly, but remains completely or partially fused from the level of rhombomere (r) 2 towards the posterior. In the absence of Zic function early hindbrain regionalization and neural crest development remain unaffected, but dorsal hindbrain progenitor cell proliferation is significantly reduced. Importantly, we find that Zic1 and Zic4 are required for development of the dorsal roof plate. In Zic morphants expression of roof plate markers, including lmx1b.1 and lmx1b.2, is disrupted. We further demonstrate that zebrafish Lmx1b function is required for both hindbrain roof plate development and 4th ventricle morphogenesis, confirming that roof plate formation is a critical component of ventricle development. Finally, we show that dorsal rhombomere boundary signaling centers depend on Zic1 and Zic4 function and on roof plate signals, and provide evidence that these boundary signals are also required for ventricle morphogenesis. In summary, we conclude that Zic1 and Zic4 control zebrafish 4th ventricle morphogenesis by regulating multiple mechanisms including cell proliferation and fate specification in the dorsal hindbrain.     

Sid4: A secreted vertebrate immunoglobulin protein with roles in zebrafish embryogenesis

The small members of the immunoglobulin superfamily (IGSF) are a molecularly diverse group of proteins composed solely of immunoglobulin domains. They may be secreted or tethered to the cell mebrane via GPI linkages and are proposed to have important functions in vivo. However, very few small IGSFs have been functionally characterized. During an ongoing in situ hybridization analysis of expressed sequence tags in zebrafish we identified secreted immunoglobulin domain 4 (sid4), a gene encoding a soluble vertebrate protein composed solely of four immunoglobulin domains. Throughout development, sid4 is expressed in regions of the embryo undergoing active cell division and migration. Functional analysis using morpholino antisense oligonucleotides demonstrates that timing of gene expression is normal in morphants, but these embryos are smaller and exhibit defects in epiboly and patterning of axial and prechordal mesoderm. Analyses of chordin, pax2, krox20, and dlx2 expression in morphants demonstrate that early brain patterning is normal but later organization of hindbrain neurons and development of cranial neural crest are perturbed. Levels of apoptosis in morphants were normal prior to 90% epiboly, but were elevated after 10 h post-fertilization (hpf). Apoptosis does not account for early patterning defects of axial mesoderm, but likely contributes to overall reduction in embryo size. Phylogenetic analysis demonstrates that Sid4 is strikingly similar to the fibronectin binding Ig domains of Perlecan/HSPG2. Overall, our data demonstrate a fundamental role for sid4, possibly as a co-factor in extracellular matrix (ECM) interactions, in processes underlying tissue patterning and organogenesis in a vertebrate.     

Prep1.1 has essential genetic functions in hindbrain development and cranial neural crest cell differentiation

In this study we analysed the function of the Meinox gene prep1.1 during zebrafish development. Meinox proteins form heterotrimeric complexes with Hox and Pbx members, increasing the DNA binding specificity of Hox proteins in vitro and in vivo. However, a role for a specific Meinox protein in the regulation of Hox activity in vivo has not been demonstrated. In situ hybridization showed that prep1.1 is expressed maternally and ubiquitously up to 24 hours post-fertilization (hpf), and restricted to the head from 48 hpf onwards. Morpholino-induced prep1.1 loss-of-function caused significant apoptosis in the CNS. Hindbrain segmentation and patterning was affected severely, as revealed by either loss or defective expression of several hindbrain markers (foxb1.2/mariposa, krox20, pax2.1 and pax6.1), including anteriorly expressed Hox genes (hoxb1a, hoxa2 and hoxb2), the impaired migration of facial nerve motor neurons, and the lack of reticulospinal neurons (RSNs) except Mauthner cells. Furthermore, the heads of prep1.1 morphants lacked all pharyngeal cartilages. This was not caused by the absence of neural crest cells or their impaired migration into the pharyngeal arches, as shown by expression of dlx2 and snail1, but by the inability of these cells to differentiate into chondroblasts. Our results indicate that prep1.1 has a unique genetic function in craniofacial chondrogenesis and, acting as a member of Meinox-Pbc-Hox trimers, it plays an essential role in hindbrain development.     

SNW1 is a critical regulator of spatial BMP activity, neural plate border formation, and neural crest specification in vertebrate embryos

Bone morphogenetic protein (BMP) gradients provide positional information to direct cell fate specification, such as patterning of the vertebrate ectoderm into neural, neural crest, and epidermal tissues, with precise borders segregating these domains. However, little is known about how BMP activity is regulated spatially and temporally during vertebrate development to contribute to embryonic patterning, and more specifically to neural crest formation. Through a large-scale in vivo functional screen in Xenopus for neural crest fate, we identified an essential regulator of BMP activity, SNW1. SNW1 is a nuclear protein known to regulate gene expression. Using antisense morpholinos to deplete SNW1 protein in both Xenopus and zebrafish embryos, we demonstrate that dorsally expressed SNW1 is required for neural crest specification, and this is independent of mesoderm formation and gastrulation morphogenetic movements. By exploiting a combination of immunostaining for phosphorylated Smad1 in Xenopus embryos and a BMP-dependent reporter transgenic zebrafish line, we show that SNW1 regulates a specific domain of BMP activity in the dorsal ectoderm at the neural plate border at post-gastrula stages. We use double in situ hybridizations and immunofluorescence to show how this domain of BMP activity is spatially positioned relative to the neural crest domain and that of SNW1 expression. Further in vivo and in vitro assays using cell culture and tissue explants allow us to conclude that SNW1 acts upstream of the BMP receptors. Finally, we show that the requirement of SNW1 for neural crest specification is through its ability to regulate BMP activity, as we demonstrate that targeted overexpression of BMP to the neural plate border is sufficient to restore neural crest formation in Xenopus SNW1 morphants. We conclude that through its ability to regulate a specific domain of BMP activity in the vertebrate embryo, SNW1 is a critical regulator of neural plate border formation and thus neural crest specification.     

Zebrafish mdk2, a novel secreted midkine, participates in posterior neurogenesis

Patterning the neural plate in vertebrates depends on complex interactions between a variety of secreted growth factors. Here we describe a novel secreted factor in zebrafish, named mdk2, related to the midkine family of heparin-binding growth factors that is involved in posterior neural development. mdk2 is expressed shortly after the onset of gastrulation in the presumptive neural plate cells of the epiblast, and this expression is enhanced by exogenous retinoic acid. Ectopic expression of mdk2 enhances neural crest cell fates at the lateral edges of the caudal neural plate, concomitant with a repression of anterior structures and mesendodermal and ectodermal markers. Reciprocally, ectopic expression of a dominant negative mdk2 results in severe deficiencies of structures posterior to the midbrain-hindbrain boundary, with negligible effects on anterior structures. In these embryos, the expression of hindbrain and neural crest markers is strongly reduced, and the formation of posterior primary moto- and sensory neurons is blocked. Analyses in mutant zebrafish embryos shows that expression of mdk2 is independent of FGF8 and nodal-related-1 signaling, but is under negative control of BMP signaling. These data support the hypothesis that mdk2 participates in posterior neural development in zebrafish.     

Expression and characterization of a brain-specific protein kinase BSK146 from zebrafish

We have previously identified a novel protein kinase, pk146, in the brain of Tetraodon. In the present study, we cloned the homologous protein kinase gene encoding a protein of 385 amino acid residues from zebrafish. The overall amino acid sequence and the kinase domain of zebrafish BSK146 shows 48% and 69% identity to that of rat sbk, a SH3-containing serine/threonine protein kinase. By whole-mount in situ hybridization and RT-PCR, the expression of bsk146 mRNA was mainly in the brain. To explore the in vivo function of BSK146 during zebrafish development, we used morpholino knockdown approach and found that BSK146 morphants displayed enlarged hindbrain ventricle and smaller eyes. Whole-mount in situ hybridization was further performed to analyze the brain defects in BSK146-MO-injected embryos. The expression of brain-specific markers, such as otx2, pax2.1, and krox20, was found normal in morphant embryos at 24hpf, while expression of pax2.1 exerted changes in midbrain-hindbrain boundary and hindbrain in morphant embryos at 48hpf. These data suggest that BSK146 may play an important role in later ventricle expansion in zebrafish brain development. Although the recombinant BSK146 protein produced in insect cells was active and could phosphorylate both histone H1 and histone 2B, the endogenous substrate of BSK146 in the embryonic brain of zebrafish is not clear at the present time and needs further investigation.     

Antisense knockdown of the beta1 integrin subunit in the chicken embryo results in abnormal neural crest cell development

Neural crest cells escape the neural tube by undergoing an epithelial to mesenchymal transition (EMT). This is followed by extensive migration along specific pathways that are lined with extracellular matrix (ECM). In this study, we have examined the roles of matrix receptors containing beta1 integrin subunits in neural crest cell morphogenesis using antisense morpholino oligos electroporated in ovo into avian neural crest cell precursors. Our results show that reduced levels of expression of beta1 integrin subunits in the dorsal neural tube results in an abnormal epithelial to mesenchymal transition. In approximately half of the experimental embryos, however, some neural crest cells filled with beta1 antisense are able to escape the neural tube and migrate ventrally, indicating that grossly normal migration of trunk neural crest cells can take place after beta1 integrin expression is reduced. This study shows the potential of this novel method for investigating the roles of genes that are required for the survival of early mouse embryos in later development events.     

The retinoic acid metabolising gene, CYP26B1, patterns the cartilaginous cranial neural crest in zebrafish

We have investigated the function of the retinoic acid metabolising enzyme, CYP26B1, by administering an antisense morpholino oligonucleotide to zebrafish embryos. The result was an alteration in the morphology of the embryo in those regions which express the gene, namely an embryo with a smaller head, correspondingly smaller hindbrain rhombomeres and severely reduced numbers of vagal brachiomotor neurons. Most strikingly, these embryos had defective or absent jaw cartilages implying a role for this enzyme in patterning or migration of the neural crest cells which give rise to this tissue type. In order to determine whether this phenotype resembles that of excess retinoic acid or a deficiency of retinoic acid, we compared the jaw defects following retinoic acid treatment or DEAB treatment, the latter being an inhibitor of retinoic acid synthesis. The effects of the inhibitor rather than excess retinoic acid most closely phenocopied the jaw defects seen with the Cyp26B1 morpholino which suggests that, at least in the zebrafish embryo, the action of CYP26B1 in the neural crest may not be simply to catabolise all-trans-RA.     

Involvement of Livertine, a hepatocyte growth factor family member, in neural morphogenesis

The formation of the nervous system in vertebrate embryos involves extensive morphogenetic movements that include the folding of the neural tube and the migration of neural crest cells. Changes in cell shape and cell movements underlie neural morphogenesis but the molecular mechanisms involved in these processes in vivo are not well understood. Here, we show that a new member of the hepatocyte growth factor family, which we name Livertine, is expressed in frog embryos in neural cells including neural crest and midline neural plate cells which are undergoing pronounced morphogenetic movements. The ectopic expression of Livertine perturbs gastrulation and leads to positional changes in injected cells without apparently changing cell type. These results suggest that one of the normal functions of Livertine is the control of neural morphogenesis in the vertebrate embryo.     

Reiterated Wnt signaling during zebrafish neural crest development

While Wnt/beta-catenin signaling is known to be involved in the development of neural crest cells in zebrafish, it is unclear which Wnts are involved, and when they are required. To address these issues we employed a zebrafish line that was transgenic for an inducible inhibitor of Wnt/beta-catenin signaling, and inhibited endogenous Wnt/beta-catenin signaling at discrete times in development. Using this approach, we defined a critical period for Wnt signaling in the initial induction of neural crest, which is distinct from the later period of development when pigment cells are specified from neural crest. Blocking Wnt signaling during this early period interfered with neural crest formation without blocking development of dorsal spinal neurons. Transplantation experiments suggest that neural crest precursors must directly transduce a Wnt signal. With regard to identifying which endogenous Wnt is responsible for this initial critical period, we established that wnt8 is expressed in the appropriate time and place to participate in this process. Supporting a role for Wnt8, blocking its function with antisense morpholino oligonucleotides eliminates initial expression of neural crest markers. Taken together, these results demonstrate that Wnt signals are critical for the initial induction of zebrafish neural crest and suggest that this signaling pathway plays reiterated roles in its development.     

Tenascin-C mRNA is expressed in cranial neural crest cells,in some placodal derivatives,and in discrete domains of the embryonic zebrafish brain

A partial zebrafish tenascin-C cDNA clone was isolated from an embryonic zebrafish cDNA library on the basis of homology to mouse tenascin-C. The expression pattern in the head of embryonic zebrafish was analyzed by in situ hybridization. Tenascin-C mRNA was detected in neural crest cells during the period of their migration and differentiation. Expression also occurred in differentiating placodal tissues and in mesodermal cells. In the developing brain, tenascin-C mRNA was expressed in specific domains. In the hindbrain the pattern of the domains was dynamic. At 18 to 22 h postfertilization, expression was widespread in rhombomeres 3, 5, and 6, confined to periventricular cells in rhombomere 2, and not detectable in rhombomere 4. At 32 h postfertilization, tenascin-C was expressed at the rhombomere boundaries. In contrast to the hindbrain, the pattern in the forebrain and midbrain did not show any major changes between 22 and 32 h postfertilization. Domains expressing tenascin-C alternated with regions devoid of it. The most anterior domain of expression was observed at the telencephalic-diencephalic border, surrounding the optic recess. A second domain, at the border between the diencephalon and the midbrain, and a third domain, in the caudal midbrain tegmentum, appeared restricted to the basal plate. Additionally, expression of tenascin-C mRNA was detected in the hypothalamus and in the developing epiphysis. These expression patterns suggest that tenascin-C may play a role in neural crest cell migration and during the differentiation of neural crest, placodal, and mesodermal derivatives. In the developing brain, tenascin-C may be involved in the consolidation of different regional identities. © 1995 John Wiley & Sons, Inc.