A role for N-cadherin in mesodermal morphogenesis during gastrulation.
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Cell adhesion molecules mediate numerous developmental processes necessary for the segregation and organization of tissues. Here we show that the zebrafish biber (bib) mutant encodes a dominant allele at the N-cadherin locus. When knocked down with antisense oligonucleotides, bib mutants phenocopy parachute (pac) null alleles, demonstrating that bib is a gain-of-function mutation. The mutant phenotype disrupts normal cell-cell contacts throughout the mesoderm as well as the ectoderm. During gastrulation stages, cells of the mesodermal germ layer converge slowly; during segmentation stages, the borders between paraxial and axial tissues are irregular and somite borders do not form; later, myotomes are fused. During neurulation, the neural tube is disorganized. Although weaker, all traits present in bib mutants were found in pac mutants. When the distribution of N-cadherin mRNA was analyzed to distinguish mesodermal from neuroectodermal expression, we found that N-cadherin is strongly expressed in the yolk cell and hypoblast in the early gastrula, just preceding the appearance of the bib mesodermal defects. Only later is N-cadherin expressed in the anlage of the CNS, where it is found as a radial gradient in the forming neural plate. Hence, besides a well-established role in neural and somite morphogenesis, N-cadherin is essential for morphogenesis of the mesodermal germ layer during gastrulation. (+info)
Initial motor axon outgrowth from the developing central nervous system.
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Rat and chick studies show that the earliest motor rootlet axon bundles emerge from all levels of the neural tube between radial glial end feet which comprise the presumptive glia limitans. The loose arrangement of the end feet at the time of emergence facilitates this passage. The points of emergence are regularly spaced in relation to the long axis of the neural tube and are not defined by any cell contact with its surface. Each rootlet carries a covering of basal lamina from the neural tube surface, which forms a sleeve around it. It is only after bundles of ventral rootlet axons have emerged that cells associate with them, forming clusters on the rootlet surface at a distance peripheral to the CNS surface of both species. A tight collar of glial end feet develops around the axon bundle at the neural tube surface shortly after initial emergence. These arrangements are in sharp contrast to those seen in the sensory rootlets, where clusters of boundary cap cells prefigure the sensory entry zones at the attachments of the prospective dorsal spinal and cranial sensory rootlets. Boundary cap cells resemble cluster cells and a neural crest origin seems the most likely for them. The study clearly demonstrates that no features resembling boundary caps are found in relation to the developing motor exit points. (+info)
Transcription factor TEAD2 is involved in neural tube closure.
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TEAD2, one of the first transcription factors expressed at the beginning of mammalian development, appears to be required during neural development. For example, Tead2 expression is greatest in the dorsal neural crest where it appears to regulate expression of Pax3, a gene essential for brain development. Consistent with this hypothesis, we found that inactivation of the Tead2 gene in mice significantly increased the risk of exencephaly (a defect in neural tube closure). However, none of the embryos exhibited spina bifida, the major phenotype of Pax3 nullizygous embryos, and expression of Pax3 in E11.5 Tead2 nullizygous embryos was normal. Thus, Tead2 plays a role in neural tube closure that is independent of its putative role in Pax3 regulation. In addition, the risk of exencephaly was greatest with Tead2 nullizygous females, and could be suppressed either by folic acid or pifithrin-alpha. These results reveal a maternal genetic contribution to neural tube closure, and suggest that Tead2-deficient mice provide a model for anencephaly, a common human birth defect that can be prevented by folic acid. (+info)
Activity-dependent neuroprotective protein constitutes a novel element in the SWI/SNF chromatin remodeling complex.
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Complete deficiency in activity-dependent neuroprotective protein (ADNP), a heterochromatin 1-binding protein, results in dramatic changes in gene expression, neural tube closure defects, and death at gestation day 9 in mice. To further understand the cellular roles played by ADNP, the HEK293 human embryonic kidney cell line that allows efficient transfection with recombinant DNA was used as a model for the identification of ADNP-interacting proteins. Recombinant green fluorescent protein (GFP)-ADNP was localized to cell nuclei. When nuclear extracts were subjected to immunoprecipitation with specific GFP antibodies followed by polyacrylamide gel electrophoresis, several minor protein bands were observed in addition to GFP-ADNP. In-gel protein digests followed by mass spectrometry identified BRG1, BAF250a, and BAF170, all components of the SWI/SNF (mating type switching/sucrose nonfermenting) chromatin remodeling complex, as proteins that co-immunoprecipitate with ADNP. These results were verified utilizing BRG1 antibodies. ADNP short hairpin RNA down-regulation resulted in microtubule reorganization and changes in cell morphology including reduction in cell process formation and cell number. These morphological changes are closely associated with the SWI/SNF complex multifunctionality. Taken together, the current study uncovers a molecular basis for the essential function of the ADNP gene and protein. (+info)
Morphogens and the control of cell proliferation and patterning in the spinal cord.
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The development of animal embryos depends on accurate coordination of the growth and specification of precursor cells. Morphogens, extracellular signals that act at a distance to control cell fate, are crucial in the patterning of embryonic tissues. One of the most extensively studied examples of a morphogen patterned tissue is the developing vertebrate spinal cord. The distribution of distinct neuronal subtypes along the dorsoventral (DV) axis of the spinal cord is determined by counteracting gradients of long-range signals. Wnt and BMP signals promote dorsal identities, while Shh signaling induces ventral identities. Simultaneous to their specification, neural progenitors proliferate, facilitating the growth of the neural tube. In this review we discuss evidence indicating that the signals governing progenitor specification also control proliferation and survival of progenitor cells. Moreover, evidence of reciprocal transcriptional interactions and cross-talk between the signaling pathways has emerged from recent studies. Together these studies suggest ways in which patterning and growth may be coordinated in the spinal cord. One level of interaction is an inhibitory regulation of repressor forms of the transcription factor Gli3 - generated in the absence of Shh - on beta-catenin activity, the transcription factor activated by Wnt signaling. This interaction may also be relevant in other tissues and situations in which the two signaling pathways are known to participate. (+info)
Ethanol teratogenesis in Japanese medaka: effects at the cellular level.
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The adverse effects of alcohol on the developing humans represent a spectrum of structural and neurobehavioral abnormalities, most appropriately termed as fetal alcohol spectrum disorder (FASD). The mechanism by which ethanol induces FASD is unknown. Human studies of FASD are very limited due to ethical constraints; however, several animal models from nematodes to mammals are utilized to understand the molecular mechanism of this disorder. We have used Japanese medaka (Oryzias latipes) embryo-larval development as a unique non-mammalian model to study the molecular mechanism of FASD. Fertilized medaka eggs were exposed to ethanol (0-400 mM) for 48 h post fertilization (hpf) and then maintained in regular embryo rearing medium without ethanol. Viable embryos were harvested on 0, 2, 4 and 6 day post fertilization (dpf) and analyzed for DNA, RNA and protein contents of the embryos. By applying semi-quantitative RT-PCR (rRT-PCR) and quantitative real-time RT-PCR (qRT-PCR), RNA samples were further analyzed for seven transcription factors, emx2, en2, iro3, otx2, shh, wnt1 and zic5 which are expressed in the neural tube of medaka embryo during early phase of development. RNA and protein contents of the embryos were significantly reduced by ethanol at 400 mM dose on 4 and 6 dpf compared to the control (no ethanol), and 100 mM ethanol treated embryos. However, significant reduction of DNA was observed only in 4 dpf embryos. Total protein contents of yolk remained unaltered after ethanol treatment. Expression pattern of emx2, en2, iro3, otx2, shh, wnt1, and zic5 mRNAs were found to be developmentally regulated, however, remained unaltered after ethanol treatment. It is therefore concluded that alteration of nucleic acid and protein contents of medaka embryo by ethanol could be used as an indicator of embryonic growth retardation which might be the result of disruption of specific gene function during development. (+info)
Labeling efficacy of superparamagnetic iron oxide nanoparticles to human neural stem cells: comparison of ferumoxides, monocrystalline iron oxide, cross-linked iron oxide (CLIO)-NH2 and tat-CLIO.
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OBJECTIVE: We wanted to compare the human neural stem cell (hNSC) labeling efficacy of different superparamagnetic iron oxide nanoparticles (SPIONs), namely, ferumoxides, monocrystalline iron oxide (MION), cross-linked iron oxide (CLIO)-NH(2) and tat-CLIO. MATERIALS AND METHODS: The hNSCs (5 x 10(5) HB1F3 cells/ml) were incubated for 24 hr in cell culture media that contained 25 microg/ml of ferumoxides, MION or CLIO-NH(2), and with or without poly-L-lysine (PLL) and tat-CLIO. The cellular iron uptake was analyzed qualitatively with using a light microscope and this was quantified via atomic absorption spectrophotometry. The visibility of the labeled cells was assessed with MR imaging. RESULTS: The incorporation of SPIONs into the hNSCs did not affect the cellular proliferations and viabilities. The hNSCs labeled with tat-CLIO showed the longest retention, up to 72 hr, and they contained 2.15+/-0.3 pg iron/cell, which are 59 fold, 430 fold and six fold more incorporated iron than that of the hNSCs labeled with ferumoxides, MION or CLIO-NH(2), respectively. However, when PLL was added, the incorporation of ferumoxides, MION or CLIO-NH(2) into the hNSCs was comparable to that of tat-CLIO. CONCLUSION: For MR imaging, hNSCs can be efficiently labeled with tat-CLIO alone or with a combination of ferumoxides, MION, CLIO-NH(2) and the transfection agent PLL. (+info)
Rab23 GTPase is expressed asymmetrically in Hensen's node and plays a role in the dorsoventral patterning of the chick neural tube.
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The mouse Rab23 protein, a Ras-like GTPase, inhibits signaling through the Sonic hedgehog pathway and thus exerts a role in the dorsoventral patterning of the spinal cord. Rab23 mouse mutant embryos lack dorsal spinal cord cell types. We cloned the chicken Rab23 gene and studied its expression in the developing nervous system. Chick Rab23 mRNA is initially expressed in the entire neural tube but retracts to the dorsal alar plate. Unlike in mouse, we find Rab23 in chick already expressed asymmetrically during gastrulation. Ectopic expression of Rab23 in ventral midbrain induced dorsal genes (Pax3, Pax7) ectopically and reduced ventral genes (Nkx2.2 and Nkx6) without influencing cell proliferation or neurogenesis. Thus, in the developing brain of chick embryos Rab23 acts in the same manner as described for the caudal spinal cord in mouse. These data indicate that Rab23 plays an important role in patterning the dorso-ventral axis by dorsalizing the neural tube. (+info)