Direct action of the nodal-related signal cyclops in induction of sonic hedgehog in the ventral midline of the CNS

The secreted molecule Sonic hedgehog (Shh) is crucial for floor plate and ventral brain development in amniote embryos. In zebrafish, mutations in cyclops (cyc), a gene that encodes a distinct signal related to the TGF(beta) family member Nodal, result in neural tube defects similar to those of shh null mice. cyc mutant embryos display cyclopia and lack floor plate and ventral brain regions, suggesting a role for Cyc in specification of these structures. cyc mutants express shh in the notochord but lack expression of shh in the ventral brain. Here we show that Cyc signalling can act directly on shh expression in neural tissue. Modulation of the Cyc signalling pathway by constitutive activation or inhibition of Smad2 leads to altered shh expression in zebrafish embryos. Ectopic activation of the shh promoter occurs in response to expression of Cyc signal transducers in the chick neural tube. Furthermore an enhancer of the shh gene, which controls ventral neural tube expression, is responsive to Cyc signal transducers. Our data imply that the Nodal related signal Cyc induces shh expression in the ventral neural tube. Based on the differential responsiveness of shh and other neural tube specific genes to Hedgehog and Cyc signalling, a two-step model for the establishment of the ventral midline of the CNS is proposed.     

Dorsal and lateral fates in the mouse neural tube require the cell-autonomous activity of the open brain gene

The processes that specify early regional identity in dorsal and lateral regions of the mammalian neural tube are not well understood. The mouse open brain (opb) gene plays an essential role in dorsal neural patterning: in the caudal spinal cord of opb mutants, dorsal cell types are absent and markers of ventral fates, including Shh, expand into dorsal regions. Analysis of the opb mutant phenotype and of opb/opb <--> wild-type chimeric embryos reveals that early in neural development, the wild-type opb gene (opb(+)) is required cell autonomously for the expression of Pax7 in dorsal cells and Pax6 in lateral cells. Thus the opb(+) gene product acts intracellularly in the reception or interpretation of signals that determine cell types in the dorsal 80% of the neural tube. At later stages, the lack of opb(+) causes a non-cell-autonomous expansion of ventral cell types into dorsal regions of the neural tube, revealing that opb(+) controls the production of a diffusible molecule that defines the domain of Shh expression. The data indicate that opb(+) could act as either a novel component of a dorsalizing pathway or a novel intracellular negative regulator of the Shh signal transduction pathway.     

The neural tube patterns vessels developmentally using the VEGF signaling pathway

Embryonic blood vessels form in a reproducible pattern that interfaces with other embryonic structures and tissues, but the sources and identities of signals that pattern vessels are not well characterized. We hypothesized that the neural tube provides vascular patterning signal(s) that direct formation of the perineural vascular plexus (PNVP) that encompasses the neural tube at mid-gestation. Both surgically placed ectopic neural tubes and ectopic neural tubes engineered genetically were able to recruit a vascular plexus, showing that the neural tube is the source of a vascular patterning signal. In mouse-quail chimeras with the graft separated from the neural tube by a buffer of host cells, graft-derived vascular cells contributed to the PNVP, indicating that the neural tube signal(s) can act at a distance. Murine neural tube vascular endothelial growth factor A (VEGFA) expression was temporally and spatially correlated with PNVP formation, suggesting it is a component of the neural tube signal. A collagen explant model was developed in which presomitic mesoderm explants formed a vascular plexus in the presence of added VEGFA. Co-cultures between presomitic mesoderm and neural tube also supported vascular plexus formation, indicating that the neural tube could replace the requirement for VEGFA. Moreover, a combination of pharmacological and genetic perturbations showed that VEGFA signaling through FLK1 is a required component of the neural tube vascular patterning signal. Thus, the neural tube is the first structure identified as a midline signaling center for embryonic vascular pattern formation in higher vertebrates, and VEGFA is a necessary component of the neural tube vascular patterning signal. These data suggest a model whereby embryonic structures with little or no capacity for angioblast generation act as a nexus for vessel patterning.     

Dynamic alterations in gene expression after Wnt-mediated induction of avian neural crest

The Wnt signaling pathway is important in the formation of neural crest cells in many vertebrates, but the downstream targets of neural crest induction by Wnt are largely unknown. Here, we examined quantitative changes in gene expression regulated by Wnt-mediated neural crest induction using quantitative PCR (QPCR). Induction was recapitulated in vitro by adding soluble Wnt to intermediate neural plate tissue cultured in collagen, and induced versus control tissue were assayed using gene-specific primers at times corresponding to premigratory (18 and 24 h) or early (36 h) stages of crest migration. The results show that Wnt signaling up-regulates in a distinct temporal pattern the expression of several genes normally expressed in the dorsal neural tube (slug, Pax3, Msx1, FoxD3, cadherin 6B) at "premigratory" stages. While slug is maintained in early migrating crest cells, Pax3, FoxD3, Msx1 and cadherin 6B all are down-regulated by the start of migration. These results differ from the temporal profile of these genes in response to the addition of recombinant BMP4, where gene expression seems to be maintained. Interestingly, expression of rhoB is unchanged or even decreased in response to Wnt-mediated induction at all times examined, though it is up-regulated by BMP signals. The temporal QPCR profiles in our culture paradigm approximate in vivo expression patterns of these genes before neural crest migration, and are consistent with Wnt being an initial neural crest inducer with additional signals like BMP and other factors maintaining expression of these genes in vivo. Our results are the first to quantitatively describe changes in gene expression in response to a Wnt or BMP signal during transformation of a neural tube cell into a migratory neural crest cell.     

Initial formation of zebrafish brain ventricles occurs independently of circulation and requires the nagie oko and snakehead/atp1a1a.1 gene products

The mechanisms by which the vertebrate brain develops its characteristic three-dimensional structure are poorly understood. The brain ventricles are a highly conserved system of cavities that form very early during brain morphogenesis and that are required for normal brain function. We have initiated a study of zebrafish brain ventricle development and show here that the neural tube expands into primary forebrain, midbrain and hindbrain ventricles rapidly, over a 4-hour window during mid-somitogenesis. Circulation is not required for initial ventricle formation, only for later expansion. Cell division rates in the neural tube surrounding the ventricles are higher than between ventricles and, consistently, cell division is required for normal ventricle development. Two zebrafish mutants that do not develop brain ventricles are snakehead and nagie oko. We show that snakehead is allelic to small heart, which has a mutation in the Na+K+ ATPase gene atp1a1a.1. The snakehead neural tube undergoes normal ventricle morphogenesis; however, the ventricles do not inflate, probably owing to impaired ion transport. By contrast, mutants in nagie oko, which was previously shown to encode a MAGUK family protein, fail to undergo ventricle morphogenesis. This correlates with an abnormal brain neuroepithelium, with no clear midline and disrupted junctional protein expression. This study defines three steps that are required for brain ventricle development and that occur independently of circulation: (1) morphogenesis of the neural tube, requiring nok function; (2) lumen inflation requiring atp1a1a.1 function; and (3) localized cell proliferation. We suggest that mechanisms of brain ventricle development are conserved throughout the vertebrates.     

Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the developing zebrafish brain (Gutzman et al, 2008). Such analysis affords a new understanding of the underlying cell biology required for development of the 3D structure of the vertebrate brain, and significantly increases our ability to study neural tube morphogenesis. The embryonic zebrafish brain is shaped beginning at 18 hours post fertilization (hpf) as the ventricles within the neuroepithelium inflate. By 24 hpf, the initial steps of neural tube morphogenesis are complete. Using the method described here, embryos at the one cell stage are injected with mRNA encoding membrane-targeted green fluorescent protein (memGFP). After injection and incubation, the embryo, now between 18 and 24 hpf, is mounted, inverted, in agarose and imaged by confocal microscopy. Notably, the zebrafish embryo is transparent making it an ideal system for fluorescent imaging. While our analyses have focused on the midbrain-hindbrain boundary and the hindbrain, this method could be extended for analysis of any region in the zebrafish to a depth of 80-100 μm.Open in a separate windowClick here to view.^{(44M, flv)}     

Analysis of CD4 gene expression in human fetal brain and neuroblasts

1. The expression of the gene codifying for CD4, the most important human immunodeficiency virus type 1 (HIV-1) receptor molecule, was analyzed in 11 fetal brains at various gestational ages and in 9 human neuroblastoma (NB) cell lines. CD4 gene expression in fetal and malignant neural cells was then compared with that observed in a hematopoietic cell line and adult hippocampus. 2. In addition, CD4 mRNA was evaluated in two NB cell lines induced to differentiate in vitro with retinoic acid (RA) or 1-(5-isoquinolinyl-sulfonyl)-2-methyl piperazine (H7), a protein kinase C inhibitor. 3. All fetal brains and NB cell lines express a 1.8-kb signal when hybridized with pT4BcDNA probe, while a 3.0-kb signal such as observed in hematopoietic human cells was found in 1 of 11 fetal brains and in 0 of 9 NB cell lines. The 1.8-kb signal was lost in all analyzed poly(A)+ mRNA samples. 4. Moreover, CD4 gene expression was not induced in either RA- or H7-treated NB cells at any tested time and dose. The analysis of NB cells by polymerase chain reaction failed to demonstrate CD4 expression in either poly(A)+ or poly(A)- RNA. 5. In conclusion, the results show that the 1.8-kb signal observed in RNA extracted from fetal or transformed human neural cells is probably due to an aspecific hybridization. However, the gene codifying for CD4 can rarely be expressed by fetal brain cells early during gestation, in still unclear circumstances.     

FGF signaling establishes the anterior border of the Ciona neural tube

The Ciona tadpole is constructed from simple, well-defined cell lineages governed by provisional gene networks that have been defined via extensive gene disruption assays. Here, we examine the patterning of the anterior neural plate, which produces placodal derivatives such as the adhesive palps and stomodeum, as well as the sensory vesicle (simple brain) of the Ciona tadpole. Evidence is presented that the doublesex-related gene DMRT is expressed throughout the anterior neural plate of neurulating embryos. It leads to the activation of FoxC and ZicL in the palp placode and anterior neural tube, respectively. This differential expression depends on FGF signaling, which inhibits FoxC expression in the anterior neural tube. Inhibition of FGF signaling leads to expanded expression of FoxC, the loss of ZicL, and truncation of the anterior neural tube.     

Misexpression of BRE gene in the developing chick neural tube affects neurulation and somitogenesis

The brain and reproductive expression (BRE) gene is expressed in numerous adult tissues and especially in the nervous and reproductive systems. However, little is known about BRE expression in the developing embryo or about its role in embryonic development. In this study, we used in situ hybridization to reveal the spatiotemporal expression pattern for BRE in chick embryo during development. To determine the importance of BRE in neurogenesis, we overexpressed BRE and also silenced BRE expression specifically in the neural tube. We established that overexpressing BRE in the neural tube indirectly accelerated Pax7⁺ somite development and directly increased HNK-1⁺ neural crest cell (NCC) migration and TuJ-1⁺ neurite outgrowth. These altered morphogenetic processes were associated with changes in the cell cycle of NCCs and neural tube cells. The inverse effect was obtained when BRE expression was silenced in the neural tube. We also determined that BMP4 and Shh expression in the neural tube was affected by misexpression of BRE. This provides a possible mechanism for how altering BRE expression was able to affect somitogenesis, neurogenesis, and NCC migration. In summary, our results demonstrate that BRE plays an important role in regulating neurogenesis and indirectly somite differentiation during early chick embryo development.     

Kif11 dependent cell cycle progression in radial glial cells is required for proper neurogenesis in the zebrafish neural tube

Radial glia serve as the resident neural stem cells in the embryonic vertebrate nervous system, and their proliferation must be tightly regulated to generate the correct number of neuronal and glial cell progeny in the neural tube. During a forward genetic screen, we recently identified a zebrafish mutant in the kif11 loci that displayed a significant increase in radial glial cell bodies at the ventricular zone of the spinal cord. Kif11, also known as Eg5, is a kinesin-related, plus-end directed motor protein responsible for stabilizing and separating the bipolar mitotic spindle. We show here that Gfap+ radial glial cells express kif11 in the ventricular zone and floor plate. Loss of Kif11 by mutation or pharmacological inhibition with S-trityl-l-cysteine (STLC) results in monoastral spindle formation in radial glial cells, which is characteristic of mitotic arrest. We show that M-phase radial glia accumulate over time at the ventricular zone in kif11 mutants and STLC treated embryos. Mathematical modeling of the radial glial accumulation in kif11 mutants not only confirmed an ~226× delay in mitotic exit (likely a mitotic arrest), but also predicted two modes of increased cell death. These modeling predictions were supported by an increase in the apoptosis marker, anti-activated Caspase-3, which was also found to be inversely proportional to a decrease in cell proliferation. In addition, treatment with STLC at different stages of neural development uncovered two critical periods that most significantly require Kif11 function for stem cell progression through mitosis. We also show that loss of Kif11 function causes specific reductions in oligodendroglia and secondary interneurons and motorneurons, suggesting these later born populations require proper radial glia division. Despite these alterations to cell cycle dynamics, survival, and neurogenesis, we document unchanged cell densities within the neural tube in kif11 mutants, suggesting that a mechanism of compensatory regulation may exist to maintain overall proportions in the neural tube. We propose a model in which Kif11 normally functions during mitotic spindle formation to facilitate the progression of radial glia through mitosis, which leads to the maturation of progeny into specific secondary neuronal and glial lineages in the developing neural tube.     

Cell lineages and early patterns of embryonic CNS vascularization

First steps of blood vessel formation and patterning in the central nervous system (CNS) of higher vertebrates are presented. Corresponding to the regional diversity of the embryonic CNS (unsegmented spinal cord vs segmented brain anlagen) and its surroundings (segmented trunk vs unsegmented head mesoderm, neural crest-derived mesenchyme), cells of different origins contribute to the endothelial and mural cell populations. The autonomous migratory potential of endothelial cells is guided by attractive and repulsive clues. Nevertheless, a common pattern in both spinal cord and forebrain vascularization appears, with primary ventral vascular sprouts supplying the periventricular vascular plexus of the neural tube, whereas dorsolateral sprouts appear later.Key words: angiogenesis, blood vessel, brain, CNS, embryo, endothelial cell, neural crest, neural tube, pattern formation, pericyte, spinal cord     

机械阻断神经管的闭合影响爪蟾神经管辐射状切入及神经嵴细胞定向迁移(英文)

神经管闭合缺陷 (NTDs)是一种严重的先天畸形疾病,在新生儿中有千分之一的发病率。神经管融合前后,多种组织参与形态发生运动。神经管一经融合,神经嵴细胞就会向背侧中线方向产生单极突出并向此方向迁移形成神经管的顶部。与此同时,神经管从腹侧开始发生辐射状切入以实现单层化。在此,我们在非洲爪蟾的移植体中机械阻断神经管的闭合以检测其细胞运动及随后的图式形成。结果显示神经管闭合缺陷的移植体不能形成单层化的神经管,并且神经嵴细胞滞留在侧面区域不能向背侧中线迁移,而对神经前体标记基因的检测显示神经管的背腹图式形成并未受到影响。以上结果表明神经管的融合对于辐射状切入和神经嵴细胞向背侧中线方向的迁移过程是必需的,而对于神经管的沿背腹轴方向的图式形成是非必需的。     

Analysis of Epigenetic Factors in Mouse Embryonic Neural Stem Cells Exposed to Hyperglycemia

Background

Maternal diabetes alters gene expression leading to neural tube defects (NTDs) in the developing brain. The mechanistic pathways that deregulate the gene expression remain unknown. It is hypothesized that exposure of neural stem cells (NSCs) to high glucose/hyperglycemia results in activation of epigenetic mechanisms which alter gene expression and cell fate during brain development.

Methods and Findings

NSCs were isolated from normal pregnancy and streptozotocin induced-diabetic pregnancy and cultured in physiological glucose. In order to examine hyperglycemia induced epigenetic changes in NSCs, chromatin reorganization, global histone status at lysine 9 residue of histone H3 (acetylation and trimethylation) and global DNA methylation were examined and found to be altered by hyperglycemia. In NSCs, hyperglycemia increased the expression of Dcx (Doublecortin) and Pafah1b1 (Platelet activating factor acetyl hydrolase, isoform 1b, subunit 1) proteins concomitant with decreased expression of four microRNAs (mmu-miR-200a, mmu-miR-200b, mmu-miR-466a-3p and mmu-miR-466 d-3p) predicted to target these genes. Knockdown of specific microRNAs in NSCs resulted in increased expression of Dcx and Pafah1b1 proteins confirming target prediction and altered NSC fate by increasing the expression of neuronal and glial lineage markers.

Conclusion/Interpretation

This study revealed that hyperglycemia alters the epigenetic mechanisms in NSCs, resulting in altered expression of some development control genes which may form the basis for the NTDs. Since epigenetic changes are reversible, they may be valuable therapeutic targets in order to improve fetal outcomes in diabetic pregnancy.     

Planar cell polarity genes and neural tube closure

Closure of the neural tube is essential for normal development of the brain and spinal cord. Failure of closure results in neural tube defects (NTDs), common and clinically severe congenital malformations whose molecular mechanisms remain poorly understood. On the other hand, it is increasingly well established that common molecular mechanisms are employed to regulate morphogenesis of multicellular organisms. For example, signaling triggered by polypeptide growth factors is highly conserved among species and utilized in multiple developmental processes. Recent studies have revealed that the Drosophila planar cell polarity (PCP) pathway, which directs position and direction of wing hairs on the surface of the fly wing, is well conserved, and orthologs of several genes encoding components of the pathway are also found in vertebrates. Interestingly, in vertebrates, this signaling pathway appears to be co-opted to regulate "convergent extension" cell movements during gastrulation. Disruption of vertebrate PCP genes in Xenopus laevis or zebrafish causes severe gastrulation defects or the shortening of the trunk, as well as mediolateral expansion of somites. In Xenopus, in which the neural tube closes by elevation and fusion of neural folds, inhibition of convergent extension can also prevent neural tube closure causing a "spina bifida-like" appearance. Furthermore, several of the genes involved in the PCP pathway have recently been shown to be required for neural tube closure in the mouse, since mutation of these genes causes NTDs. Therefore, understanding the mechanisms underlying the establishment of cell polarity in Drosophila may provide important clues to the molecular basis of NTDs.     

Keratin and vimentin expression in early organogenesis of the rabbit embryo

Summary The expression of vimentin and keratins is analysed in the early postimplantation embryo of the rabbit at 11 days post conceptionem (d.p.c.) using a panel of monoclonal antibodies specific for single intermediate filament polypeptides (keratins 7, 8, 18, 19 and vimentin) and a pan-epithelial monoclonal keratin antibody. Electrophoretic separation of cytoskeletal preparations obtained from embryonic tissues, in combination with immunoblotting of the resulting polypeptide bands, demonstrates the presence of the rabbit equivalents of human keratins 8, 18, and vimentin in 11-day-old rabbit embryonic tissues. Immunohistochemical staining shows that several embryonic epithelia such as notochord, surface ectoderm, primitive intestinal tube, and mesonephric duct, express keratins, while others (neural tube, dermomyotome) express vimentin, and a third group (coelomic epithelia) can express both. Similarly, of the mesenchymal tissues sclerotomal mesenchyme expresses vimentin, while somatopleuric mesenchyme (abdominal wall) expresses keratins, and splanchnopleuric mesenchyme (dorsal mesentery) expresses both keratins and vimentin. While these results are in accordance with most results of keratin and vimentin expression in embryos of other species, they stand against the common concept of keratin and vimentin specificity in adult vertebrate tissues. Furthermore, keratin and vimentin are not expressed in accordance with germ layer origin of tissues in the mammalian embryo; rather the expression of these proteins seems to be related to cellular function during embryonic development.Supported by the Deutsche Forschungsgemeinschaft and by the Netherlands Cancer Foundation