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Here, we define a gene regulatory network for Hoxa2, responsible for temporal and spatial expression in hindbrain development. Hoxa2 plays an important role in regulating the regional identity of rhombomere 2 (r2) and is the only Hox gene expressed in this segment. In this study, we found that a Hoxa2 cis-regulatory module consists of five elements that direct expression in r2 of the developing hindbrain. Surprisingly, the module is imbedded in the second coding exon of Hoxa2 and therefore may be constrained by both protein coding and gene regulatory requirements. This highly conserved enhancer consists of two consensus Sox binding sites and several additional elements that act in concert to direct strong r2 specific expression. Our findings provide important insight into the regulation of segmental identity in the anterior hindbrain. Furthermore, they have broader implications in designing arrays and interpreting data from global analyses of gene regulation because regulatory input from coding regions needs to be considered. cis elements | hindbrain segmentation | Hox genes | regulatory network

Neural tube defects (NTDs) are very frequent congenital abnormalities in humans. Recently, we have documented independent association of Vangl1 and Vangl2 gene mutations with NTDs. In the Looptail mouse, homozygosity (but not heterozygosity) for lossof-function alleles at Vangl2 causes the severe NTD craniorachischisis, whereas heterozygosity for mutant variants of VANGL1 is associated with NTDs in a human cohort of sporadic and familial cases. To understand the role of Vangl1 in normal development, we created a mouse mutant with an inactivating mutation at Vangl1 ([Vangl1.sup.gt]). Vangl1 shows a dynamic pattern of expression in the developing neural tube and notochord at the time of neural tube closure. [Vangl1.sup.gt/+] heterozygotes and [Vangl1.sup.gt/gt] homozygotes are viable and fertile, although [Vangl1.sup.gt/gt] display subtle alterations in polarity of inner hair cells of the cochlea. Remarkably, and as opposed to healthy [Vangl1.sup.gt/+] and [Vangl2.sup.lp/+] heterozygotes, [Vangl1.sup.gt/+];[Vangl2.sup.lp/+] double heterozygotes show profound developmental defects that include severe craniorachischisis, inner ear defects (disorganization of the stereociliary bundles of hair cells of the organ of Corti), and cardiac abnormality (aberrant right subclavian artery). These results show that genetic interaction between Vangl1 and Vangl2 genes causes neural tube defects and raise the possibility that interaction between individual Vangl genes and other genetic loci and/or environmental factors may additionally contribute to the etiology of NTDs. convergent extension | Looptail mice | planar cell polarity | mutant mouse

A study identified three mutations in the VANGL1 gene in patients with familial types (V239I and R274Q) and a sporadic type (M328T) of the disease, as well as a spontaneous mutation (V239I) appearing in a familial setting. Results demonstrate that, in a protein-protein interaction assay, V239I abolished interaction of VANGL1 protein with its binding partners, disheveled-1, -2, and -3, which implicate VANGL1 as a risk factor in human neural-tube defects.

Using a sudy on Xenopus embryos, the role of NF-protocadherin and its partner TAF1/Set in cell-cell interactions and adhesions in neural folds and hence in neurulation, is discussed.

Using a study on mice, the role of Ovo genes, especially Oval2, in neuroectoderm patterning, neural development regulation, and mammalian embryogenesis, is examined.

The axial midline is an important source of patterning and morphogenesis cues in the vertebrate embryo. The midline derives from a small group of cells in the gastrulating embryo, known as 'the organizer' in recognition of its ability to organize an entire body plan. The mammalian organizer, the node, gives rise to axial midline structures: the notochord, dorsal foregut, and part of the floor plate of the neural tube. Only some of the genes that direct midline development are known. In this study, we present the complete coding sequence for a novel gene, cordon-bleu (cobl), expressed specifically in the node and its derivatives until organogenesis stages. The deduced sequence does not resemble any gene of known function. However, cobl is widely conserved: apparent orthologs and paralogs are found in many vertebrate species, with several sequence domains of high conservation but unknown function. We find that chicken cordon-bleu is similarly expressed in the node and its derivatives, suggesting functional conservation. We also report the sequence and nonoverlapping expression of a related mouse gene, Coblrl. Finally, we show that cobl interacts with the neurulation gene Vangl2 to facilitate midbrain neural tube closure, demonstrating roles for both cobl and Vangl2 in midbrain neurulation. Keywords: Cordon-bleu; Organizer; Node; Notochord; Loop-tail; Vangl2; Neural tube defect; Neurulation; Coblrl

The floor plate is an organising centre that controls neural differentiation and axonogenesis in the neural tube. The axon guidance molecule Netrin1 is expressed in the floor plate of zebrafish embryos. To elucidate the regulatory mechanisms underlying expression in the floor plate, we scanned the netrin1 locus for regulatory regions and identified an enhancer that drives expression in the floor plate and hypochord of transgenic embryos. The expression of the transgene is ectopically activated by Cyclops (Nodal) signals but does not respond to Hedgehog signals. The winged-helix transcription factor foxA2 (also HNF3[beta], axial) is expressed in the notochord and floor plate. We show that knock-down of FoxA2 leads to loss of floor plate, while notochord and hypochord development is unaffected, suggesting a specific requirement of FoxA2 in the floor plate. The transgene is ectopically activated by FoxA2, and expression of FoxA2 leads to rescue of floor plate differentiation in mutant embryos that are deficient in Cyclops signalling. Zebrafish and mouse use different signalling systems to specify floor plate. The zebrafish netrin1 regulatory region also drives expression in the floor plate of mouse and chicken embryos. This suggests that components of the regulatory circuits controlling expression in the floor plate are conserved and that FoxA2--given its importance for midline development also in the mouse--may be one such component. Key Words: cyclops; Nodal; floor plate; hypochord; netrin1; foxA2 (HNF3[beta], axial).

Research has been conducted on the Gli3 repressor activity in the neuronal tube. Results suggest that Gli proteins are common mediators integrating sonic hedgehog signals and controlling patterning in the ventral neural tube.

Research has been conducted on the Hedgehog signaling pathway. Results suggest that Hedgehog signaling is important for organizing ventral cell pattern via the differential cell affinity control.

Research has been conducted on the transcription factor Pax-3 which is expressed in the neural tube. Results demonstrate that p53 deficiency caused by the germ-line mutation or by pifithrin-alpha rescues apoptosis and the neural tube defects in the Splotch embryos.

The purpose of this minireview is to discuss the transcription code that governs cell fate specification in the ventral spinal cord. The mechanisms coordinating the sequential events leading from the mitotic progenitor cell to the mature neuron are explored.

The mucosa-associated lymphoid tissue protein Bcl10, containing an N-terminal caspase recruitment domain, regulates lymphocyte proliferation, connecting antigen receptor signaling in B and T lymphocytes to NF-kappaB activation. Data point out that Bcl10 is also involved in neural tube closure.

The role of Nkx genes, transcription factors, in the ventral spinal cord as it relates to neural patterning is discussed in this perspective article. Three central questions can be seen from the general model. One deals with how the Sonic hedgehog (Shh) concentration gradient is generated, another with how the gradient is translated into distinct transcriptional responses, and the third with what the transcriptional regulators that lock in a particular neuronal or glial fate are. Evidence indicates cell type diversity in the dorsal and ventral central nervous system depends on the starting action of qualitatively distinct signals that act in a concentration-dependent way to set up autonomous subdomains of homeodomain protein activity along the D-V axis of the neural tube.

Study results show that Mash1 and Neugenins 1 and 2 (Ngns) have a role in specification of dorsal-ventral neuronal identity as well as acting as neuronal determinants. They link pathways of neurogenesis and regional patterning in the forebrain directly. The replacement of the Ngns by Mash1 in dorsal prgenitors was used to look at the role of the neural detmination genes in neruonal-type specification in the telencephalon.

A PDZ domain-containing actin-binding protein, shroom (shrm) is necessary for neural tube mouse morphogenesis. A mutation that causes mouse exencephaly, acrania, spina bifida, and facial clefting has been found through use of gene trap mutagenesis. It appears that Shrm is a critical determination of the cellular architecture necessary for proper neurulation.

The stage has been set for significant advances in understanding relative to the notochord, its differentiation and the molecular mechanisms that relate to its structure/function. Up until the H. Takahashi group used embryos of the ascidian, a primitive chordate, to identify a large number of notochord-specific genes by subtractive cloning, information was lacking, but the efficacy of the Takahashi approach is remarkable and progress has begun.

Neural tube patterning in vertebrates is controlled in part by locally secreted factors that act in a paracrine manner on nearby cells to regulate proliferation and gene expression. We show here by in situ hybridization that genes for the neuropeptide pituitary adenylate cyclase-activating peptide (PACAP) and one of its high-affinity receptors ([PAC.sub.1]) are widely expressed in the mouse neural tube on embryonic day (E) 10.5. Transcripts for the ligand are present in differentiating neurons in much of the neural tube, whereas the receptor gene is expressed in the underlying ventricular zone, most prominently in the alar region and floor plate. PACAP potently increased cAMP levels more than 20-fold in cultured E10.5 hindbrain neuroepithelial cells, suggesting that PACAP activates protein kinase A (PKA) in the neural tube and might act in the process of patterning. Consistent with this possibility, PACAP down-regulated expression of the sonic hedgehog- and PKA-dependent target gene gli-1 in cultured neuroepithelial cells, concomitant with a decrease in DNA synthesis. PACAP is thus an early inducer of cAMP levels in the embryo and may act in the neural tube during patterning to control cell proliferation and gene expression.

A second common mutation in the methylenetetrahydrofolate reductase (MTHFR) gene has been identified as a possible risk factor for neural-tube defects. The novel mutation, 1298( A to C) mutation, with an allele frequency of .33, involves a change from glutamate into alanine, destroying an MboII recognition site. Combined heterozygosity of this mutation and the 677(C to T) mutation apparently accounts for some folate-related neural-tube defects.

Mammalian cadherin-6 (K-cadherin, cad6) was originally identified by means of the polymerase chain reaction, but its biological functions have not yet been determined. We analyzed the expression pattern of the mouse homologue of this cadherin during development and found that it was transiently expressed in restricted rhombomeres and in other subdivisions of the neural plate and tube. In the midbrain and anterior hindbrain of E8.0-8.5 embryos, cad6 was expressed only in neural crest-generating regions. In contrast, in the posterior hindbrain and contiguous spinal cord of these embryos, cad6 occurred throughout the neural plate, forming a sharp anterior limit at the future rhombomere 4 and 5 boundary. Subsequently, this neural plate expression became confined to rhombomere 6, although most of the neural crest-generating areas remained positive throughout the body. Neural crest cells expressing cad6 migrated out of the neural tube, and subsequently accumulated mainly along peripheral nerves. We then studied the effect of Hoxa-1 mutation on the expression of cad6, as their expressions spatiotemporally overlapped with each other in the early posterior hindbrain. In E8.0-8.5 Hoxa-1 mutants, cad6 expression was suppressed in the region of rhombomeres 4 to 6, although that in the other regions was not essentially affected. At later stages, however, cad6-positive crest cells appeared and migrated out of rhombomeres 4 to 6, indicating that the suppression of cad6 expression was transient and restricted to early stages. Importantly, this effect of the Hoxa-1 mutation concurred with the timing of the expression of this gene. We also studied Hoxa-3 mutants, but found no effect of this mutation on the cad6 expression pattern. These findings suggest that cad6 may contribute to the formation of the segmental structure of the early brain through its ability to confer specific adhesiveness on cells and that Hoxa-1 may be required for early cad6 expression in the posterior hindbrain.

Research has previously shown that, without methionine supplements, neural tube proteins of rat embryos cultured on bovine sera were hypomethylated and neural tubes failed to close. In the present study, to identify the proteins that became methylated during neurulation, rat embryos were first cultured on methionine-deficient bovine serum for 40 hr, then incubated with puromycin for 1 hr, and, finally, incubated with [methyl-14C]methionine and puromycin for 5 hr. On the basis of molecular weights, isoelectric points, and Western immunoblots, the methyl-14C-labeled proteins were identified as actin, [Alpha][Beta]-tubulin, and neurofilament L. Indirect immunofluorescence studies indicated that without the addition of methionine to the culture, localization of actin and [Alpha][Beta]-tubulin in the basal cytoplasm did not occur and these neuroepithelial cells lost their columnar morphology.

Analysis of mice embryonic motor neuron differentiation revealed the importance of the expression of the LIM homeodomain transcription factor, Isl1. Isl1 is responsible for motor neuron differentiation in the neural tube embryos. The significance of Isl1 expression was confirmed with the findings that motor neurons send signals that initiate differentiation of interneurons.

Characterization of the homeoprotein-containing regulatory complexes, when it comes, will be helpful to elucidation of molecular mechanisms that underlie the control of morphological diversity by Hox genes in animal development. Effects of hexapeptide mutations in vivo on targets that are well defined will make the investigation of the HOX proteins possible in the absence of cooperativity. Mutations in homeodomains that get rid of DNA binding may give some insight into composition of the complex on in vivo targets.

We have previously isolated human/mouse CRIM/Crim1, which encodes a putative transmembrane protein, containing multiple repeats of a cysteine-rich domain found in the extracellular TGF-[Beta]-regulating proteins chordin, short gastrulation, and crossveinless-2. Expression of Crim1 in the mouse developing spinal cord suggests a role in cell patterning. To further analyse the function of Crim1 we endeavor to utilize the chicken model system. We report here the isolation of the full-length cDNA sequence of chicken Crim1 and its expression analyzed by RNA in situ hybridization within the developing chick spinal cord. Chicken Crim1 is very similar to human/mouse Crim1, containing an identical domain structure and 81% homology at the DNA level. Expression of Crim1 initiates at embryonic day (E) 2.5 with expression in the notochord and the floor plate of the developing hindbrain and spinal cord and at later stages in the developing motor neurons and roof plate. To determine the possible function of CRIM1 in neural development, we have overexpressed human CRIM1 in the developing chicken spinal cord. Expression by in ovo electroporation of a truncated form of CRIM1 results in a decrease in the number of EN-1-positive ventral interneurons and ISl-1-positive motor neurons. These results suggest a role for Crim1 in neural tube patterning in vertebrates.

Sonic Hedgehog (Shh) is required for specification of ventral cell types in the vertebrate neural tube. In Shh mutant mouse embryos cells of the ventral neural tube do not adopt ventral identities and, instead, express markers of dorsal identity. Mutations in the mouse open brain (opb) gene produce an opposing phenotype: 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 ??WT chimeras reveals two components of the opb phenotype in the spinal cord. First, the wild type opb gene (opb+) is required cell autonomously for the initial specification of dorsal neural fates. At a later stage, opb+ acts nonautonomously to prevent the expansion of ventral fates. Although BMPs can induce dorsal neural fates, opb mutant cells can respond to BMP signals, indicating the opb+ does not act within the BMP signaling pathway. In the ventral neural tube a critical function of opb+ is to restrict the domain of Shh expression. opb+ may antagonize Shh activity as either a component of a novel dorsalizing pathway or as an intracellular negative regulator of the Shh signal transduction pathway. We will present data from Shh/opb double mutants to help discriminate between these possibilities.

Cell fate determining gene mab-21 regulates the proper establishment of neural cell fate and sensory organ identity in Caenorhabditis elegans. Murine homologs of mab-21 have also been implicated to participate in central nervous system and craniofacial differentiation. We report here the isolation of XMab21l2 from Xenopus. At the amino acid level, this gene shares a similarity of 97% and 55% with the respective homologs in mouse and worm. Whole-mount in situ hybridization showed that XMab21l2 was first expressed during early neurulation. Signal was detected at the optic vesicles, hindbrain, anterior paraxial mesoderm, and the brachial pouches. To define its biological function, we have performed loss-of-function analysis by introducing into early embryos Xmab21l2 specific antisense RNA or antisense oligodeoxynucleotides. We have also tested dsRNA interference for gene inhibition. All the gene suppression approaches have generated abnormal embryos with neural tube closure defect and diminished eyes phenotype. For embryos that survived the neural tube defect, retarded development of posterior structures were observed. Our report is the first to demonstrate effective and potent gene-specific suppression by dsRNA in Xenopus laevis. The results strongly argue for a functional importance of XMab21l2 in the CNS and PNS development. (This study was funded by Research Grants Council, Hong Kong.)

Inductive signals from the midline play an important role in the patterning of the CNS and somites. One of the key midline signals is the secreted molecule sonic hedgehog (shh). It is still unclear how the shh signal is transmitted and interpreted in responding tissues and if other signaling pathways are also involved in midline function. Zebrafish mutations affecting midline development (midline mutants) provide cues for dissecting the signaling pathway at the midline. We have carried out a detailed analysis of the chameleon(con) mutant. Mutant embryos develop medial floor plate, but have defects in the neural tube and somite patterning, con mutants lack muscle pioneer cells in the somite and nk2.2-positive cells in the ventral neural tube. Accordingly, pax6 gene expression in the neural tube is expanded ventrally. No defect is observed in the medial and dorsal parts of the neural tube. Reduced expression of patched around the midline of con mutants indicates that shh signal transduction is impaired. Injection of shh mRNA into con embryos rescues mutant phenotypes, suggesting a possible upstream or parallel function of con in relation to shh. We have initiated the molecular analysis of the con gene and are currently trying to identify the responsible gene by positional cloning.

Byline: Sushil Budhiraja (1), Pushpa Dahiya (2), Manish Ghei (1), Geeta Gathwala (3) Keywords: Keywords Congenital malformation; Neural-tube defect; Twins Abstract: Despite the strong association between twinning and neural-tube defects (NTD), concordance of twins with NTDs is very rare. A case of concordant NTDs in dizygotic twins is reported in view of its extreme rarity. Author Affiliation: (1) Department of Paediatric Surgery, B.D. Sharma Postgraduate Institute of Medical Sciences, Rohtak-124001 Haryana, India, IN (2) Department of Obstetrics and Gynaecology, B.D. Sharma Postgraduate Institute of Medical Sciences, Rohtak-124001, Haryana, India, IN (3) Department of Paediatrics, B.D. Sharma Postgraduate Institute of Medical Sciences, Rohtak-124001, Haryana, India, IN (4) 8 FM/6, Medical Enclave, Rohtak-124001, Haryana, India, IN Article note: Accepted: 23 March 2001

The expressions of two related LIM homeodomain (LIM-HD) genes, Lhx3 and Lhx4, were characterized during motor neuron development utilizing CRE-mediated lineage tracing in the mouse. LHx3 and Lhx4 were expressed in all motor neuron types where axons emerged ventrally from the neural tube but not in the motor neurons that extend axons dorsally from the neural tube. It was revealed that the expressed genes functioned independently from the factors that activated motor neuron differentiation to regulate the selection of motor neuron axon exit point from the neural tube.