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Neurulation is a highly synchronized biomechanical process leading to the formation of the brain and spinal cord, and its failure leads to neural tube defects (NTDs). Although we are rapidly learning the genetic mechanisms underlying NTDs, the biomechanical aspects are largely unknown. To understand the correlation between NTDs and tissue stiffness during neural tube closure (NTC), we imaged an NTD murine model using optical coherence tomography (OCT), Brillouin microscopy and confocal fluorescence microscopy. Here, we associate structural information from OCT with local stiffness from the Brillouin signal of embryos undergoing neurulation. The stiffness of neuroepithelial tissues in Mthfd1l null embryos was significantly lower than that of wild-type embryos. Additionally, exogenous formate supplementation improved tissue stiffness and gross embryonic morphology in nullizygous and heterozygous embryos. Our results demonstrate the significance of proper tissue stiffness in normal NTC and pave the way for future studies on the mechanobiology of normal and abnormal embryonic development., Competing Interests: Competing interests M.S. and K.V.L. have a financial interest in ElastEye, which is unrelated to this work. All other authors declare they have no competing interests., (© 2024. Published by The Company of Biologists Ltd.)

Coordinated migration of the mesoderm is essential for accurate organization of the body plan during embryogenesis. However, little is known about how mesoderm migration influences posterior neural tube closure in mammals. Here, we show that spinal neural tube closure and lateral migration of the caudal paraxial mesoderm depend on transmembrane protein 132A (TMEM132A), a single-pass type I transmembrane protein, the function of which is not fully understood. Our study in Tmem132a-null mice and cell models demonstrates that TMEM132A regulates several integrins and downstream integrin pathway activation as well as cell migration behaviors. Our data also implicates mesoderm migration in elevation of the caudal neural folds and successful closure of the caudal neural tube. These results suggest a requirement for paraxial mesodermal cell migration during spinal neural tube closure, disruption of which may lead to spina bifida., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

Disruptions in neural tube (NT) closure result in neural tube defects (NTDs). To understand the molecular processes required for mammalian NT closure, we investigated the role of Snx3 , a sorting nexin gene. Snx3 -/- mutant mouse embryos display a fully-penetrant cranial NTD. In vivo , we observed decreased canonical WNT target gene expression in the cranial neural epithelium of the Snx3 -/- embryos and a defect in convergent extension of the neural epithelium. Snx3 -/- cells show decreased WNT secretion, and live cell imaging reveals aberrant recycling of the WNT ligand-binding protein WLS and mis-trafficking to the lysosome for degradation. The importance of SNX3 in WNT signaling regulation is demonstrated by rescue of NT closure in Snx3 -/- embryos with a WNT agonist. The potential for SNX3 to function in human neurulation is revealed by a point mutation identified in an NTD-affected individual that results in functionally impaired SNX3 that does not colocalize with WLS and the degradation of WLS in the lysosome. These data indicate that Snx3 is crucial for NT closure via its role in recycling WLS in order to control levels of WNT signaling., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

The transcription factor grainyhead-like 2 (GRHL2) is expressed in non-neural ectoderm (NNE) and Grhl2 loss results in fully penetrant cranial neural tube defects (NTDs) in mice. GRHL2 activates expression of several epithelial genes; however, additional molecular targets and functional processes regulated by GRHL2 in the NNE remain to be determined, as well as the underlying cause of the NTDs in Grhl2 mutants. Here, we find that Grhl2 loss results in abnormal mesenchymal phenotypes in the NNE, including aberrant vimentin expression and increased cellular dynamics that affects the NNE and neural crest cells. The resulting loss of NNE integrity contributes to an inability of the cranial neural folds to move toward the midline and results in NTD. Further, we identified Esrp1, Sostdc1, Fermt1, Tmprss2 and Lamc2 as novel NNE-expressed genes that are downregulated in Grhl2 mutants. Our in vitro assays show that they act as suppressors of the epithelial-to-mesenchymal transition (EMT). Thus, GRHL2 promotes the epithelial nature of the NNE during the dynamic events of neural tube formation by both activating key epithelial genes and actively suppressing EMT through novel downstream EMT suppressors., (© 2016. Published by The Company of Biologists Ltd.)

Non-canonical Wnt/planar cell polarity (PCP) signaling plays a primary role in the convergent extension that drives neural tube closure and body axis elongation. PCP signaling gene mutations cause severe neural tube defects (NTDs). However, the role of canonical Wnt/β-catenin signaling in neural tube closure and NTDs remains poorly understood. This study shows that conditional gene targeting of β-catenin in the dorsal neural folds of mouse embryos represses the expression of the homeobox-containing genes Pax3 and Cdx2 at the dorsal posterior neuropore (PNP), and subsequently diminishes the expression of the Wnt/β-catenin signaling target genes T, Tbx6 and Fgf8 at the tail bud, leading to spina bifida aperta, caudal axis bending and tail truncation. We demonstrate that Pax3 and Cdx2 are novel downstream targets of Wnt/β-catenin signaling. Transgenic activation of Pax3 cDNA can rescue the closure defect in the β-catenin mutants, suggesting that Pax3 is a key downstream effector of β-catenin signaling in the PNP closure process. Cdx2 is known to be crucial in posterior axis elongation and in neural tube closure. We found that Cdx2 expression is also repressed in the dorsal PNPs of Pax3-null embryos. However, the ectopically activated Pax3 in the β-catenin mutants cannot restore Cdx2 mRNA in the dorsal PNP, suggesting that the presence of both β-catenin and Pax3 is required for regional Cdx2 expression. Thus, β-catenin signaling is required for caudal neural tube closure and elongation, acting through the transcriptional regulation of key target genes in the PNP.

Neural tube defects (NTDs) are some of the most common birth defects observed in humans. The incidence of NTDs can be reduced by peri-conceptional folic acid supplementation alone and reduced even further by supplementation with folic acid plus a multivitamin. Here, we present evidence that iron maybe an important nutrient necessary for normal development of the neural tube. Following implantation of the mouse embryo, ferroportin 1 (Fpn1) is essential for the transport of iron from the mother to the fetus and is expressed in the visceral endoderm, yolk sac and placenta. The flatiron (ffe) mutant mouse line harbors a hypomorphic mutation in Fpn1 and we have created an allelic series of Fpn1 mutations that result in graded developmental defects. A null mutation in the Fpn1 gene is embryonic lethal before gastrulation, hypomorphic Fpn1(ffe/ffe) mutants exhibit NTDs consisting of exencephaly, spina bifida and forebrain truncations, while Fpn1(ffe/KI) mutants exhibit even more severe NTDs. We show that Fpn1 is not required in the embryo proper but rather in the extra-embryonic visceral endoderm. Our data indicate that loss of Fpn1 results in abnormal morphogenesis of the anterior visceral endoderm (AVE). Defects in the development of the forebrain in Fpn1 mutants are compounded by defects in multiple signaling centers required for maintenance of the forebrain, including the anterior definitive endoderm (ADE), anterior mesendoderm (AME) and anterior neural ridge (ANR). Finally, we demonstrate that this loss of forebrain maintenance is due in part to the iron deficiency that results from the absence of fully functional Fpn1.

Among the cellular properties that are essential for the organization of tissues during animal development, the importance of cell polarity in the plane of epithelial sheets has become increasingly clear in the past decades. Planar cell polarity (PCP) signaling in vertebrates has indispensable roles in many aspects of their development, in particular, controlling alignment of various types of epithelial cells. Disrupted PCP has been linked to developmental defects in animals and to human pathology. Neural tube closure defects (NTD) and disorganization of the mechanosensory cells of the organ of Corti are commonly known consequences of disturbed PCP signaling in mammals. We report here a typical PCP phenotype in a mouse mutant for the Sec24b gene, including the severe NTD craniorachischisis, abnormal arrangement of outflow tract vessels and disturbed development of the cochlea. In addition, we observed genetic interaction between Sec24b and the known PCP gene, scribble. Sec24b is a component of the COPII coat protein complex that is part of the endoplasmic reticulum (ER)-derived transport vesicles. Sec24 isoforms are thought to be directly involved in cargo selection, and we present evidence that Sec24b deficiency specifically affects transport of the PCP core protein Vangl2, based on experiments in embryos and in cultured primary cells.

In this study we show in mice that Ftm (Rpgrip1l) is located at the ciliary basal body. Our data reveal that Ftm is necessary for developmental processes such as the establishment of left-right asymmetry and patterning of the neural tube and the limbs. The loss of Ftm affects the ratio of Gli3 activator to Gli3 repressor, suggesting an involvement of Ftm in Shh signalling. As Ftm is not essential for cilia assembly but for full Shh response, Ftm can be considered as a novel component for cilium-related Hh signalling. Furthermore, the absence of Ftm in arthropods underlines the divergence between vertebrate and Drosophila Hh pathways.

Planar-cell-polarity (PCP) signalling is necessary for initiation of neural tube closure in higher vertebrates. In mice with PCP gene mutations, a broad embryonic midline prevents the onset of neurulation through wide spacing of the neural folds. In order to evaluate the role of convergent extension in this defect, we vitally labelled the midline of loop-tail (Lp) embryos mutant for the PCP gene Vangl2. Injection of DiI into the node, and electroporation of a GFP expression vector into the midline neural plate, revealed defective convergent extension in both axial mesoderm and neuroepithelium, before the onset of neurulation. Chimeras containing both wild-type and Lp-mutant cells exhibited mainly wild-type cells in the midline neural plate and notochordal plate, consistent with a cell-autonomous disturbance of convergent extension. Inhibitor studies in whole-embryo culture demonstrated a requirement for signalling via RhoA-Rho kinase, but not jun N-terminal kinase, in convergent extension and the onset of neural tube closure. These findings identify a cell-autonomous defect of convergent extension, requiring PCP signalling via RhoA-Rho kinase, during the development of severe neural tube defects in the mouse.

Through the direct analysis of cell behaviors, we address the mechanisms underlying anterior neural tube morphogenesis in the zebrafish and the role of the cell adhesion molecule N-cadherin (N-cad) in this process. We demonstrate that although the mode of neurulation differs at the morphological level between amphibians and teleosts, the underlying cellular mechanisms are conserved. Contrary to previous reports, the zebrafish neural plate is a multi-layered structure, composed of deep and superficial cells that converge medially while undergoing radial intercalation, to form a single cell-layered neural tube. Time-lapse recording of individual cell behaviors reveals that cells are polarized along the mediolateral axis and exhibit protrusive activity. In N-cad mutants, both convergence and intercalation are blocked. Moreover, although N-cad-depleted cells are not defective in their ability to form protrusions, they are unable to maintain them stably. Taken together, these studies uncover key cellular mechanisms underlying neural tube morphogenesis in teleosts, and reveal a role for cadherins in promoting the polarized cell behaviors that underlie cellular rearrangements and shape the vertebrate embryo.

The neural crest is a multipotent, migratory cell population that contributes to a variety of tissues and organs during vertebrate embryogenesis. Here, we focus on the function of Msx1 and Msx2, homeobox genes implicated in several disorders affecting craniofacial development in humans. We show that Msx1/2 mutants exhibit profound deficiencies in the development of structures derived from the cranial and cardiac neural crest. These include hypoplastic and mispatterned cranial ganglia, dysmorphogenesis of pharyngeal arch derivatives and abnormal organization of conotruncal structures in the developing heart. The expression of the neural crest markers Ap-2alpha, Sox10 and cadherin 6 (cdh6) in Msx1/2 mutants revealed an apparent retardation in the migration of subpopulations of preotic and postotic neural crest cells, and a disorganization of neural crest cells paralleling patterning defects in cranial nerves. In addition, normally distinct subpopulations of migrating crest underwent mixing. The expression of the hindbrain markers Krox20 and Epha4 was altered in Msx1/2 mutants, suggesting that defects in neural crest populations may result, in part, from defects in rhombomere identity. Msx1/2 mutants also exhibited increased Bmp4 expression in migratory cranial neural crest and pharyngeal arches. Finally, proliferation of neural crest-derived mesenchyme was unchanged, but the number of apoptotic cells was increased substantially in neural crest-derived cells that contribute to the cranial ganglia and the first pharyngeal arch. This increase in apoptosis may contribute to the mispatterning of the cranial ganglia and the hypoplasia of the first arch.

The hedgehog (Hh) pathway is conserved from Drosophila to humans and plays a key role in embryonic development. In addition, activation of the pathway in somatic cells contributes to cancer development in several tissues. Suppressor of fused is a negative regulator of Hh signaling. Targeted disruption of the murine suppressor of fused gene (Sufu) led to a phenotype that included neural tube defects and lethality at mid-gestation (9.0-10.5 dpc). This phenotype resembled that caused by loss of patched (Ptch1), another negative regulator of the Hh pathway. Consistent with this finding, Ptch1 and Sufu mutants displayed excess Hh signaling and resultant altered dorsoventral patterning of the neural tube. Sufu mutants also had abnormal cardiac looping, indicating a defect in the determination of left-right asymmetry. Marked expansion of nodal expression in 7.5 dpc embryos and variable degrees of node dysmorphology in 7.75 dpc embryos suggested that the pathogenesis of the cardiac developmental abnormalities was related to node development. Other mutants of the Hh pathway, such as Shh, Smo and Shh/Ihh compound mutants, also have laterality defects. In contrast to Ptch1 heterozygous mice, Sufu heterozygotes had no developmental defects and no apparent tumor predisposition. The resemblance of Sufu homozygotes to Ptch1 homozygotes is consistent with mouse Sufu being a conserved negative modulator of Hh signaling.

Dyneins have been implicated in left-right axis determination during embryonic development and in a variety of human genetic syndromes. In this paper, we study the recently discovered mouse dynein 2 light intermediate chain (mD2LIC), which is believed to be involved in retrograde intraflagella transport and which, like left-right dynein, is expressed in the node of the mouse embryo. Cells of the ventral node of mouse embryos lacking mD2LIC have an altered morphology and lack monocilia, and expression of Foxa2 and Shh in this structure is reduced or completely absent. At later stages, consistent with the absence of nodal cilia, mD2LIC is required for the establishment of the left-right axis and for normal expression of Nodal, and the ventral neural tube fails to express Shh, Foxa2 and Ebaf. mD2LIC also functions indirectly in the survival of anterior definitive endoderm and in the maintenance of the anterior neural ridge, probably through maintenance of Foxa2/Hnf3beta expression. Together, our results indicate that mD2LIC is required to maintain or establish ventral cell fates and for correct signalling by the organiser and midline, and they identify the first embryonic function of a vertebrate cytoplasmic dynein.

In Xenopus, Dishevelled (Xdsh) signaling is required for both neural tube closure and neural convergent extension, but the connection between these two morphogenetic processes remains unclear. Indeed normal neurulation requires several different cell polarity decisions, any of which may require Xdsh signaling. In this paper we address two issues: (1) which aspects of normal neurulation require Xdsh function; and (2) what role convergent extension plays in the closure of the neural tube. We show that Xdsh signaling is not required for neural fold elevation, medial movement or fusion. Disruption of Xdsh signaling therefore provides a specific tool for uncoupling convergent extension from other processes of neurulation. Using disruption of Xdsh signaling, we demonstrate that convergent extension is crucial to tube closure. Targeted injection revealed that Xdsh function was required specifically in the midline for normal neural tube closure. We suggest that the inherent movement of the neural folds can accomplish only a finite amount of medial progress and that convergent extension of the midline is necessary to reduce the distance between the nascent neural folds, allowing them to meet and fuse. Similar results with Xenopus strabismus implicate the planar cell polarity (PCP) signaling cascade in neural convergent extension and tube closure. Together, these data demonstrate that PCP-mediated convergent extension movements are crucial to proper vertebrate neurulation.

The Spemann organizer plays a central role in neural induction, patterning of the neuroectoderm and mesoderm, and morphogenetic movements during early embryogenesis. By seeking genes whose expression is activated by the organizer-specific LIM homeobox gene Xlim-1 in Xenopus animal caps, we isolated the receptor tyrosine kinase Xror2. Xror2 is expressed initially in the dorsal marginal zone, then in the notochord and the neuroectoderm posterior to the midbrain-hindbrain boundary. mRNA injection experiments revealed that overexpression of Xror2 inhibits convergent extension of the dorsal mesoderm and neuroectoderm in whole embryos, as well as the elongation of animal caps treated with activin, whereas it does not appear to affect cell differentiation of neural tissue and notochord. Interestingly, mutant constructs in which the kinase domain was point-mutated or deleted (named Xror2-TM) also inhibited convergent extension, and did not counteract the wild-type, suggesting that the ectodomain of Xror2 per se has activities that may be modulated by the intracellular domain. In relation to Wnt signaling for planar cell polarity, we observed: (1) the Frizzled-like domain in the ectodomain is required for the activity of wild-type Xror2 and Xror2-TM; (2) co-expression of Xror2 with Xwnt11, Xfz7, or both, synergistically inhibits convergent extension in embryos; (3) inhibition of elongation by Xror2 in activin-treated animal caps is reversed by co-expression of a dominant negative form of Cdc42 that has been suggested to mediate the planar cell polarity pathway of Wnt; and (4) the ectodomain of Xror2 interacts with Xwnts in co-immunoprecipitation experiments. These results suggest that Xror2 cooperates with Wnts to regulate convergent extension of the axial mesoderm and neuroectoderm by modulating the planar cell polarity pathway of Wnt.

Brief ectopic expression of Gcm1 in mouse embryonic tail bud profoundly affects the development of the nervous system. All mice from 5 independently derived transgenic lines exhibited either one or both of two types of congenital spinal cord pathologies: failure of the neural tube to close (spina bifida) and multiple neural tubes (diastematomyelia). Because the transgene is expressed only in a restricted caudal region and only for a brief interval (E8.5 to E13.5), there was no evidence of embryonic lethality. The dysraphisms develop during the period and within the zone of transgene expression. We present evidence that these dysraphisms result from an inhibition of neuropore closure and a stimulation of secondary neurulation. After transgene expression ceases, the spinal dysraphisms are progressively resolved and the neonatal animals, while showing signs of scarring and tissue resorption, have a closed vertebral column. The multiple spinal cords remain but are enclosed in a single spinal column as in the human diastematomyelia. The animals live a normal life time, are fertile and do not exhibit any obvious weakness or motor disabilities.

Tumorhead (TH) is a novel maternal gene product from Xenopus laevis containing several basic domains and a weak coiled-coil. Overexpression of wild-type TH resulted in increased proliferation of neural plate cells, causing expansion of the neural field followed by neural tube and craniofacial abnormalities. Overexpressed TH protein repressed neural differentiation and neural crest markers, but did not inhibit the neural inducers, pan-neural markers or mesodermal markers. Loss of function by injection of anti-TH antibody inhibited cell proliferation. Our data are consistent with a model in which tumorhead functions in regulating differentiation of the neural tissues but not neural induction or determination through its effect on cell proliferation.

Rho GTPases direct actin rearrangements in response to a variety of extracellular signals. P190 RhoGAP (GTPase activating protein) is a potent Rho regulator that mediates integrin-dependent adhesion signaling in cultured cells. We have determined that p190 RhoGAP is specifically expressed at high levels throughout the developing nervous system. Mice lacking functional p190 RhoGAP exhibit several defects in neural development that are reminiscent of those described in mice lacking certain mediators of neural cell adhesion. The defects reflect aberrant tissue morphogenesis and include abnormalities in forebrain hemisphere fusion, ventricle shape, optic cup formation, neural tube closure, and layering of the cerebral cortex. In cells of the neural tube floor plate of p190 RhoGAP mutant mice, polymerized actin accumulates excessively, suggesting a role for p190 RhoGAP in the regulation of +Rho-mediated actin assembly within the neuroepithelium. Significantly, several of the observed tissue fusion defects seen in the mutant mice are also found in mice lacking MARCKS, the major substrate of protein kinase C (PKC), and we have found that p190 RhoGAP is also a PKC substrate in vivo. Upon either direct activation of PKC or in response to integrin engagement, p190 RhoGAP is rapidly translocated to regions of membrane ruffling, where it colocalizes with polymerized actin. Together, these results suggest that upon activation of neural adhesion molecules, the action of PKC and p190 RhoGAP leads to a modulation of Rho GTPase activity to direct several actin-dependent morphogenetic processes required for normal neural development.

A series of gain- or loss-of-function experiments performed in different vertebrate species have demonstrated that the Engrailed genes play multiple roles during brain development. In particular, they have been implicated in the determination of the mid/hindbrain domain, in cell proliferation and survival, in neurite formation, tissue polarization and axonal pathfinding. We have analyzed the consequences of a local gain of En function within or adjacent to the endogenous expression domain in mouse and chick embryos. In WEXPZ.En1 transgenic mice (Danielian, P. S. and McMahon, A. P. (1996) Nature 383, 332-334) several genes are induced as a consequence of ectopic expression of En1 in the diencephalic roof (but in a pattern inconsistent with a local di- to mes-encephalon fate change). The development of several structures with secretory function, generated from the dorsal neuroepithelium, is severely compromised. The choroid plexus, subcommissural organ and pineal gland either fail to form or are atrophic. These defects are preceded by an increase in cell death at the dorsal midline. Comparison with the phenotype of Wnt1(sw/sw) (swaying) mutants suggests that subcommissural organ failure is the main cause of prenatal hydrocephalus observed in both strains. The formation of the posterior commissure is also delayed, and errors in axonal pathfinding are frequent. In chick, ectopic expression of En by in ovo electroporation, affects growth and differentiation of the choroid plexus.

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.

Hedgehog (Hh) proteins control many developmental events by inducing specific cell fates or regulating cell proliferation. The Patched1 (Ptc1) protein, a binding protein for Hh molecules, appears to oppose Hh signals by repressing transcription of genes that can be activated by Hh. Sonic hedgehog (Shh), one of the vertebrate homologs of Hh, controls patterning and growth of the limb but the early embryonic lethality of ptc1(-)(/)(-) mice obscures the roles of ptc1 in later stages of development. We partially rescued ptc1 homozygous mutant embryos using a metallothionein promoter driving ptc1. In a wild-type background, the transgene causes a marked decrease in animal size starting during embryogenesis, and loss of anterior digits. In ptc1 homozygotes, a potent transgenic insert allowed survival to E14 and largely normal morphology except for midbrain overgrowth. A less potent transgene gave rise to partially rescued embryos with massive exencephaly, and polydactyly and branched digits in the limbs. The polydactyly was preceded by unexpected anterior limb bud transcription of Shh, so one function of ptc1 is to repress Shh expression in the anterior limb bud.

The rae28 gene is a mouse homologue of the Drosophila polyhomeotic gene (Nomura, M., Takihara, Y. and Shimada, K. (1994) Differentiation 57, 39-50), which is a member of the Polycomb group (Pc-G) of genes (DeCamillis, M., Cheng, N., Pierre, D. and Brock, H.W. (1992) Genes Dev. 6, 223-232). The Pc-G genes are required for the correct expression of the Homeotic complex genes and segment specification during Drosophila embryogenesis and larval development. To study the role of the rae28 gene in mouse development, we generated rae28-deficient mice by gene targeting in embryonic stem cells. The rae28-/- homozygous mice exhibited perinatal lethality, posterior skeletal transformations and defects in neural crest-related tissues, including ocular abnormalities, cleft palate, parathyroid and thymic hypoplasia and cardiac anomalies. The anterior boundaries of Hoxa-3, a-4, a-5, b-3, b-4 and d-4 expression were shifted rostrally in the paraxial mesoderm of the rae28-/- homozygous embryos, and those of Hoxb-3 and b-4 expression were also similarly altered in the rhombomeres and/or pharyngeal arches. These altered Hox codes were presumed to be correlated with the posterior skeletal transformations and neural crest defects observed in the rae28-/- homozygous mice. These results indicate that the rae28 gene is involved in the regulation of Hox gene expression and segment specification during paraxial mesoderm and neural crest development.

The mechanisms underlying neuronal specification and axonogenesis in the vertebrate hindbrain are poorly understood. To address these questions, we have employed anatomical methods and mutational analysis to characterize the branchiomotor neurons in the zebrafish embryo. The zebrafish branchiomotor system is similar to those in the chick and mouse, except for the location of the nVII and nIX branchiomotor neurons. Developmental analyses of genes expressed by branchiomotor neurons suggest that the different location of the nVII neurons in the zebrafish may result from cell migration. To gain insight into the mechanisms underlying the organization and axonogenesis of these neurons, we examined the development of the branchiomotor pathways in neuronal mutants. The valentino b337 mutation blocks the formation of rhombomeres 5 and 6, and severely affects the development of the nVII and nIX motor nuclei. The cyclops b16 mutation deletes ventral midline cells in the neural tube, and leads to a severe disruption of most branchiomotor nuclei and axon pathways. These results demonstrate that rhombomere-specific cues and ventral midline cells play important roles in the development of the branchiomotor pathways.

Transgenic mice were generated containing a cytomegaloviral promoter driven construct (CMV43) expressing the gap junction polylpeptide connexin 43. RNA and protein analysis confirmed that the transgene was being expressed. In situ hybridization analysis of embryo sections revealed that transgene expression was targeted to the dorsal neural tube and in subpopulations of neural crest cells. This expression pattern was identical to that seen in transgenic mice harboring other constructs driven by the cytomegaloviral promoter (Kothary, R., Barton, S. C., Franz, T., Norris, M. L., Hettle, S. and Surani, M. A. H. (1991) Mech. Develop. 35, 25-31; Koedood, M., Fitchel, A., Meier, P. and Mitchell, P. (1995) J. Virol. 69, 2194-2207), and corresponded to a subset of the endogenous Cx43 expression domains. Significantly, dye injection studies showed that transgene expression resulted in an increase in gap junctional communication. Though viable and fertile, these transgenic mice exhibited reduced postnatal viability. Examination of embryos at various stages of development revealed developmental perturbations consisting of cranial neural tube defects (NTD) and heart malformations. Interestingly, breeding of the CMV43 transgene into the Cx43 knockout mice extended postnatal viability of mice homozygote for the Cx43 knockout allele, indicating that the CMV43 trangsene may partially complement the Cx43 deletion. Both the Cx43 knockout and the CMV43 transgenic mice exhibit heart defects associated with malformations in the conotruncus, a region of the heart in which neural crest derivatives are known to have important roles during development. Together with our results indicating neural-crest-specific expression of the transgene in our CMV-based constructs, these observations strongly suggest a role for Cx43-mediated gap junctional communication in neural crest development. Furthermore, these observations indicate that the precise level of Cx43 function may be of critical importance in downstream events involving these migratory cell populations. As such, the CMV43 mouse may represent a powerful new model system for examining the role of extracardiac cell populations in cardiac morphogenesis and other developmental processes.

The differentiation of somite derivatives is dependent on signals from neighboring axial structures. While ventral signals have been described extensively, little is known about dorsal influences, especially those from the dorsal half of the neural tube. Here, we describe severe phenotypic alterations in dorsal somite derivatives of homozygous open brain (opb) mutant mouse embryos which suggest crucial interactions between dorsal neural tube and dorsal somite regions. At Theiler stage 17 (day 10.5 post coitum) of development, strongly altered expression patterns of Pax3 and Myf5 were observed in dorsal somite regions indicating that the dorsal myotome and dermomyotome were not differentiating properly. These abnormalities were later followed by the absence of epaxial (dorsal) musculature; whereas, body wall and limb musculature formed normally. Analysis of Mox1 and Pax1 expression in opb embryos revealed additional defects in the differentiation of the dorsal sclerotome. The observed abnormalities coincided with defects in differentiation of dorsal neural tube regions. The implications of our findings for interactions between dorsal neural tube, surface ectoderm and dorsomedial somite regions in specifying epaxial musculature are discussed.

The mouse mutations splotch (Sp) and curly tail (ct) both produce spinal neural tube defects with closely similar morphology, but achieve this by different embryonic mechanisms. To determine whether the mutants may interact during development, we constructed mice carrying both mutations. Double heterozygotes exhibited tail defects in 10% of cases, although the single heterozygotes do not express this phenotype. Backcrosses of double heterozygotes to ct/ct produced offspring with an elevated incidence of neural tube defects, both spina bifida and tail defects, compared with a control backcross in which Sp was not involved. Use of the deletion allele Sp2H permitted embryos carrying a splotch mutation to be recognised by polymerase chain reaction assay. This experiment showed that only embryos carrying Sp2H develop spina bifida in the backcross with ct/ct, suggesting that the genotype Sp2H/+, ct/ct is usually lethal around the time of birth as a result of severe disturbance of neurulation. The interaction between Sp and ct was investigated further by examining embryos in the backcross for developmental markers of the Sp/Sp and ct/ct genotypes. Sp/Sp embryos characteristically lack neural crest derivatives, such as dorsal root ganglia, and die on day 13 of gestation. Double mutant embryos from the backcross did not exhibit either of these characteristics suggesting that homozygosity for ct does not cause Sp/+ embryos to develop as if they were of genotype Sp/Sp. The angle of ventral curvature of the posterior neuropore region is enhanced in affected ct/ct embryos whereas it was found to be reduced in Sp/Sp embryos compared with their normal littermates.(ABSTRACT TRUNCATED AT 250 WORDS)

Mouse embryos homozygous for the mutant gene Loop-tail (Lp) are characterized by craniorachischisis, an open neural tube extending from the midbrain to the tail. In the present study, experimental chimeric mice containing mixtures of genetically mutant (from Lp/+ x Lp/+ matings) and genetically normal cells were produced. Our aim was to determine whether a 'rescue,' phenotypic gradient, or intermediate expression (i.e. alternating areas of open and closed neural tube) would be observed in these chimeras. We report our analyses of Loop-tail mutant chimeras (n = 82) by gross examination, progeny testing and quantitative analysis of glucose phosphate isomerase (GPI) isozyme levels. An all-or-none craniorachischisis in Loop-tail mutant chimeras was observed. Two multicolored adult chimeras, without any gross evidence of a neural tube defect, were shown to be homozygous Loop-tail chimeras (Lp/Lp in equilibrium +/+) by progeny testing. These results indicate that the normal phenotype can be expressed in the presence of mutant cells. Conversely, six neonates with craniorachischisis were shown to be chimeras by GPI analyses. These results show that the full mutant phenotype can be expressed even when one-third to one-half of the cells are genotypically wild-type. This study did not determine which tissue is primarily responsible for the defective neurulation in this mutant, but suggests that a 'threshold' mechanism underlies the Loop-tail mutant phenotype. In some chimeras that threshold is not reached and the neural tube remains open, whereas in other chimeras the threshold is reached and the neural tube closes completely.