Defining a PARticular Pathway of Neural Tube Closure

Mammalian neurulation is completed when the dorsolateral neural folds bend inwards, their tips make adhesive contacts across the midline, and the epithelia remodel to create a closed neural tube. Two recent papers (one by Camerer et al. in this issue of Developmental Cell) demonstrate a vital role for protease-activated G protein-coupled receptor signaling in these late closure events, opening up new avenues for exploring the molecular basis of mammalian neural tube morphogenesis

Rib Truncations and Fusions in the Sp2HMouse Reveal a Role for Pax3 in Specification of the Ventro-lateral and Posterior Parts of the Somite

AbstractThesplotch (Pax3)mouse mutant serves as a model for developmental defects of several types, including defective migration of dermomyotomal cells to form the limb musculature. Here, we describe abnormalities of the ribs, neural arches, and acromion inSp2Hhomozygous embryos, indicating a widespread dependence of lateral somite development onPax3function. Moreover, the intercostal and body wall muscles, derivatives of the ventrolateral myotome, are also abnormal inSp2Hhomozygotes.Pax3is expressed in the dermomyotome, but not in either the sclerotome or the myotome, raising the possibility thatPax3-dependent inductive influences from the dermomyotome are necessary for early specification of lateral sclerotome and myotome. Support for this idea comes from analysis of gene expression markers of lateral sclerotome (tenascin-Candscleraxis) and myotome (myogenin, MyoD,andMyf5). All exhibit ventrally truncated domains of expression inSp2Hhomozygotes, potentially accounting for the rib and intercostal muscle truncations. In contrast, the medial sclerotomal markerPax1is expressed normally in mutant embryos, arguing thatPax3is not required for development of the medial sclerotome. Most of the somitic markers show ectopic expression in anteroposterior and mediolateral dimensions, suggesting a loss of definition of somite boundaries insplotchand explaining the rib and muscle fusions. An exception isMyf5,which is not ectopically expressed inSp2Hhomozygotes, consistent with the previous suggestion thatPax3andMyf5function in different pathways of skeletal myogenesis. PDGFα and its receptor are candidates for mediating signalling between myotome and sclerotome. We find that both genes are misexpressed inSp2Hembryos, suggesting that PDGFα/PDGFRα may function downstream ofPax3,accounting for the close similarities between thesplotchandPatchmutant phenotypes. Our findings point to additional regulatory functions for the Pax3 transcription factor, apart from those already demonstrated for development of the neural tube, neural crest, and dermomyotome

Morphological phenotyping after mouse whole embryo culture

Morphological phenotyping of the mouse embryo is described at neurulation stages, primarily as a guide to evaluating the outcome of whole embryo cultures between embryonic days 8.5 and 9.5. During this period, neural tube closure is initiated and progresses to completion in the cranial region. Spinal closure is still underway at the end of the culture period. The focus of this article is particularly on phenotyping that can be performed at the bench, using a stereomicroscope. This involves assessment of embryonic health, through observation and scoring of yolk sac blood circulation, measurement of developmental stage by somite counting, and determination of crown-rump length as a measure of growth. Axial rotation (“turning”) can also be assessed using a simple scoring system. Neural tube closure assessment includes: 1) determining whether closure has been initiated at the Closure 1 site; 2) evaluating the complex steps of cranial neurulation including initiation at Closure sites 2 and 3, and completion of closure at the anterior and hindbrain neuropores; 3) assessment of spinal closure by measurement of posterior neuropore length. Interpretation of defects in neural tube closure requires an appreciation of, first, the stages that particular events are expected to be completed and, second, the correspondence between embryonic landmarks, for example, somite position, and the resulting adult axial levels. Detailed embryonic phenotyping, as described in this article, when combined with the versatile method of whole embryo culture, can form the basis for a wide range of experimental studies in early mouse neural development

The surface ectoderm exhibits spatially heterogenous tension that correlates with YAP localisation during spinal neural tube closure in mouse embryos

The single cell layer of surface ectoderm (SE) which overlies the closing neural tube (NT) plays a crucial biomechanical role during mammalian NT closure (NTC), challenging previous assumptions that it is only passive to the force-generating neuroepithelium (NE). Failure of NTC leads to congenital malformations known as NT defects (NTDs), including spina bifida (SB) and anencephaly in the spine and brain respectively. In several mouse NTD models, SB is caused by misexpression of SE-specific genes and is associated with disrupted SE mechanics, including loss of rostrocaudal cell elongation believed to be important for successful closure. In this study, we asked how SE mechanics affect NT morphology, and whether the characteristic rostrocaudal cell elongation at the progressing closure site is a response to tension anisotropy in the SE. We show that blocking SE-specific E-cadherin in ex utero mouse embryo culture influences NT morphology, as well as the F-actin cable. Cell border ablation shows that cell shape is not due to tension anisotropy, but that there are regional differences in SE tension. We also find that YAP nuclear translocation reflects regional tension heterogeneity, and that its expression is sensitive to pharmacological reduction of tension. In conclusion, our results confirm that the SE is a biomechanically important tissue for spinal NT morphogenesis and suggest a possible role of spatial regulation of cellular tension which could regulate downstream gene expression via mechanically-sensitive YAP activity

Laminin and integrin expression in the ventral ectodermal ridge of the mouse embryo: Implications for regulation of BMP signalling

PMCID: PMC3629792.[Background]: The ventral ectodermal ridge (VER) is an important signalling centre in the mouse tail-bud following completion of gastrulation. BMP regulation is essential for VER function, but how these signals are transmitted between adjacent tissues is unclear. [Results]: We investigated the idea that extracellular matrix components might be involved, using immunohistochemistry and in situ hybridisation to detect all known α, β, and γ laminin chains and their mRNAs in the early tail bud. We identified an apparently novel laminin variant, comprising α5, β3 and γ2 chains, as a major component of the VER basement membrane at E9.5. Strikingly, only the mRNAs for these chains were co-expressed in VER cells, suggesting that lamin532 may be the sole basement membrane laminin at this stage. Since α6 integrin was also expressed in VER cells, this raises the possibility of cell-matrix interactions regulating BMP signalling at this site of caudal morphogenesis. [Conclusions]: Laminin532 could interact with α6-containing integrin to direct differentiation of the specialised VER cells from surface ectoderm.This work was supported by de Instituto de Salud Carlos III project CP08/00111 and PS09/00050 (to P.Y.-G.), la Consejería de Salud de la Junta de Andalucia project PI-0438-2010 (to P.Y.-G.), and the Wellcome Trust (to A.J.C.).Peer reviewe

Dual mechanism underlying failure of neural tube closure in the Zic2 mutant mouse

Understanding the molecular mechanisms that lead to birth defects is an important step towards improved primary prevention. Mouse embryos homozygous for the Kumba (Ku) mutant allele of Zic2 develop severe spina bifida with complete lack of dorsolateral hinge points (DLHPs) in the neuroepithelium. Bone morphogenetic protein (BMP) signalling is over-activated in Zic2Ku/Ku embryos, and the BMP inhibitor dorsomorphin partially rescues neural tube closure in cultured embryos. RhoA signalling is also over-activated, with accumulation of actomyosin in the Zic2Ku/Ku neuroepithelium, and the myosin inhibitor Blebbistatin partially normalises neural tube closure. However, dorsomorphin and Blebbistatin differ in their effects at tissue and cellular levels: DLHP formation is rescued by dorsomorphin but not Blebbistatin, whereas abnormal accumulation of actomyosin is rescued by Blebbistatin but not dorsomorphin. These findings suggest a dual mechanism of spina bifida origin in Zic2Ku/Ku embryos: BMP-dependent formation of DLHPs is faulty, together with RhoA-dependent F-actin accumulation in the neuroepithelium. Hence, we identify a multi-pathway origin of spina bifida in a mammalian system that may provide a developmental basis for understanding the corresponding multifactorial human defects

Vangl2 disruption alters the biomechanics of late spinal neurulation leading to spina bifida in mouse embryos

peer-reviewedHuman mutations in the planar cell polarity component VANGL2 are associated with the neural tube defect spina bifida. Homozygous Vangl2 mutation in mice prevents initiation of neural tube closure, precluding analysis of its subsequent roles in neurulation. Spinal neurulation involves rostral-to-caudal ‘zippering’ until completion of closure is imminent, when a caudal-to-rostral closure point, ‘Closure 5’, arises at the caudal-most extremity of the posterior neuropore (PNP). Here, we used Grhl3Cre to delete Vangl2 in the surface ectoderm (SE) throughout neurulation and in an increasing proportion of PNP neuroepithelial cells at late neurulation stages. This deletion impaired PNP closure after the ∼25-somite stage and resulted in caudal spina bifida in 67% of Grhl3Cre/+Vangl2Fl/Fl embryos. In the dorsal SE, Vangl2 deletion diminished rostrocaudal cell body orientation, but not directional polarisation of cell divisions. In the PNP, Vangl2 disruption diminished mediolateral polarisation of apical neuroepithelial F-actin profiles and resulted in eversion of the caudal PNP. This eversion prevented elevation of the caudal PNP neural folds, which in control embryos is associated with formation of Closure 5 around the 25-somite stage. Closure 5 formation in control embryos is associated with a reduction in mechanical stress withstood at the main zippering point, as inferred from the magnitude of neural fold separation following zippering point laser ablation. This stress accommodation did not happen in Vangl2-disrupted embryos. Thus, disruption of Vangl2-dependent planar-polarised processes in the PNP neuroepithelium and SE preclude zippering point biomechanical accommodation associated with Closure 5 formation at the completion of PNP closure

Caudal Fgfr1 disruption produces localised spinal mis-patterning and a terminal myelocystocele-like phenotype in mice

Closed spinal dysraphisms are poorly understood malformations classified as neural tube (NT) defects. Several, including terminal myelocystocele, affect the low spine. We previously identified a NT closure-initiating point, Closure 5, in the distal spine of mice. Here we document equivalent morphology of the caudal-most closing posterior neuropore (PNP) in mice and humans. Closure 5 forms in a region of active FGF signalling and pharmacological FGF receptor blockade impairs its formation in cultured mouse embryos. Conditional genetic deletion of Fgfr1 in caudal embryonic tissues with Cdx2Cre diminishes neuroepithelial proliferation, impairs Closure 5 formation and delays PNP closure. After closure, the distal NT of Fgfr1-disrupted embryos dilates to form a fluid-filled sac overlying ventrally flattened spinal cord. This phenotype resembles terminal myelocystocele. Histological analysis reveals regional and progressive loss of SHH and FOXA2-positive ventral NT domains, resulting in OLIG2-labelling of the ventral-most NT. The OLIG2-domain is also subsequently lost, eventually producing a NT entirely positive for the dorsal marker PAX3. Thus, a terminal myelocystocele-like phenotype can arise after completion of NT closure with localised spinal mis-patterning caused by disruption of FGFR1 signalling

Perturbation of Retinoid Homeostasis Increases Malformation Risk in Embryos Exposed to Pregestational Diabetes

Funding. This work was supported by funding from Hong Kong Research Grants Council General Research Fund project reference 441606 and 474109 to A.S.W.S., Y.C.L., C.C.W., P.J.M. and A.J.C..Peer reviewedPostprin