Neurulation of the cynomolgus monkey embryo achieved from 3D blastocyst culture.

Neural tube (NT) defects arise from abnormal neurulation and result in the most common birth defects worldwide. Yet, mechanisms of primate neurulation remain largely unknown due to prohibitions on human embryo research and limitations of available model systems. Here, we establish a three-dimensional (3D) prolonged in vitro culture (pIVC) system supporting cynomolgus monkey embryo development from 7 to 25 days post-fertilization. Through single-cell multi-omics analyses, we demonstrate that pIVC embryos form three germ layers, including primordial germ cells, and establish proper DNA methylation and chromatin accessibility through advanced gastrulation stages. In addition, pIVC embryo immunofluorescence confirms neural crest formation, NT closure, and neural progenitor regionalization. Finally, we demonstrate that the transcriptional profiles and morphogenetics of pIVC embryos resemble key features of similarly staged in vivo cynomolgus and human embryos. This work therefore describes a system to study non-human primate embryogenesis through advanced gastrulation and early neurulation.

Embryo model completes gastrulation to neurulation and organogenesis.

Embryonic stem (ES) cells can undergo many aspects of mammalian embryogenesis in vitro1-5, but their developmental potential is substantially extended by interactions with extraembryonic stem cells, including trophoblast stem (TS) cells, extraembryonic endoderm stem (XEN) cells and inducible XEN (iXEN) cells6-11. Here we assembled stem cell-derived embryos in vitro from mouse ES cells, TS cells and iXEN cells and showed that they recapitulate the development of whole natural mouse embryo in utero up to day 8.5 post-fertilization. Our embryo model displays headfolds with defined forebrain and midbrain regions and develops a beating heart-like structure, a trunk comprising a neural tube and somites, a tail bud containing neuromesodermal progenitors, a gut tube, and primordial germ cells. This complete embryo model develops within an extraembryonic yolk sac that initiates blood island development. Notably, we demonstrate that the neurulating embryo model assembled from Pax6-knockout ES cells aggregated with wild-type TS cells and iXEN cells recapitulates the ventral domain expansion of the neural tube that occurs in natural, ubiquitous Pax6-knockout embryos. Thus, these complete embryoids are a powerful in vitro model for dissecting the roles of diverse cell lineages and genes in development. Our results demonstrate the self-organization ability of ES cells and two types of extraembryonic stem cells to reconstitute mammalian development through and beyond gastrulation to neurulation and early organogenesis.

Optical coherence tomography-guided Brillouin microscopy highlights regional tissue stiffness differences during anterior neural tube closure in the Mthfd1l murine mutant.

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.

A time-resolved single-cell roadmap of the logic driving anterior neural crest diversification from neural border to migration stages.

Neural crest cells exemplify cellular diversification from a multipotent progenitor population. However, the full sequence of early molecular choices orchestrating the emergence of neural crest heterogeneity from the embryonic ectoderm remains elusive. Gene-regulatory-networks (GRN) govern early development and cell specification toward definitive neural crest. Here, we combine ultradense single-cell transcriptomes with machine-learning and large-scale transcriptomic and epigenomic experimental validation of selected trajectories, to provide the general principles and highlight specific features of the GRN underlying neural crest fate diversification from induction to early migration stages using Xenopus frog embryos as a model. During gastrulation, a transient neural border zone state precedes the choice between neural crest and placodes which includes multiple converging gene programs. During neurulation, transcription factor connectome, and bifurcation analyses demonstrate the early emergence of neural crest fates at the neural plate stage, alongside an unbiased multipotent-like lineage persisting until epithelial-mesenchymal transition stage. We also decipher circuits driving cranial and vagal neural crest formation and provide a broadly applicable high-throughput validation strategy for investigating single-cell transcriptomes in vertebrate GRNs in development, evolution, and disease.

TMEM132A regulates mouse hindgut morphogenesis and caudal development.

Caudal developmental defects, including caudal regression, caudal dysgenesis and sirenomelia, are devastating conditions affecting the skeletal, nervous, digestive, reproductive and excretory systems. Defects in mesodermal migration and blood supply to the caudal region have been identified as possible causes of caudal developmental defects, but neither satisfactorily explains the structural malformations in all three germ layers. Here, we describe caudal developmental defects in transmembrane protein 132a (Tmem132a) mutant mice, including skeletal, posterior neural tube closure, genitourinary tract and hindgut defects. We show that, in Tmem132a mutant embryos, visceral endoderm fails to be excluded from the medial region of early hindgut, leading directly to the loss or malformation of cloaca-derived genitourinary and gastrointestinal structures, and indirectly to the neural tube and kidney/ureter defects. We find that TMEM132A mediates intercellular interaction, and physically interacts with planar cell polarity (PCP) regulators CELSR1 and FZD6. Genetically, Tmem132a regulates neural tube closure synergistically with another PCP regulator Vangl2. In summary, we have identified Tmem132a as a new regulator of PCP, and hindgut malformation as the underlying cause of developmental defects in multiple caudal structures.

A new atlas to study embryonic cell types in Xenopus.

This paper introduces a single-cell atlas for pivotal developmental stages in Xenopus, encompassing gastrulation, neurulation, and early tailbud. Notably surpassing its predecessors, the new atlas enhances gene mapping, read counts, and gene/cell type nomenclature. Leveraging the latest Xenopus tropicalis genome version, alongside advanced alignment pipelines and machine learning for cell type assignment, this release maintains consistency with previous cell type annotations while rectifying nomenclature issues. Employing an unbiased approach for cell type assignment proves especially apt for embryonic contexts, given the considerable number of non-terminally differentiated cell types. An alternative cell type attribution here adopts a fuzzy, non-deterministic stance, capturing the transient nature of early embryo progenitor cells by presenting an ensemble of types in superposition. The value of the new resource is emphasized through numerous examples, with a focus on previously unexplored germ cell populations where we uncover novel transcription onset features. Offering interactive exploration via a user-friendly web portal and facilitating complete data downloads, this atlas serves as a comprehensive and accessible reference.

A simple method for gene expression in endo- and ectodermal cells in mouse embryos before neural tube closure.

The lack of a widely accessible method for expressing genes of interest in wild-type embryos is a fundamental obstacle to understanding genetic regulation during embryonic development. In particular, only a few methods are available for introducing gene expression vectors into cells prior to neural tube closure, which is a period of drastic development for many tissues. In this study, we present a simple technique for injecting vectors into the amniotic cavity and allowing them to reach the ectodermal cells and the epithelia of endodermal organs of mouse embryos at E8.0 via in utero injection, using only a widely used optical fiber with an illuminator. Using this technique, retroviruses can be introduced to facilitate the labeling of cells in various tissues, including the brain, spinal cord, epidermis, and digestive and respiratory organs. We also demonstrated in utero electroporation of plasmid DNA into E7.0 and E8.0 embryos. Taking advantage of this method, we reveal the association between Ldb1 and the activity of the Neurog2 transcription factor in the mouse neocortex. This technique can aid in analyzing the roles of genes of interest during endo- and ectodermal development prior to neural tube closure.

Distinct spatiotemporal contribution of morphogenetic events and mechanical tissue coupling during Xenopus neural tube closure.

Neural tube closure (NTC) is a fundamental process during vertebrate development and is indispensable for the formation of the central nervous system. Here, using Xenopus laevis embryos, live imaging, single-cell tracking, optogenetics and loss-of-function experiments, we examine the roles of convergent extension and apical constriction, and define the role of the surface ectoderm during NTC. We show that NTC is a two-stage process with distinct spatiotemporal contributions of convergent extension and apical constriction at each stage. Convergent extension takes place during the first stage and is spatially restricted at the posterior tissue, whereas apical constriction occurs during the second stage throughout the neural plate. We also show that the surface ectoderm is mechanically coupled with the neural plate and its movement during NTC is driven by neural plate morphogenesis. Finally, we show that an increase in surface ectoderm resistive forces is detrimental for neural plate morphogenesis.

Hinge point emergence in mammalian spinal neurulation.

Neurulation is the process in early vertebrate embryonic development during which the neural plate folds to form the neural tube. Spinal neural tube folding in the posterior neuropore changes over time, first showing a median hinge point, then both the median hinge point and dorsolateral hinge points, followed by dorsolateral hinge points only. The biomechanical mechanism of hinge point formation in the mammalian neural tube is poorly understood. Here we employ a mechanical finite element model to study neural tube formation. The computational model mimics the mammalian neural tube using microscopy data from mouse and human embryos. While intrinsic curvature at the neural plate midline has been hypothesized to drive neural tube folding, intrinsic curvature was not sufficient for tube closure in our simulations. We achieved neural tube closure with an alternative model combining mesoderm expansion, nonneural ectoderm expansion, and neural plate adhesion to the notochord. Dorsolateral hinge points emerged in simulations with low mesoderm expansion and zippering. We propose that zippering provides the biomechanical force for dorsolateral hinge point formation in settings where the neural plate lateral sides extend above the mesoderm. Together, these results provide a perspective on the biomechanical and molecular mechanism of mammalian spinal neurulation.

The Phylotypic Brain of Vertebrates, from Neural Tube Closure to Brain Diversification.

BACKGROUND: The phylotypic or intermediate stages are thought to be the most evolutionary conserved stages throughout embryonic development. The contrast with divergent early and later stages derived from the concept of the evo-devo hourglass model. Nonetheless, this developmental constraint has been studied as a whole embryo process, not at organ level. In this review, we explore brain development to assess the existence of an equivalent brain developmental hourglass. In the specific case of vertebrates, we propose to split the brain developmental stages into: (1) Early: Neurulation, when the neural tube arises after gastrulation. (2) Intermediate: Brain patterning and segmentation, when the neuromere identities are established. (3) Late: Neurogenesis and maturation, the stages when the neurons acquire their functionality. Moreover, we extend this analysis to other chordates brain development to unravel the evolutionary origin of this evo-devo constraint. SUMMARY: Based on the existing literature, we hypothesise that a major conservation of the phylotypic brain might be due to the pleiotropy of the inductive regulatory networks, which are predominantly expressed at this stage. In turn, earlier stages such as neurulation are rather mechanical processes, whose regulatory networks seem to adapt to environment or maternal geometries. The later stages are also controlled by inductive regulatory networks, but their effector genes are mostly tissue-specific and functional, allowing diverse developmental programs to generate current brain diversity. Nonetheless, all stages of the hourglass are highly interconnected: divergent neurulation must have a vertebrate shared end product to reproduce the vertebrate phylotypic brain, and the boundaries and transcription factor code established during the highly conserved patterning will set the bauplan for the specialised and diversified adult brain. KEY MESSAGES: The vertebrate brain is conserved at phylotypic stages, but the highly conserved mechanisms that occur during these brain mid-development stages (Inducing Regulatory Networks) are also present during other stages. Oppositely, other processes as cell interactions and functional neuronal genes are more diverse and majoritarian in early and late stages of development, respectively. These phenomena create an hourglass of transcriptomic diversity during embryonic development and evolution, with a really conserved bottleneck that set the bauplan for the adult brain around the phylotypic stage.

Exploring the association between congenital vertebral malformations and neural tube defects.

Congenital vertebral malformations (CVMs) and neural tube defects (NTDs) are common birth defects affecting the spine and nervous system, respectively, due to defects in somitogenesis and neurulation. Somitogenesis and neurulation rely on factors secreted from neighbouring tissues and the integrity of the axial structure. Crucial signalling pathways like Wnt, Notch and planar cell polarity regulate somitogenesis and neurulation with significant crosstalk. While previous studies suggest an association between CVMs and NTDs, the exact mechanism underlying this relationship remains unclear. In this review, we explore embryonic development, signalling pathways and clinical phenotypes involved in the association between CVMs and NTDs. Moreover, we provide a summary of syndromes that exhibit occurrences of both CVMs and NTDs. We aim to provide insights into the potential mechanisms underlying the association between CVMs and NTDs, thereby facilitating clinical diagnosis and management of these anomalies.

Intramedullary spinal capillary hemangioma with secondary neurulation defect in children.

Intramedullary spinal capillary hemangioma is a rare occurrence in pediatric patients, and only limited cases have been reported. This study presents the first two cases of spinal capillary hemangioma co-present with retained medullary cord and one case of spinal capillary hemangioma with lumbosacral lipomatous malformation. Previous literature on ten patients with this pathology was reviewed. We speculated pathogenesis, imaging features, and histopathologic findings of the disease.

In vivo high-content imaging and regression analysis reveal non-cell autonomous functions of Shroom3 during neural tube closure.

During neural tube closure, neural ectoderm cells constrict their apical surfaces to bend and fold the tissue into a tube that will become the central nervous system. Recent data from mice and humans with neural tube defects suggest that key genes required for neural tube closure can exert non-cell autonomous effects on cell behavior, but the nature of these effects remains obscure. Here, we coupled tissue-scale, high-resolution time-lapse imaging of the closing neural tube of Xenopus to multivariate regression modeling, and we show that medial actin accumulation drives apical constriction non-autonomously in neighborhoods of cells, rather than solely in individual cells. To further explore this effect, we examined mosaic crispant embryos and identified both autonomous and non-autonomous effects of the apical constriction protein Shroom3.

Planar cell polarity and the pathogenesis of Tourette Disorder: New hypotheses and perspectives.

Planar cell polarity (PCP) signaling plays a fundamental role in shaping the development and ongoing function of the nervous system, beginning from early stages of neural tube closure and spanning the maintenance of functional synapses in adults. While mutations in core PCP signaling proteins have long been suspected to underlie neural tube closure defects in humans, recent findings also implicate their potential involvement in neurodevelopmental and neuropsychiatric disorders. Missense and loss-of-function mutations in CELSR3, a core component of PCP signaling complexes, are highly associated with Tourette Disorder. Although the functional significance of these mutations has yet to be elucidated in animal and cell models, the expression patterns of Celsr3 in mice point to alterations in cortico-striato-thalamo-cortical circuits. Here, we briefly review the known functions of Celsr3 for nervous system development. We also propose circuit models for Tourette Disorder by hypothesizing roles for Celsr3 in controlling striatal neuromodulation via effects on cholinergic interneurons, and thalamic inhibition through its functions in thalamic reticular nuclei. Testing these and related hypotheses in animal and cell models will move us closer to unraveling the neuropathogenesis of Tourette Disorder, with the ultimate goal of developing more efficacious treatments for both motor and cognitive symptoms.

Expression atlas of avian neural crest proteins: Neurulation to migration.

Neural crest (NC) cells are a dynamic population of embryonic stem cells that create various adult tissues in vertebrate species including craniofacial bone and cartilage and the peripheral and enteric nervous systems. NC development is thought to be a conserved and complex process that is controlled by a tightly-regulated gene regulatory network (GRN) of morphogens, transcription factors, and cell adhesion proteins. While multiple studies have characterized the expression of several GRN factors in single species, a comprehensive protein analysis that directly compares expression across development is lacking. To address this lack in information, we used three closely related avian models, Gallus gallus (chicken), Coturnix japonica (Japanese quail), and Pavo cristatus (Indian peafowl), to compare the localization and timing of four GRN transcription factors, PAX7, SNAI2, SOX9, and SOX10, from the onset of neurulation to migration. While the spatial expression of these factors is largely conserved, we find that quail NC cells express SNAI2, SOX9, and SOX10 proteins at the equivalent of earlier developmental stages than chick and peafowl. In addition, quail NC cells migrate farther and more rapidly than the larger organisms. These data suggest that despite a conservation of NC GRN players, differences in the timing of NC development between species remain a significant frontier to be explored with functional studies.

An efficient miRNA knockout approach using CRISPR-Cas9 in Xenopus.

In recent years CRISPR-Cas9 knockouts (KO) have become increasingly ultilised to study gene function. MicroRNAs (miRNAs) are short non-coding RNAs, 20-22 nucleotides long, which affect gene expression through post-transcriptional repression. We previously identified miRNAs-196a and -219 as implicated in the development of Xenopus neural crest (NC). The NC is a multipotent stem-cell population, specified during early neurulation. Following EMT, NC cells migrate to various points in the developing embryo where they give rise to a number of tissues including parts of the peripheral nervous system, pigment cells and craniofacial skeleton. Dysregulation of NC development results in many diseases grouped under the term neurocristopathies. As miRNAs are so small, it is difficult to design CRISPR sgRNAs that reproducibly lead to a KO. We have therefore designed a novel approach using two guide RNAs to effectively 'drop out' a miRNA. We have knocked out miR-196a and miR-219 and compared the results to morpholino knockdowns (KD) of the same miRNAs. Validation of efficient CRISPR miRNA KO and phenotype analysis included use of whole-mount in situ hybridization of key NC and neural plate border markers such as Pax3, Xhe2, Sox10 and Snail2, q-RT-PCR and Sanger sequencing. To show specificity we have also rescued the knockout phenotype using miRNA mimics. MiRNA-219 and miR-196a KO's both show loss of NC, altered neural plate and hatching gland phenotypes. Tadpoles show gross craniofacial and pigment phenotypes.

Neural tube development depends on notochord-derived sonic hedgehog released into the sclerotome.

Sonic hedgehog (Shh), produced in the notochord and floor plate, is necessary for both neural and mesodermal development. To reach the myotome, Shh has to traverse the sclerotome and a reduction of sclerotomal Shh affects myotome differentiation. By investigating loss and gain of Shh function, and floor-plate deletions, we report that sclerotomal Shh is also necessary for neural tube development. Reducing the amount of Shh in the sclerotome using a membrane-tethered hedgehog-interacting protein or Patched1, but not dominant active Patched, decreased the number of Olig2+ motoneuron progenitors and Hb9+ motoneurons without a significant effect on cell survival or proliferation. These effects were a specific and direct consequence of Shh reduction in the mesoderm. In addition, grafting notochords in a basal but not apical location, vis-à-vis the tube, profoundly affected motoneuron development, suggesting that initial ligand presentation occurs at the basal side of epithelia corresponding to the sclerotome-neural tube interface. Collectively, our results reveal that the sclerotome is a potential site of a Shh gradient that coordinates the development of mesodermal and neural progenitors.

Wnt/planar cell polarity signaling controls morphogenetic movements of gastrulation and neural tube closure.

Gastrulation and neurulation are successive morphogenetic processes that play key roles in shaping the basic embryonic body plan. Importantly, they operate through common cellular and molecular mechanisms to set up the three spatially organized germ layers and to close the neural tube. During gastrulation and neurulation, convergent extension movements driven by cell intercalation and oriented cell division generate major forces to narrow the germ layers along the mediolateral axis and elongate the embryo in the anteroposterior direction. Apical constriction also makes an important contribution to promote the formation of the blastopore and the bending of the neural plate. Planar cell polarity proteins are major regulators of asymmetric cell behaviors and critically involved in a wide variety of developmental processes, from gastrulation and neurulation to organogenesis. Mutations of planar cell polarity genes can lead to general defects in the morphogenesis of different organs and the co-existence of distinct congenital diseases, such as spina bifida, hearing deficits, kidney diseases, and limb elongation defects. This review outlines our current understanding of non-canonical Wnt signaling, commonly known as Wnt/planar cell polarity signaling, in regulating morphogenetic movements of gastrulation and neural tube closure during development and disease. It also attempts to identify unanswered questions that deserve further investigations.

Secondary Neurulation Defects: Retained Medullary Cord.

Retained medullary cord (RMC) is a defect resulting from impaired secondary neurulation. Intraoperatively, RMC is recognizable as an elongated cord-like structure caudal to the conus, that contains histologically confirmed neuroglial components and a lumen with an ependymal lining. It characteristically does not possess neurological function. This chapter aims to summarize (1) the mechanisms that lead to the occurrence of RMC; (2) the various forms of RMC, such as cystic RMC and 'possible RMC', and (3) the treatment strategies, especially untethering through limited exposure.

Secondary Neurulation Defect: Terminal Myelocystocele, a Biological Leviathan.

Terminal myelocystocele (TMC) has been a puzzling entity of spinal dysraphism. It is found in the sacrococcygeal region usually forming a subcutaneous hump of various sizes. The wide variation of its morphology has been clarified by defining the essential and nonessential features as described in this chapter. Although it is not a common entity, TMC is attractive in that a highly plausible hypothesis on its pathoembryogenesis has been proposed based on observations on the secondary neurulation of the chick embryo. In this chapter, the embryology will be described, followed by the surgical strategy in accordance with the embryogenesis. The clinical features and prognosis will also be presented in detail.