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.