Glucose oxidation drives trunk neural crest cell development and fate.

Bioenergetic metabolism is a key regulator of cellular function and signaling, but how it can instruct the behavior of cells and their fate during embryonic development remains largely unknown. Here, we investigated the role of glucose metabolism in the development of avian trunk neural crest cells (NCCs), a migratory stem cell population of the vertebrate embryo. We uncovered that trunk NCCs display glucose oxidation as a prominent metabolic phenotype, in contrast to what is seen for cranial NCCs, which instead rely on aerobic glycolysis. In addition, only one pathway downstream of glucose uptake is not sufficient for trunk NCC development. Indeed, glycolysis, mitochondrial respiration and the pentose phosphate pathway are all mobilized and integrated for the coordinated execution of diverse cellular programs, epithelial-to-mesenchymal transition, adhesion, locomotion, proliferation and differentiation, through regulation of specific gene expression. In the absence of glucose, the OXPHOS pathway fueled by pyruvate failed to promote trunk NCC adaptation to environmental stiffness, stemness maintenance and fate-decision making. These findings highlight the need for trunk NCCs to make the most of the glucose pathway potential to meet the high metabolic demands appropriate for their development.

Human spinal cord in vitro differentiation pace is initially maintained in heterologous embryonic environments.

Species-specific differentiation pace in vitro indicates that some aspects of neural differentiation are governed by cell intrinsic properties. Here we describe a novel in vitro human neural-rosette assay that recapitulates dorsal spinal cord differentiation but proceeds more rapidly than in the human embryo, suggesting that it lacks endogenous signalling dynamics. To test whether in vitro conditions represent an intrinsic differentiation pace, human iPSC-derived neural rosettes were challenged by grafting into the faster differentiating chicken embryonic neural tube iso-chronically, or hetero-chronically into older embryos. In both contexts in vitro differentiation pace was initially unchanged, while long-term analysis revealed iso-chronic slowed and hetero-chronic conditions promoted human neural differentiation. Moreover, hetero-chronic conditions did not alter the human neural differentiation programme, which progressed to neurogenesis, while the host embryo advanced into gliogenesis. This study demonstrates that intrinsic properties limit human differentiation pace, and that timely extrinsic signals are required for progression through an intrinsic human neural differentiation programme.

A lateral protrusion latticework connects neuroepithelial cells and is regulated during neurogenesis.

Dynamic contacts between cells within the developing neuroepithelium are poorly understood but play important roles in cell and tissue morphology and cell signalling. Here, using live-cell imaging and electron microscopy we reveal multiple protrusive structures in neuroepithelial apical endfeet of the chick embryonic spinal cord, including sub-apical protrusions that extend laterally within the tissue, and observe similar structures in human neuroepithelium. We characterise the dynamics, shape and cytoskeleton of these lateral protrusions and distinguish them from cytonemes, filopodia and tunnelling nanotubes. We demonstrate that lateral protrusions form a latticework of membrane contacts between non-adjacent cells, depend on actin but not microtubule dynamics, and provide a lamellipodial-like platform for further extending fine actin-dependent filipodia. We find that lateral protrusions depend on the actin-binding protein WAVE1 (also known as WASF1): misexpression of mutant WAVE1 attenuated protrusion and generated a round-ended apical endfoot morphology. However, this did not alter apico-basal cell polarity or tissue integrity. During normal neuronal delamination, lateral protrusions were withdrawn, but precocious protrusion loss induced by mutant WAVE1 was insufficient to trigger neurogenesis. This study uncovers a new form of cell-cell contact within the developing neuroepithelium, regulation of which prefigures neuronal delamination. This article has an associated First Person interview with the first author of the paper.

Wnt regulates amino acid transporter Slc7a5 and so constrains the integrated stress response in mouse embryos.

Amino acids are essential for cellular metabolism, and it is important to understand how nutrient supply is coordinated with changing energy requirements during embryogenesis. Here, we show that the amino acid transporter Slc7a5/Lat1 is highly expressed in tissues undergoing morphogenesis and that Slc7a5-null mouse embryos have profound neural and limb bud outgrowth defects. Slc7a5-null neural tissue exhibited aberrant mTORC1 activity and cell proliferation; transcriptomics, protein phosphorylation and apoptosis analyses further indicated induction of the integrated stress response as a potential cause of observed defects. The pattern of stress response gene expression induced in Slc7a5-null embryos was also detected at low level in wild-type embryos and identified stress vulnerability specifically in tissues undergoing morphogenesis. The Slc7a5-null phenotype is reminiscent of Wnt pathway mutants, and we show that Wnt/ß-catenin loss inhibits Slc7a5 expression and induces this stress response. Wnt signalling therefore normally supports the metabolic demands of morphogenesis and constrains cellular stress. Moreover, operation in the embryo of the integrated stress response, which is triggered by pathogen-mediated as well as metabolic stress, may provide a mechanistic explanation for a range of developmental defects.

Neural differentiation, selection and transcriptomic profiling of human neuromesodermal progenitor-like cells in vitro.

Robust protocols for directed differentiation of human pluripotent cells are required to determine whether mechanisms operating in model organisms are relevant to our own development. Recent work in vertebrate embryos has identified neuromesodermal progenitors as a bipotent cell population that contributes to paraxial mesoderm and spinal cord. However, precise protocols for in vitro differentiation of human spinal cord progenitors are lacking. Informed by signalling in amniote embryos, we show here that transient dual-SMAD inhibition, together with retinoic acid (dSMADi-RA), provides rapid and reproducible induction of human spinal cord progenitors from neuromesodermal progenitor-like cells. Using CRISPR-Cas9 to engineer human embryonic stem cells with a GFP-reporter for neuromesodermal progenitor-associated gene Nkx1.2 we facilitate selection of this cell population. RNA-sequencing was then used to identify human and conserved neuromesodermal progenitor transcriptional signatures, to validate this differentiation protocol and to reveal new pathways/processes in human neural differentiation. This optimised protocol, novel reporter line and transcriptomic data are useful resources with which to dissect molecular mechanisms regulating human spinal cord generation and allow the scaling-up of distinct cell populations for global analyses, including proteomic, biochemical and chromatin interrogation.

Cadherin interplay during neural crest segregation from the non-neural ectoderm and neural tube in the early chick embryo.

BACKGROUND: In the avian embryo, neural crest (NC) progenitors arise in the neuroectoderm during gastrulation, long before their dissemination. Although the gene regulatory network involved in NC specification has been deciphered, the mechanisms involved in their segregation from the other neuroectoderm-derived progenitors, notably the epidermis and neural tube, are unknown. Because cadherins mediate cell recognition and sorting, we scrutinized their expression profiles during NC specification and delamination. RESULTS: We found that the NC territory is defined precociously by the robust expression of Cadherin-6B in cells initially scattered among other cells uniformly expressing E-cadherin, and that NC progenitors are progressively sorted and regrouped into a discrete domain between the prospective epidermis and neural tube. At completion of NC specification, the epidermis, NC, and neural tube are fully segregated in contiguous compartments characterized by distinct cadherin repertoires. We also found that Cadherin-6B down-regulation constitutes a major event during NC delamination and that, with the exception of the caudal part of the embryo, N-cadherin is unlikely to control NC emigration. CONCLUSIONS: Our results indicate that partition of the neuroectoderm is mediated by cadherin interplays and ascribes a key role to Cadherin-6B in the specification and delamination of the NC population. Developmental Dynamics 246:550-565, 2017. © 2017 Wiley Periodicals, Inc.

Resolving time and space constraints during neural crest formation and delamination.

A striking feature of neural crest development in vertebrates is that all the specification, delamination, migration, and differentiation steps occur consecutively in distinct areas of the embryo and at different timings of development. The significance and consequences of this partition into clearly separated events are not fully understood yet, but it ought to be related to the necessity of controlling precisely and independently each step, given the wide array of cell types and tissues derived from the neural crest and the long duration of their development spanning almost the entire embryonic life. In this chapter, using the examples of early neural crest induction and delamination, we discuss how time and space constraints influence their development and describe the molecular and cellular responses that are employed by cells to adapt. In the first example, we analyze how cell sorting and cell movements cooperate to allow nascent neural crest cells, which are initially mingled with other neurectodermal progenitors after induction, to segregate from the neural tube and ectoderm populations and settle at the apex of the neural tube prior to migration. In the second example, we examine how cadherins drive the entire process of neural crest segregation from the rest of the neurectoderm by their dual role in mediating first cell sorting and cohesion during specification and later in promoting their delamination. In the third example, we describe how the expression and activity of the transcription factors known to drive epithelium-to-mesenchyme transition (EMT) are regulated timely and spatially by the cellular machinery so that they can alternatively and successively regulate neural crest specification and delamination. In the last example, we briefly tackle the problem of how factors triggering EMT may elicit different cell responses in neural tube and neural crest progenitors.

Junctional neurulation: a unique developmental program shaping a discrete region of the spinal cord highly susceptible to neural tube defects.

In higher vertebrates, the primordium of the nervous system, the neural tube, is shaped along the rostrocaudal axis through two consecutive, radically different processes referred to as primary and secondary neurulation. Failures in neurulation lead to severe anomalies of the nervous system, called neural tube defects (NTDs), which are among the most common congenital malformations in humans. Mechanisms causing NTDs in humans remain ill-defined. Of particular interest, the thoracolumbar region, which encompasses many NTD cases in the spine, corresponds to the junction between primary and secondary neurulations. Elucidating which developmental processes operate during neurulation in this region is therefore pivotal to unraveling the etiology of NTDs. Here, using the chick embryo as a model, we show that, at the junction, the neural tube is elaborated by a unique developmental program involving concerted movements of elevation and folding combined with local cell ingression and accretion. This process ensures the topological continuity between the primary and secondary neural tubes while supplying all neural progenitors of both the junctional and secondary neural tubes. Because it is distinct from the other neurulation events, we term this phenomenon junctional neurulation. Moreover, the planar-cell-polarity member, Prickle-1, is recruited specifically during junctional neurulation and its misexpression within a limited time period suffices to cause anomalies that phenocopy lower spine NTDs in human. Our study thus provides a molecular and cellular basis for understanding the causality of NTD prevalence in humans and ascribes to Prickle-1 a critical role in lower spinal cord formation.

Timing and kinetics of E- to N-cadherin switch during neurulation in the avian embryo.

BACKGROUND: During embryonic development, cadherin switches are correlated with tissue remodelings, such as epithelium-to-mesenchyme transition (EMT). An E- to N-cadherin switch also occurs during neurogenesis, but this is not accompanied with EMT. The biological significance of this switch is currently unknown. RESULTS: We analyzed the timing and kinetics of the E- to N-cadherin switch during early neural induction and neurulation in the chick embryo, in relation to the patterns of their transcriptional regulators. We found that deployment of the E- to N-cadherin switch program varies considerably along the embryonic axis. Rostrally in regions of primary neurulation, it occurs progressively both in time and space in a manner that appears neither in connection with morphological transformation of neural epithelial cells nor in synchrony with movements of neurulation. Caudally, in regions of secondary neurulation, neurogenesis was not associated with cadherin switch as N-cadherin pre-existed before formation of the neural tube. We also found that, during neural development, cadherin switch is orchestrated by a set of transcriptional regulators distinct from those involved in EMT. CONCLUSIONS: Our results indicate that cadherin switch correlates with the partition of the neurectoderm into its three main populations: ectoderm, neural crest, and neural tube.