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.

Disruption of CXCR4 signaling in pharyngeal neural crest cells causes DiGeorge syndrome-like malformations.

DiGeorge syndrome (DGS) is a congenital disease causing cardiac outflow tract anomalies, craniofacial dysmorphogenesis, thymus hypoplasia, and mental disorders. It results from defective development of neural crest cells (NCs) that colonize the pharyngeal arches and contribute to lower jaw, neck and heart tissues. Although TBX1 has been identified as the main gene accounting for the defects observed in human patients and mouse models, the molecular mechanisms underlying DGS etiology are poorly identified. The recent demonstrations that the SDF1/CXCR4 axis is implicated in NC chemotactic guidance and impaired in cortical interneurons of mouse DGS models prompted us to search for genetic interactions between Tbx1, Sdf1 (Cxcl12) and Cxcr4 in pharyngeal NCs and to investigate the effect of altering CXCR4 signaling on the ontogeny of their derivatives, which are affected in DGS. Here, we provide evidence that Cxcr4 and Sdf1 are genetically downstream of Tbx1 during pharyngeal NC development and that reduction of CXCR4 signaling causes misrouting of pharyngeal NCs in chick and dramatic morphological alterations in the mandibular skeleton, thymus and cranial sensory ganglia. Our results therefore support the possibility of a pivotal role for the SDF1/CXCR4 axis in DGS etiology.

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.

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.

Misregulation of SDF1-CXCR4 signaling impairs early cardiac neural crest cell migration leading to conotruncal defects.

RATIONALE: Cardiac neural crest cells (NCs) contribute to heart morphogenesis by giving rise to a variety of cell types from mesenchyme of the outflow tract, ventricular septum, and semilunar valves to neurons of the cardiac ganglia and smooth muscles of the great arteries. Failure in cardiac NC development results in outflow and ventricular septation defects commonly observed in congenital heart diseases. Cardiac NCs derive from the vagal neural tube, which also gives rise to enteric NCs that colonize the gut; however, so far, molecular mechanisms segregating these 2 populations and driving cardiac NC migration toward the heart have remained elusive. OBJECTIVE: Stromal-derived factor-1 (SDF1) is a chemokine that mediates oriented migration of multiple embryonic cells and mice deficient for Sdf1 or its receptors, Cxcr4 and Cxcr7, exhibit ventricular septum defects, raising the possibility that SDF1 might selectively drive cardiac NC migration toward the heart via a chemotactic mechanism. METHODS AND RESULTS: We show in the chick embryo that Sdf1 expression is tightly coordinated with the progression of cardiac NCs expressing Cxcr4. Cxcr4 loss-of-function causes delayed migration and enhanced death of cardiac NCs, whereas Sdf1 misexpression results in their diversion from their normal pathway, indicating that SDF1 acts as a chemoattractant for cardiac NCs. These alterations of SDF1 signaling result in severe cardiovascular defects. CONCLUSIONS: These data identify Sdf1 and its receptor Cxcr4 as candidate genes responsible for cardiac congenital pathologies in human.

Buckling along boundaries of elastic contrast as a mechanism for early vertebrate morphogenesis.

We have investigated the mechanism of formation of the body of a typical vertebrate, the chicken. We find that the body forms initially by folding at boundaries of stiffness contrast. These boundaries are dynamic lines, separating domains of different cell sizes, that are advected in a deterministic thin-film visco-elastic flow. While initially roughly circular, the lines of elastic contrast form large "peanut" shapes evoking a slender figure-8 at the moment of formation of the animal body, due to deformation and flow in a quadrupolar stretch caused by mesoderm migration. Folding of these "peanut" or "figure-8" motives along the lines of stiffness contrast creates the global pattern of the animal, and segregates several important territories. The main result is that the pattern of cell texture in the embryo serves simultaneously two seemingly different purposes: it regionalizes territories that will differentiate to different cell types and it also locks the folds that physically segregate these territories. This explains how the different cellular types segregate in physically separated domains.

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.

Sonic hedgehog regulates integrin activity, cadherin contacts, and cell polarity to orchestrate neural tube morphogenesis.

In vertebrates, the embryonic nervous system is shaped and patterned by a series of temporally and spatially regulated cell divisions, cell specifications, and cell adhesions and movements. Morphogens of the Hedgehog, Wnt, and bone morphogenetic protein families have been shown to play a crucial role in the control of cell division and specification in the trunk neural tube, but their possible implication in the regulation of adhesive events has been poorly documented. In the present study, we demonstrate that Sonic hedgehog regulates neural epithelial cell adhesion and polarity through regulation of integrin activity, cadherin cell-cell contact, and cell polarity genes in immature neural epithelial cells before the specification of neuronal cells. We propose that Sonic hedgehog orchestrates neural tube morphogenesis by coordinating adhesive and motility events with cell proliferation and differentiation.

Establishing Primary Cultures of Trunk Neural Crest Cells.

Neural crest cells constitute a unique population of progenitor cells with extensive stem cell capacities able to navigate throughout various environments in the embryo and are a source of multiple cell types, including neurons, glia, melanocytes, smooth muscles, endocrine cells, cardiac cells, and also skeletal and supportive tissues in the head. Neural crest cells are not restricted to the embryo but persist as well in adult tissues where they provide a reservoir of stem cells with great therapeutic promise. Many fundamental questions in cell, developmental, and stem cell biology can be addressed using this system. During the last decades there has been an increased availability of elaborated techniques, animal models, and molecular tools to tackle neural crest cell development. However, these approaches are often very challenging and difficult to establish and they are not adapted for rapid functional investigations of mechanisms driving cell migration and differentiation. In addition, they are not adequate for collecting pure populations of neural crest cells usable in large scale analyses and for stem cell studies. Transferring and adapting the neural crest system in tissue culture may then represent an attractive alternative, opening up numerous prospects. Here we describe a simple method for establishing primary cultures of neural crest cells derived from trunk neural tubes using the avian embryo as a source of cells. This protocol is suited for producing pure populations of neural crest cells that can be processed for cytological, cellular, and functional approaches aimed at characterizing their phenotype, behavior, and potential. © 2020 Wiley Periodicals LLC. Basic Protocol: Primary cultures of avian trunk neural crest cells Support Protocol: Adaptations for immunofluorescence labeling and videomicroscopy.

Spatio-temporal control of neural epithelial cell migration and epithelium-to-mesenchyme transition during avian neural tube development.

As opposed to the neural crest, the neural epithelium is generally viewed as a static and cohesive structure. Here, using an ex vivo system free of the environmental influences and physical constraints encountered in the embryo, we show that neural epithelial cells are on the contrary intrinsically motile, although they do not undergo spontaneous epithelium-to-mesenchyme transition and display molecular and cellular characteristics distinct from those of neural crest cells. However, they can be instructed to undergo epithelium-to-mesenchyme conversion independently of the acquisition of neural crest traits. Migration potentialities of neural epithelial cells are transient and are progressively restricted during neural tube development. Restriction of cell migration is irreversible and can be in part accounted for by increase in N-cadherin in cellular junctions and in cell polarity. In conclusion, our study reveals that the neural epithelium is a highly flexible tissue in which cells are maintained cohesive under the control of a combination of extrinsic factors and physical constraints.

Neural crest delamination and migration: integrating regulations of cell interactions, locomotion, survival and fate.

During the entire process of neural crest development from specification till final differentiation, delamination and migration are critical steps where nascent crest cells face multiple challenges: within a relatively short period of time that does not exceed several hours, they have to change drastically their cell- and substrate-adhesion properties, lose cell polarity and activate the locomotory machinery, while keeping proliferating, surviving and maintaining a pool of precursors in the neural epithelium. Then, as soon as they are released from the neural tube, neural crest cells have to adapt to a new, rapidly-changing environment and become able to interpret multiple cues which guide them to appropriate target sites and prevent them from distributing in aberrant locations. It appears from recent studies that, behind an apparent linearity and unity, neural crest development is subdivided into several independent steps, each being governed by a multiplicity of rules and referees. Here resides probably one of the main reasons of the success of neural crest cells to accomplish their task.

SDF1-CXCR4 signaling: A new player involved in DiGeorge/22q11-deletion syndrome.

The DiGeorge/22q11-deletion syndrome (22q11DS), also known as velocardiofacial syndrome, is a congenital disease causing numerous structural and behavioral disorders, including cardiac outflow tract anomalies, craniofacial dysmorphogenesis, parathyroid and thymus hypoplasia, and mental disorders. It results from a unique chromosomal microdeletion on the 22q11.2 region in which the transcriptional activator TBX1 is decisive for the occurrence of the disease. During embryogenesis, Tbx1 is required for patterning of pharyngeal region giving rise to structures of the face, neck and chest. Genetic and developmental studies demonstrated that the severity and variability of the syndrome are determined by Tbx1 targets involved in pharyngeal neural crest cell migration and survival. Recently, we demonstrated that the chemokine Sdf1/Cxcl12 and its receptor Cxcr4 are genetically downstream of Tbx1 during pharyngeal development and that reduction of CXCR4 signaling results in defects which recapitulate the major morphological anomalies of 22q11DS, supporting the possibility of a pivotal role for the SDF1/CXCR4 axis in its etiology.

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.

Hyperactivation of Alk induces neonatal lethality in knock-in AlkF1178L mice.

The ALK (Anaplastic Lymphoma Kinase) gene encodes a tyrosine kinase receptor preferentially expressed in the central and peripheral nervous systems. A syndromic presentation associating congenital neuroblastoma with severe encephalopathy and an abnormal shape of the brainstem has been described in patients harbouring de novo germline F1174V and F1245V ALK mutations. Here, we investigated the phenotype of knock-in (KI) mice bearing the AlkF1178L mutation (F1174L in human). Although heterozygous KI mice did not reproduce the severe breathing and feeding difficulties observed in human patients, behavioral tests documented a reduced activity during dark phases and an increased anxiety of mutated mice. Matings of heterozygotes yielded the expected proportions of wild-type, heterozygotes and homozygotes at birth but a high neonatal lethality was noticed for homozygotes. We documented Alk expression in several motor nuclei of the brainstem involved in the control of sucking and swallowing. Evaluation of basic physiological functions 12 hours after birth revealed slightly more apneas but a dramatic reduced milk intake for homozygotes compared to control littermates. Overall, our data demonstrate that Alk activation above a critical threshold is not compatible with survival in mice, in agreement with the extremely severe phenotype of patients carrying aggressive de novo ALK germline mutations.

Diversity in the molecular and cellular strategies of epithelium-to-mesenchyme transitions: Insights from the neural crest.

Although epithelial to mesenchymal transitions (EMT) are often viewed as a unique event, they are characterized by a great diversity of cellular processes resulting in strikingly different outcomes. They may be complete or partial, massive or progressive, and lead to the complete disruption of the epithelium or leave it intact. Although the molecular and cellular mechanisms of EMT are being elucidated owing chiefly from studies on transformed epithelial cell lines cultured in vitro or from cancer cells, the basis of the diversity of EMT processes remains poorly understood. Clues can be collected from EMT occuring during embryonic development and which affect equally tissues of ectodermal, endodermal or mesodermal origins. Here, based on our current knowledge of the diversity of processes underlying EMT of neural crest cells in the vertebrate embryo, we propose that the time course and extent of EMT do not depend merely on the identity of the EMT transcriptional regulators and their cellular effectors but rather on the combination of molecular players recruited and on the possible coordination of EMT with other cellular processes.

Ets-1 confers cranial features on neural crest delamination.

Neural crest cells (NCC) have the particularity to invade the environment where they differentiate after separation from the neuroepithelium. This process, called delamination, is strikingly different between cranial and trunk NCCs. If signalings controlling slow trunk delamination start being deciphered, mechanisms leading to massive and rapid cranial outflow are poorly documented. Here, we show that the chick cranial NCCs delamination is the result of two events: a substantial cell mobilization and an epithelium to mesenchyme transition (EMT). We demonstrate that ets-1, a transcription factor specifically expressed in cranial NCCs, is responsible for the former event by recruiting massively cranial premigratory NCCs independently of the S-phase of the cell cycle and by leading the gathered cells to straddle the basal lamina. However, it does not promote the EMT process alone but can cooperate with snail-2 (previously called slug) to this event. Altogether, these data lead us to propose that ets-1 plays a pivotal role in conferring specific cephalic characteristics on NCC delamination.

alpha1beta1-integrin engagement to distinct laminin-1 domains orchestrates spreading, migration and survival of neural crest cells through independent signaling pathways.

Integrin engagement regulates cell adhesion, shape, migration, growth, and differentiation, but molecular mechanisms coordinating these functions in cells remain unclear. Because of their migratory and differentiation potential, neural crest cells constitute a powerful paradigm to address this question. Here, we describe that laminin-1, a major component of their migration routes, promotes crest cell spreading, migration and survival through two distinct integrin-binding domains that are situated on both sides of its alpha1 subunit and can be separated in the LN-1 elastase proteolytic fragments E1' and E8. Interaction with either domain was mediated by the same integrin alpha1beta1 but produced distinct, complementary responses through specific signaling cascades. FAK activation upon E8 binding induced spreading, formation of actin bundles and focal adhesions, stimulated oriented migration, but failed to support survival. Conversely, Erk activation upon E1' binding promoted long-term survival and random migration without actin reorganization. Consistent with this, interaction with laminin-5 or laminin-10/11, which do not harbor integrin-binding domains in the N-terminal side of their alpha chains, failed to support survival. Thus, the signaling activity and function of integrins might depend on binding domains in their ligands, thereby revealing ligand control of integrin function as a possible mechanism for the modulation and coordination of cell response to adhesive signals.

Cellular localization and signaling activity of beta-catenin in migrating neural crest cells.

In the vertebrate embryo, development of the neural crest is accompanied by sequential changes in cellular adhesiveness, allowing cells to delaminate from the neural epithelium, to undergo migration through extracellular matrix material, and to coalesce into ganglia of the peripheral nervous system. Because of its dual role in cell adhesion, as a link between cadherins and the actin cytoskeleton, and in cell signaling, as a key mediator of the Wnt-signaling pathway, beta-catenin is a good candidate to play a central role in the control of neural crest cell development. In the present study, we analyzed, by using an in vitro culture system, whether the cellular localization and the signaling activity of beta-catenin are regulated in conjunction with cell migration during ontogeny of trunk neural crest cells in the avian embryo. beta-Catenin molecules were found primarily in association with N-cadherin in the regions of intercellular contacts in most migrating neural crest cells, and only early-migrating cells situated in proximity with the dorsal side of the neural tube showed detectable beta-catenin in their nuclei. This finding indicates that beta-catenin may be recruited for signaling in neural crest cells only transiently at the onset of migration and that sustained beta-catenin signals are not necessary for the progression of migration. The nuclear distribution of beta-catenin within crest cells was not affected upon modification of the N-cadherin-mediated cell-cell contacts, revealing that recruitment of beta-catenin for signaling is not driven by changes in intercellular cohesion during migration. Overstimulation of beta-catenin signals in neural crest cells at the time of their migration, using LiCl treatment or coculture with Wnt-1-producing cells, induced nuclear translocation of beta-catenin and Lef-1 up-regulation in neural crest cells and provoked a marked inhibition of cell delamination and migration. The effect of LiCl and exogenous Wnt-1 on neural crest cells could be essentially attributed to a dramatic decrease in integrin-mediated cell-matrix adhesion as well as a massive reduction of cell proliferation. In addition, although it apparently did not affect expression of neural crest markers, Wnt-1 exposure dramatically affected signaling events involving Notch-Delta, presumably also accounting for the strong reduction in cell delamination. In conclusion, our data indicate that beta-catenin functions primarily in cell adhesion events during migration and may be recruited transiently for signaling during delamination possibly to regulate the balance between cell proliferation and cell differentiation.

A dual role for Sonic hedgehog in regulating adhesion and differentiation of neuroepithelial cells.

In vertebrates, the nervous system arises from a flat sheet of epithelial cells, the neural plate, that gradually transforms into a hollow neural tube. This process, called neurulation, involves sequential changes in cellular interactions that are precisely coordinated both spatially and temporally by the combined actions of morphogens. To gain further insight into the molecular events regulating cell adhesion during neurulation, we investigated whether the adhesive and migratory capacities of neuroepithelial cells might be modulated by Sonic hedgehog (Shh), a signaling molecule involved in the control of cell differentiation in the ventral neural tube. When deposited onto extracellular matrix components in vitro, neural plates explanted from avian embryos at early neurulation readily dispersed into monolayers of spread cells, thereby revealing their intrinsic ability to migrate. In the presence of Shh added in solution to the culture medium, the explants still exhibited the same propensity to disperse. In contrast, when Shh was immobilized to the substrate or produced by neuroepithelial cells themselves after transfection, neural plate explants failed to disperse and instead formed compact structures. Changes in the adhesive capacities of neuroepithelial cells caused by Shh could be accounted for by inactivation of surface beta1-integrins combined with an increase in N-cadherin-mediated cell adhesion. Furthermore, immobilized Shh promoted differentiation of neuroepithelial cells into motor neurons and floor plate cells with the same potency as soluble Shh. However, the effect of Shh on the neuroepithelial cell adhesion was discernible and apparently independent from its differentiation effect and was not mediated by the signaling cascade elicited by the Patched-Smoothened receptor and involving the Gli transcription factors. Thus, our experiments indicate that Shh is able to control sequentially adhesion and differentiation of neuroepithelial cells through different mechanisms, leading to a coordinated regulation of the various cell interactions essential for neural tube morphogenesis.