Premigratory Cranial Neural Crest Cells Research Articles

MicroRNAs promote neural crest multipotency

Restriction of developmental potential occurs when pluripotent cells undergo gastrulation and germ layer specification. Although cranial neural crest cells are specified from the ectoderm, they are capable of generating both ectodermal and mesodermal lineages. How mammalian neural crest give rise to multiple lineages is not well understood. Here, we use single‐cell ATAC sequencing to characterize chromatin accessibility of cranial neural crest and identify a transient increase in accessibility during specification. This increase in chromatin accessibility coincides with co‐expression of both pluripotency and neural crest transcriptional programs. Analysis of the differentiation trajectory during neural crest specification using single‐cell mRNA sequencing revealed that pluripotent cells of the anterior epiblast resolve into two populations of premigratory cranial neural crest corresponding to ectoderm and mesoderm fates. Our data identify two populations of premigratory cranial neural crest cells that deploy distinct pluripotency programs and rely upon post‐transcriptional gene regulation to expand developmental potential and direct cell fate.

Connexin 43 Regulates Multiple Aspects of Chick Cranial Neural Crest Cell Development

Connexins are transmembrane proteins that cluster to form intercellular channels called gap junctions, which allow direct diffusion of small molecules and ions between adjacent cells. Connexin 43 (Cx43) is one of the predominant components of gap junctions and is expressed in multiple organisms and their tissues. To date, however, very little is known about the role of gap junctions, and specifically Cx43, in the neural crest. Our prior immunohistochemical analyses revealed that Cx43 is expressed in premigratory cranial neural crest cells both prior to and during their epithelial‐to‐mesenchymal transition (EMT), and in migratory neural crest cells. Given the robust expression of Cx43 in premigratory neural crest cells, we sought to elucidate the role of Cx43, and gap junctions in particular, during neural crest cell EMT. To this end, we evaluated the presence of gap junctions in the neural crest and performed perturbation experiments to alter the levels of Cx43 in both premigratory and migratory neural crest cells. Our fixed and live imaging results reveal that gap junction formation occurs within both the premigratory and migratory neural crest cell populations. Moreover, morpholino‐mediated reduction in Cx43 protein levels prevents neural crest cells from forming functional gap junctions, as assessed by live imaging of transfer of the gap junction‐specific dye Calcein in the embryo. Additionally, depletion of Cx43 from premigratory neural crest cells reduces the size of the premigratory neural crest cell domain and also delays the emergence of neural crest cells from the dorsal neural tube. This delayed neural tube exit phenotype can be rescued by introduction of full‐length rat Cx43 that is refractory to the morpholino. These data suggest that gap junctions, and Cx43 in particular, are involved in early neural crest cell specification and in the emergence of neural crest cells from the neural tube.Support or Funding InformationThe work was supported by an American Association for Anatomy Postdoctoral Fellowship to K.J. and grants to L.A.T. (NIH R01DE024217, American Cancer Society RSG‐15‐023‐01‐CSM).

The gap junction protein connexin 43 controls multiple aspects of cranial neural crest cell development.

Gap junctions are intercellular channels between cells that facilitate cell-cell communication. Connexin 43 (Cx43; also known as GJA1), the predominant gap junction protein in vertebrates, is expressed in premigratory cranial neural crest cells and is maintained throughout the neural crest cell epithelial-to-mesenchymal transition (EMT), but its function in these cells is unknown. To this end, we used a combination of in vivo and ex vivo experiments to assess gap junction formation, and Cx43 function, in chick cranial neural crest cells. Our results demonstrate that gap junctions exist between premigratory and migratory cranial neural crest cells and depend on Cx43 for their function. In the embryo, Cx43 knockdown just prior to EMT delays the emergence of Cx43-depleted neural crest cells from the neural tube, but these cells eventually successfully emigrate and join the migratory stream. This delay can be rescued by introduction of full-length Cx43 into Cx43-depleted cells. Furthermore, Cx43 depletion reduces the size of the premigratory neural crest cell domain through an early effect on neural crest cell specification. Collectively, these data identify new roles for Cx43 in chick cranial neural crest cell development.

Connexin 43 Function in the Cranial Neural Crest

Connexins are transmembrane proteins that cluster to form intercellular channels called gap junctions, which function to allow direct diffusion of small molecules and ions between adjacent cells. Gap junctions are composed of two connexons (one on each cell which are docked head‐to‐head) consisting of either homomeric or heteromeric connexin proteins. Connexin 43 (Cx43) is one of the predominant components of gap junctions and is expressed in multiple organisms and their tissues. To date, however, very little is known about the role of gap junctions, and specifically Cx43, in the neural crest. Our prior immunohistochemical analyses revealed that Cx43 is expressed in premigratory cranial neural crest cells both prior to and during their epithelial‐to‐mesenchymal transition (EMT), and in migratory neural crest cells. Given the robust expression of Cx43 in premigratory neural crest cells, we sought to elucidate the role of Cx43, and gap junctions in particular, during neural crest cell EMT. To this end, we evaluated the presence of gap junctions in the neural crest and performed molecular and chemical perturbation experiments to alter Cx43 and general gap junction function. Our results reveal that gap junctions form between migratory neural crest cells, demonstrated in the embryo and in explants of premigratory neural crest tissue cultured ex vivo. Live‐imaging of embryos initially possessing Calcein‐AM (a dye that only traverses gap junctions) in a small number of premigratory neural crest cells demonstrates passage of dye to adjacent cells over time. Additionally, depletion of Cx43 from premigratory neural crest cells reveals changes in some neural crest cell molecular markers. In ex vivo explants, Cx43 knockdown in migratory neural crest cells precludes transfer of Calcein‐AM dye, and abrogation of gap junction function through chemical inhibition hinders neural crest cell EMT and/or migration. In future experiments, we will ascertain the effects of Cx43 knockdown and overexpression on neural crest cell EMT and migration in vivo by examining passage of Calcein‐AM dye to assess gap junction function and through immunohistochemistry to evaluate changes in neural crest cells. These data will provide insight into how neural crest cells interact and communicate during EMT and migration to allow for proper embryonic patterning throughout vertebrate development.Support or Funding InformationThe work was supported by grants to L.A.T. (NIH R01DE024217, American Cancer Society RSG‐15‐023‐01‐CSM).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Cadherin-6B proteolytic N-terminal fragments promote chick cranial neural crest cell delamination by regulating extracellular matrix degradation

During epithelial-to-mesenchymal transitions (EMTs), chick cranial neural crest cells simultaneously delaminate from the basement membrane and segregate from the epithelia, in part, via multiple protease-mediated mechanisms. Proteolytic processing of Cadherin-6B (Cad6B) in premigratory cranial neural crest cells by metalloproteinases not only disassembles cadherin-based junctions but also generates shed Cad6B ectodomains or N-terminal fragments (NTFs) that may possess additional roles. Here we report that Cad6B NTFs promote delamination by enhancing local extracellular proteolytic activity around neural crest cells undergoing EMT en masse. During EMT, Cad6B NTFs of varying molecular weights are observed, indicating that Cad6B may be cleaved at different sites by A Disintegrin and Metalloproteinases (ADAMs) 10 and 19 as well as by other matrix metalloproteinases (MMPs). To investigate Cad6B NTF function, we first generated NTF constructs that express recombinant NTFs with similar relative mobilities to those NTFs shed in vivo. Overexpression of either long or short Cad6B NTFs in premigratory neural crest cells reduces laminin and fibronectin levels within the basement membrane, which then facilitates precocious neural crest cell delamination. Zymography assays performed with supernatants of neural crest cell explants overexpressing Cad6B long NTFs demonstrate increased MMP2 activity versus controls, suggesting that Cad6B NTFs promote delamination through a mechanism involving MMP2. Interestingly, this increase in MMP2 does not involve up-regulation of MMP2 or its regulators at the transcriptional level but instead may be attributed to a physical interaction between shed Cad6B NTFs and MMP2. Taken together, these results highlight a new function for Cad6B NTFs and provide insight into how cadherins regulate cellular delamination during normal developmental EMTs as well as aberrant EMTs that underlie human disease.

Connexin 43 Function in the Cranial Neural Crest

Connexins are transmembrane proteins that cluster to form intercellular channels called gap junctions, which function to allow direct diffusion of small molecules and ions between adjacent cells. Gap junctions are composed of two connexons (one on each cell which are docked head‐to‐head) consisting either of homomeric or heteromeric connexin proteins, although only certain combinations of connexins are possible. Connexin 43 is one of the predominant components of gap junctions and is expressed in multiple organisms and their tissues. To date, however, very little is known about the role of gap junctions, and specifically Connexin 43, in the neural crest. Immunohistochemical analyses revealed that Connexin 43 is expressed in premigratory cranial neural crest cells as well as during the epithelial‐to‐mesenchymal transition (EMT), migration, and coalescence of neural crest cells with placodal neurons as the trigeminal ganglion assembles. Interestingly, Connexin 43 is also observed in the cranial mesenchyme, but placodal neurons lack Connexin 43. Given the robust expression of Connexin 43 in the neural crest, we sought to elucidate the role of Connexin 43, and gap junctions in particular, during neural crest cell EMT and migration. To this end, we are assessing the presence of gap junctions in the neural crest and performing molecular and chemical perturbation experiments to alter Connexin 43 and general gap junction function. Our initial results reveal that gap junctions form between migratory neural crest cells generated from explants of premigratory neural crest tissue in an ex vivo culture assay. In future experiments, we will inhibit gap junctions using pharmacological reagents and modulate levels of Connexin 43 through knockdown and overexpression experiments, evaluating effects on neural crest cell EMT and migration in all approaches. Importantly, these assays will confirm that Connexin 43 is critical for gap junction function in neural crest cells. These data will provide insight into how neural crest cells interact and communicate during EMT and migration to allow for proper embryonic patterning throughout vertebrate development.Support or Funding InformationThis research was funded by ACS RSG‐1502301‐CSM and NIH R01DE024217 (L.A.T.).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Cadherin‐6B Proteolysis Controls The Neural Crest Cell Epithelial‐to‐Mesenchymal Transition Through Transcriptional Regulation

During epithelial‐to‐mesenchymal transitions (EMTs), cells disassemble cadherin‐based adherens junctions to segregate from the epithelia. Chick premigratory cranial neural crest cells reduce Cadherin‐6B (Cad6B) levels through several mechanisms, including proteolysis, to permit their EMT and migration. Serial processing of Cad6B by ADAMs and γ‐secretase generates an intracellular C‐terminal fragment (CTF2) that could acquire additional functions independent of full‐length Cad6B. Here we report that Cad6B CTF2 possesses a novel pro‐EMT role by up‐regulating EMT effector genes in vivo. Following proteolysis, CTF2 remains associated with β‐catenin, which stabilizes and redistributes both proteins to the cytosol and nucleus, leading to an increase in β‐catenin, CyclinD1, Snail2, and Snail2 promoter‐based GFP expression in vivo. A CTF2 β‐catenin‐binding mutant, however, fails to alter gene expression, indicating that CTF2 modulates β‐catenin‐responsive EMT effector genes. Notably, CTF2 associates with the endogenous Snail2 promoter in the cranial neural crest, providing, for the first time, evidence that a cadherin CTF2 can bind to chromatin in vivo. Furthermore, this association with chromatin is dependent upon an intact β‐catenin‐binding domain within CTF2. Collectively, our data reveal how Cad6B proteolysis orchestrates multiple pro‐EMT regulatory inputs, including a reduction in adhesion within the premigratory neural crest cell population and up‐regulation of the Cad6B repressor Snail2, to ensure proper cranial neural crest cell EMT and appropriate patterning of the vertebrate embryo.Support or Funding InformationThis work was supported by NIH grants R01DE024217 and F32DE022990 and a Research Scholar Grant, RSG‐15‐023‐01‐CSM, from the American Cancer Society.

Cadherin-6B proteolysis promotes the neural crest cell epithelial-to-mesenchymal transition through transcriptional regulation.

During epithelial-to-mesenchymal transitions (EMTs), cells disassemble cadherin-based junctions to segregate from the epithelia. Chick premigratory cranial neural crest cells reduce Cadherin-6B (Cad6B) levels through several mechanisms, including proteolysis, to permit their EMT and migration. Serial processing of Cad6B by a disintegrin and metalloproteinase (ADAM) proteins and γ-secretase generates intracellular C-terminal fragments (CTF2s) that could acquire additional functions. Here we report that Cad6B CTF2 possesses a novel pro-EMT role by up-regulating EMT effector genes in vivo. After proteolysis, CTF2 remains associated with β-catenin, which stabilizes and redistributes both proteins to the cytosol and nucleus, leading to up-regulation of β-catenin, CyclinD1, Snail2, and Snail2 promoter-based GFP expression in vivo. A CTF2 β-catenin-binding mutant, however, fails to alter gene expression, indicating that CTF2 modulates β-catenin-responsive EMT effector genes. Notably, CTF2 association with the endogenous Snail2 promoter in the neural crest is β-catenin dependent. Collectively, our data reveal how Cad6B proteolysis orchestrates multiple pro-EMT regulatory inputs, including CTF2-mediated up-regulation of the Cad6B repressor Snail2, to ensure proper cranial neural crest EMT.

Cadherin-6B undergoes macropinocytosis and clathrin-mediated endocytosis during cranial neural crest cell EMT.

The epithelial-to-mesenchymal transition (EMT) is important for the formation of migratory neural crest cells during development and is co-opted in human diseases such as cancer metastasis. Chick premigratory cranial neural crest cells lose intercellular contacts, mediated in part by Cadherin-6B (Cad6B), migrate extensively, and later form a variety of adult derivatives. Importantly, modulation of Cad6B is crucial for proper neural crest cell EMT. Although Cad6B possesses a long half-life, it is rapidly lost from premigratory neural crest cell membranes, suggesting the existence of post-translational mechanisms during EMT. We have identified a motif in the Cad6B cytoplasmic tail that enhances Cad6B internalization and reduces the stability of Cad6B upon its mutation. Furthermore, we demonstrate for the first time that Cad6B is removed from premigratory neural crest cells through cell surface internalization events that include clathrin-mediated endocytosis and macropinocytosis. Both of these processes are dependent upon the function of dynamin, and inhibition of Cad6B internalization abrogates neural crest cell EMT and migration. Collectively, our findings reveal the significance of post-translational events in controlling cadherins during neural crest cell EMT and migration.

Follow-the-leader cell migration requires biased cell–cell contact and local microenvironmental signals

Directed cell migration often involves at least two types of cell motility that include multicellular streaming and chain migration. However, what is unclear is how cell contact dynamics and the distinct microenvironments through which cells travel influence the selection of one migratory mode or the other. The embryonic and highly invasive neural crest (NC) are an excellent model system to study this question since NC cells have been observed in vivo to display both of these types of cell motility. Here, we present data from tissue transplantation experiments in chick and in silico modeling that test our hypothesis that cell contact dynamics with each other and the microenvironment promote and sustain either multicellular stream or chain migration. We show that when premigratory cranial NC cells (at the pre-otic level) are transplanted into a more caudal region in the head (at the post-otic level), cells alter their characteristic stream behavior and migrate in chains. Similarly, post-otic NC cells migrate in streams after transplantation into the pre-otic hindbrain, suggesting that local microenvironmental signals dictate the mode of NC cell migration. Simulations of an agent-based model (ABM) that integrates the NC cell behavioral data predict that chain migration critically depends on the interplay of biased cell–cell contact and local microenvironment signals. Together, this integrated modeling and experimental approach suggests new experiments and offers a powerful tool to examine mechanisms that underlie complex cell migration patterns.

Tetraspanin18 is a FoxD3-responsive antagonist of cranial neural crest epithelial-to-mesenchymal transition that maintains cadherin-6B protein.

During epithelial-to-mesenchymal transition (EMT), tightly associated, polarized epithelial cells become individual mesenchymal cells capable of migrating. Here, we investigate the role of the transmembrane protein tetraspanin18 (Tspan18) in chick cranial neural crest EMT. Tspan18 mRNA is expressed in premigratory cranial neural crest cells, but is absent from actively migrating neural crest cells. Tspan18 knockdown leads to a concomitant loss of cadherin-6B (Cad6B) protein, whereas Cad6B protein persists when Tspan18 expression is extended. The temporal profile of Cad6B mRNA downregulation is unaffected in these embryos, which indicates that Tspan18 maintains Cad6B protein levels and reveals that Cad6B is regulated by post-translational mechanisms. Although downregulation of Tspan18 is necessary, it is not sufficient for neural crest migration: the timing of neural crest emigration, basal lamina breakdown and Cad7 upregulation proceed normally in Tspan18-deficient cells. This emphasizes the need for coordinated transcriptional and post-translational regulation of Cad6B during EMT and illustrates that Tspan18-antagonized remodeling of cell-cell adhesions is only one step in preparation for cranial neural crest migration. Unlike Cad6B, which is transcriptionally repressed by Snail2, Tspan18 expression is downstream of the winged-helix transcription factor FoxD3, providing a new transcriptional input into cranial neural crest EMT. Together, our data reveal post-translational regulation of Cad6B protein levels by Tspan18 that must be relieved by a FoxD3-dependent mechanism in order for cranial neural crest cells to migrate. These results offer new insight into the molecular mechanisms of cranial neural crest EMT and expand our understanding of tetraspanin function relevant to metastasis.

The fate of cranial neural crest cells in the Australian lungfish, Neoceratodus forsteri

The cranial neural crest has been shown to give rise to a diversity of cells and tissues, including cartilage, bone and connective tissue, in a variety of tetrapods and in the zebrafish. It has been claimed, however, that in the Australian lungfish these tissues are not derived from the cranial neural crest, and even that no migrating cranial neural crest cells exist in this species. We have earlier documented that cranial neural crest cells do migrate, although they emerge late, in the Australian lungfish. Here, we have used the lipophilic fluorescent dye, DiI, to label premigratory cranial neural crest cells and follow their fate until stage 43, when several cranial skeletal elements have started to differentiate. The timing and extent of their migration was investigated, and formation of mandibular, hyoid and branchial streams documented. Cranial neural crest was shown to contribute cells to several parts of the head skeleton, including the trabecula cranii and derivatives of the mandibular arch (e.g., Meckel's cartilage, quadrate), the hyoid arch (e.g., the ceratohyal) and the branchial arches (ceratobranchials I-IV), as well as to the connective tissue surrounding the myofibers in cranial muscles. We conclude that cranial neural crest migration and fate in the Australian lungfish follow the stereotyped pattern documented in other vertebrates.

Expression of cardiac neural crest and heart genes isolated by modified differential display

The invasion of the cardiac neural crest (CNC) into the outflow tract (OFT) and subsequent outflow tract septation are critical events during vertebrate heart development. We have performed four modified differential display screens in the chick embryo to identify genes that may be involved in CNC, OFT, secondary heart field, and heart development. The screens included differential display of RNA isolated from three different axial segments containing premigratory cranial neural crest cells; of RNA from distal outflow tract, proximal outflow tract, and atrioventricular tissue of embryonic chick hearts; and of RNA isolated from left and right cranial tissues, including the early heart fields. These screens have resulted in the identification of the five cDNA clones presented here, which are expressed in the cardiac neural crest, outflow tract and developing heart in patterns that are unique in heart development.

Timing of odontogenic neural crest cell migration and tooth-forming capability in mice.

The mammalian tooth develops through sequential and reciprocal interactions between cranial neural crest (CNC)- derived ectomesenchymal cells and the stomadial epithelium. Classic tissue recombination studies demonstrated that premigratory CNC cells and CNC-derived ectomesenchymal cells possess odontogenic capacity and can respond to oral epithelial signals to form a tooth, suggesting that the CNC cells contributing to odontogenic tissue are not prespecified. Here we show that, in mice, CNC cells have populated the forming first branchial arch before the 9-somite stage and continue to migrate into the arch by the 13-somite stage. Grafts of the first arch from the 10-somite embryo or earlier yielded membranous bone and cysts but no teeth after subrenal culture. However, grafts of the first arch with its dorsally adjacent tissue containing migrating neural crest cells from the same age embryos gave rise to teeth. In contrast, teeth formed in first arch grafts that do not contain migrating neural crest cells from embryos with 12 or more somites. Interestingly, the acquisition of tooth forming capability in the first arch coincides with the onset of Fgf8 expression in the oral epithelium. These results suggest that there exists a population of odontogenic neural crest cells that migrates into the first arch between the 10- and 12-somite stages. These cells either possess odontogenic potential and are able to initiate tooth development, or can respond to odontogenic signals derived from the oral epithelium to support tooth formation.

Ectodermal markers delineate the neural fold interface during avian neurulation.

The formation and morphogenesis of the neural folds are important processes underlying neurulation. We showed previously that these processes comprise four key events in avian embryos: epithelial ridging, kinking, delamination, and apposition. Collectively, these events establish the paired, bilaminar neural folds, which fuse in the dorsal midline during late neurulation to close the neural groove and to establish the neural tube. Here, we use an antisense riboprobe for a new gene called Plato, as well as an antibody for a previously cloned transcription factor, AP-2, as markers to identify critical subpopulations of ectodermal cells during the formation and morphogenesis of the avian neural folds. Plato antisense riboprobe marks the cranial neural ectoderm and premigratory cranial neural crest cells, whereas AP-2 antibody marks the epidermal ectoderm and the early migratory neural crest. We show that subpopulations of ectodermal cells at the forebrain and midbrain levels undergo considerable rearrangement within the neural fold transition zone, which redistributes incipient neural crest cells from the neural ectodermal side of the forming neural fold interface to the epidermal ectodermal side. Additionally, we show that Plato and AP-2 provide useful markers for delineating the incipient neural fold interface.

Nerve growth factor (NGF) supports tooth morphogenesis in mouse first branchial arch explants.

Posterior midbrain and anterior hindbrain neuroectoderm trans-differentiate into cranial neural crest cells (CNCC), emigrate from the neural folds, and become crest-derived ectomesenchyme within the mandibular and maxillary processes. To investigate the growth factor requirement specific for the initiation of tooth morphogenesis, we designed studies to test whether nerve growth factor (NGF) can support odontogenesis in a first branchial arch (FBA) explant culture system. FBA explants containing neural-fold tissues before CNCC emigration and the anlagen of the FBA were microdissected from embryonic day 8 (E8) mouse embryos, and cultured for 8 days in medium supplemented with 10% fetal calf serum only, or serum-containing medium further supplemented with either NGF or epidermal growth factor (EGF) at three different concentrations: 50, 100, or 200 ng/ml. Morphological, morphometric, and total protein analyses indicated that growth and development in all groups were comparable. Meckel's cartilage and tongue formation were also observed in all groups. However, odontogenesis was only detected in explants cultured in the presence of exogenous NGF. NGF-supplemented cultures were permissive for bud stage (50 ng/ml) as well as cap stage of tooth morphogenesis (100 and 200 ng/ml). Morphometric analyses of the volume of tooth organs showed a significant dose-dependent increase in tooth volume as the concentration of NGF increased. Whole-mount in situ hybridization and semiquantitative reverse transcription-polymerase chain reaction for Pax9, a molecular marker of dental mesenchyme, further supported and confirmed the morphological data of the specificity and dose dependency of NGF on odontogenesis. We conclude that (1) E8 FBA explants contain premigratory CNCC that are capable of emigration, proliferation, and differentiation in vitro; (2) serum-supplemented medium is permissive for CNCC differentiation into tongue myoblasts and chondrocytes in FBA explants; and (3) NGF controls CNCC cell fate specification and differentiation into tooth organs.

High glucose concentration inhibits migration of rat cranial neural crest cells in vitro

Cranial neural crest cells give rise to a large part of the facial structures, and disturbed development of these cells may therefore cause congenital malformations affecting the head and face. We studied the effects of increased glucose concentration on the migration and development of cranial neural crest cells, maintained in vitro for 48 h. Pre-migratory cranial neural crest cells were removed from embryos of normal and diabetic rats on gestational day 9. After 24 h in 10 mmol/l glucose the cells were exposed to glucose concentrations of 10, 30, or 50 mmol/l for another 24 h. The cultures were photographed at 24 h and 48 h in a phase-contrast microscope to evaluate cell morphology, cell number, and cell migration. Exposure to 50 mmol/l glucose reduced the total number of neural crest cells, their mean migratory distance and migratory area expansion compared to cells cultured in 10 mmol/l glucose. To investigate the effect of antioxidant agents, high glucose cultures were studied after addition of N-acetylcysteine (NAC), or superoxide dismutase (SOD). Addition of NAC diminished the inhibitory effect of high glucose, whereas SOD did not offer any improvement in cell development. Neural crest cell culture from embryos of diabetic rats showed reduced cell migration in vitro at all glucose concentrations compared to normal cells. In addition, the cells from embryos of diabetic rats showed reduced migratory area expansion after culture in the basal 10 mmol/l glucose concentration, indicating that maternal diabetes permanently influences the future development of premigratory cranial neural crest cells. These findings indicate that high glucose concentration inhibits cranial neural crest development in vitro, and that antioxidant therapy may diminish this inhibition. Free radical oxygen species may be involved in the induction of malformations and antioxidants may therefore have a role in future attempts to block the teratogenic effects of diabetic pregnancy.

Vital dye analysis of cranial neural crest cell migration in the mouse embryo.

The spatial and temporal aspects of cranial neural crest cell migration in the mouse are poorly understood because of technical limitations. No reliable cell markers are available and vital staining of embryos in culture has had limited success because they develop normally for only 24 hours. Here, we circumvent these problems by combining vital dye labelling with exo utero embryological techniques. To define better the nature of cranial neural crest cell migration in the mouse embryo, premigratory cranial neural crest cells were labelled by injecting DiI into the amniotic cavity on embryonic day 8. Embryos, allowed to develop an additional 1 to 5 days exo utero in the mother before analysis, showed distinct and characteristic patterns of cranial neural crest cell migration at the different axial levels. Neural crest cells arising at the level of the forebrain migrated ventrally in a contiguous stream through the mesenchyme between the eye and the diencephalon. In the region of the midbrain, the cells migrated ventrolaterally as dispersed cells through the mesenchyme bordered by the lateral surface of the mesencephalon and the ectoderm. At the level of the hindbrain, neural crest cells migrated ventrolaterally in three subectodermal streams that were segmentally distributed. Each stream extended from the dorsal portion of the neural tube into the distal portion of the adjacent branchial arch. The order in which cranial neural crest cells populate their derivatives was determined by labelling embryos at different stages of development. Cranial neural crest cells populated their derivatives in a ventral-to-dorsal order, similar to the pattern observed at trunk levels. In order to confirm and extend the findings obtained with exo utero embryos, DiI (1,1-dioctadecyl-3,3,3',3'-tetramethylindo-carbocyanine perchlorate) was applied focally to the neural folds of embryos, which were then cultured for 24 hours. Because the culture technique permitted increased control of the timing and location of the DiI injection, it was possible to determine the duration of cranial neural crest cell emigration from the neural tube. Cranial neural crest cell emigration from the neural folds was completed by the 11-somite stage in the region of the rostral hindbrain, the 14-somite stage in the regions of the midbrain and caudal hindbrain and not until the 16-somite stage in the region of the forebrain. At each level, the time between the earliest and latest neural crest cells to emigrate from the neural tube appeared to be 9 hours.(ABSTRACT TRUNCATED AT 400 WORDS)

Distribution and function of tenascin during cranial neural crest development in the chick

Tenascin is a glycoprotein associated with the extracellular matrix and the surface of some cell types. Here, the distribution and possible function of tenascin have been examined along the pathways followed by cranial neural crest cells. During early stages of neural crest migration, tenascin was observed in a dense matrix surrounding premigratory cranial neural crest cells. Along the neural tube, tenascin immunoreactivity was observed in a dorsoventral gradient and was also noted under the ectoderm and around the notochord. During advanced neural crest migration, tenascin immunoreactivity colocalized with and appeared to be on the surface of migrating neural crest cells. At later stages, tenascin was present around the otic vesicles, retina, lens, and in an interstitial matrix in the region of the branchial arches. At the level of the occipital somites, tenascin immunoreactivity was observed around the neural tube, notochord, dermamyotome, and on the basal surface of the ectoderm. Tenascin was also observed in an interstitial matrix within the sclerotome. At early stages of vagal neural crest migration, immunoreactivity was uniform within the sclerotome, whereas at later stages tenascin colocalized with vagal neural crest cells within the rostral half of each sclerotome. The possible function of tenascin was tested by injecting antitenascin antibodies lateral to the mesencephalic neural tube. Two predominant defects were noted in injected embryos: 1) ectopic aggregates of cranial neural crest cells external to the neural tube and sometimes located on the apical side of the ectoderm; and 2) open and deformed neural tubes. Both the distribution and results of the perturbation experiment suggest that tenascin is required for proper cranial neural crest migration.

Hemodynamic characteristics in neural crest cell-excised chick embryo.

Damage to premigratory cranial neural crest cells results in cardiovascular anomalies of so-called conotruncal anomaly. In one report, it was suggested that hemodynamic alteration would precede abnormal cardiovascular morphogenesis. We repeated this hemodynamic study in neural crest cell-excised chick embryos and found that there was no difference in heart rate and blood pressure between the treated embryos (n = 11; 164 +/- 3 beats per min (BPM) and 0.75 +/- 0.03 mmHg, respectively) and control embryos (n = 7; 160 +/- 6 BPM and 0.72 +/- 0.07 mmHg, respectively). The response of heart rate to acetylcholine was less (P less than 0.05) in the treated embryos (-13% +/- 2%) than in the control (-20% +/- 2%). Thus, the present data do not support the hypothesis that alterations in blood pressure and heart rate are causally related to abnormal cardiovascular morphogenesis. The developmental significance of subtle functional changes in neural crest-extirpated embryos in response to cholinergic challenge is unclear.