Premigratory Neural Crest Research Articles

The role of the Notch signalling pathway in regulating the balance between neuronal and nonneuronal cells in sympathetic ganglia and the adrenal gland.

Sympathetic neurons and endocrine chromaffin cells of the adrenal medulla are catecholaminergic cells that derive from the neural crest. According to the classic model, they develop from a common sympathoadrenal (SA) progenitor that has the ability to differentiate into both sympathetic neurons and chromaffin cells depending on signals provided by their final environment. Our previous data revealed that a single premigratory neural crest cell can give rise to both sympathetic neurons and chromaffin cells, indicating that the fate decision between these cell types occurs after delamination. A more recent study demonstrated that at least half of chromaffin cells arise from a later contribution by Schwann cell precursors. Since Notch signalling is known to be implicated in the regulation of cell fate decisions, we investigated the early role of Notch signalling in regulating the development of neuronal and non-neuronal SA cells within sympathetic ganglia and the adrenal gland. To this end, we implemented both gain and loss of function approaches. Electroporation of premigratory neural crest cells with plasmids encoding Notch inhibitors revealed an elevation in the number of SA cells expressing the catecholaminergic enzyme tyrosine-hydroxylase, with a concomitant reduction in the number of cells expressing the glial marker P0 in both sympathetic ganglia and adrenal gland. As expected, gain of Notch function had the opposite effect. Numbers of neuronal and non-neuronal SA cells were affected differently by Notch inhibition depending on the time of its onset. Together our data show that Notch signalling can regulate the ratio of glial cells, neuronal SA cells and nonneuronal SA cells in both sympathetic ganglia and the adrenal gland.

Post-transcriptional regulation in cranial neural crest cells expands developmental potential

Developmental potential is progressively restricted after germ layer specification during gastrulation. However, cranial neural crest cells challenge this paradigm, as they develop from anterior ectoderm, yet give rise to both ectodermal derivatives of the peripheral nervous system and ectomesenchymal bone and cartilage. How cranial neural crest cells differentiate into multiple lineages is poorly understood. Here, we demonstrate that cranial neural crest cells possess a transient state of increased chromatin accessibility. We profile the spatiotemporal emergence of premigratory neural crest and find evidence of lineage bias toward either a neuronal or ectomesenchymal fate, with each expressing distinct factors from earlier stages of development. We identify the miR-302 miRNA family to be highly expressed in cranial neural crest cells and genetic deletion leads to precocious specification of the ectomesenchymal lineage. Loss of mir-302 results in reduced chromatin accessibility in the neuronal progenitor lineage of neural crest and a reduction in peripheral neuron differentiation. Mechanistically, we find that mir-302 directly targets Sox9 to slow the timing of ectomesenchymal neural crest specification and represses multiple genes involved in chromatin condensation to promote accessibility required for neuronal differentiation. Our findings reveal a posttranscriptional mechanism governed by miRNAs to expand developmental potential of cranial neural crest.

424 Perivascular niches dictate developmental cellular hierarchies during melanoma growth

Cellular plasticity endows cancer cells with capacity towards a phenotypic state. Except from the genetic origin of cellular plasticity, cancer cells can transit to reversible phenotypic states as a result of microenvironmental cues and are often driven by stochastic epigenetic and/or transcriptional fluctuations. Melanoma is a prime example of heterogeneous and plastic tumors where the origin and magnitude of cell state diversity remains poorly understood. Combining lineage tracing, 3D imaging and quantitative mathematical modelling in a clinically-relevant mouse model of melanoma, we demonstrated that tumors follow a hierarchical model of growth supported by a population of Melanoma Stem-like Cells (MSCs) that exhibit a transcriptomic signature of pre-migratory neural crest cells established transiently during embryonic development.

Recent advances in understanding cell type transitions during dorsal neural tube development.

The vertebrate neural tube is a representative example of a morphogen-patterned tissue that generates different cell types with spatial and temporal precision. More specifically, the development of the dorsal region of the neural tube is of particular interest because of its highly dynamic behavior. First, early premigratory neural crest progenitors undergo an epithelial-to-mesenchymal transition, exit the neural primordium, and generate, among many derivatives, most of the peripheral nervous system. Subsequently, the dorsal neural tube becomes populated by definitive roof plate cells that constitute an organizing center for dorsal interneurons and guide axonal patterning. In turn, roof plate cells transform into dorsal radial glia that contributes to and shapes the formation of the dorsal ependyma of the central nervous system.To form a normal functional spinal cord, these extraordinary transitions should be tightly regulated in time and space. Thus far, the underlying cellular changes and molecular mechanisms are only beginning to be uncovered. In this review, we discuss recent results that shed light on the end of neural crest production and delamination, the early formation of the definitive roof plate, and its further maturation into radial glia. The last of these processes culminate in the formation of the dorsal ependyma, a component of the stem cell niche of the central nervous system. We highlight how similar mechanisms operate throughout these transitions, which may serve to reveal common design principles applicable to the ontogeny of epithelial tissues.

TFAP2 Paralogs Regulate Alx Genes During Midface Cranial Neural Crest Development

Craniofacial anomalies are the 2nd leading structural malformation present at birth. Those in the midface, such as orofacial clefts and hypertelorism, can arise from mutations in the Transcription Factor Activating Protein 2 genes TFAP2A and TFAP2B (abbreviated as TFAP2). These mutations do not reduce their gene expression, but instead generate mutant proteins that disrupt the activity of other wild‐type and functionally redundant paralogs. Previous studies showed that both paralogs are co‐expressed in the embryonic midface, hinting at their cooperative role during face development. Yet, how TFAP2 regulate midface development at the cellular and molecular level is poorly understood. To this end, we leveraged CRE‐based approaches in mice to delete both Tfap2 members in the neural crest (NC), a cell lineage that gives rise to craniofacial bone and cartilage. We show that NC‐specific knockout of Tfap2 (Tfap2NCKO) results in a fully penetrant midface cleft compared to controls and single paralog mutants. Lineage tracing analyses in Tfap2NCKO embryos suggest that cranial NC migration into the embryonic face is not severely impaired. Corroborating this result, we find that Tfap2 deletion in either pre‐migratory or late‐migratory NC cells still results in a midface cleft. This suggests this phenotype stems from disrupted TFAP2 activity in post‐migratory NC cells. Moreover, when examining Tfap2NCKO craniofacial skeletons, we observe severe bone malformations and ectopic structures in the frontonasal regions when compared to controls. Single‐cell RNA sequencing analysis reveals that loss of Tfap2 results in reduced cranial NC cell numbers and dysregulated Alx1/3/4 (Alx) gene expression. This is supported by the literature, as the Tfap2NCKO midface clefts phenocopy those with loss of various Alxgene combinations. Our ongoing efforts aim to test how TFAP2 regulates Alx genes, if loss of Tfap2 compromises midface NC survival or proliferation, and whether Tfap2 and Alx genetically interact. In sum, we propose the model that TFAP2 regulates Alx gene expression in NC cells during midface development.

Single-cell atlas of early chick development reveals gradual segregation of neural crest lineage from the neural plate border during neurulation.

The epiblast of vertebrate embryos is comprised of neural and non-neural ectoderm, with the border territory at their intersection harboring neural crest and cranial placode progenitors. Here, we a generate single-cell atlas of the developing chick epiblast from late gastrulation through early neurulation stages to define transcriptional changes in the emerging 'neural plate border' as well as other regions of the epiblast. Focusing on the border territory, the results reveal gradual establishment of heterogeneous neural plate border signatures, including novel genes that we validate by fluorescent in situ hybridization. Developmental trajectory analysis infers that segregation of neural plate border lineages only commences at early neurulation, rather than at gastrulation as previously predicted. We find that cells expressing the prospective neural crest marker Pax7 contribute to multiple lineages, and a subset of premigratory neural crest cells shares a transcriptional signature with their border precursors. Together, our results suggest that cells at the neural plate border remain heterogeneous until early neurulation, at which time progenitors become progressively allocated toward defined neural crest and placode lineages. The data also can be mined to reveal changes throughout the developing epiblast.

The double-edged sword role of TGF-β signaling pathway between intrauterine inflammation and cranial neural crest development.

Intrauterine infection would harm a developing embryo/fetus, thereby increasing the risk of developmental malformation. But, whether or not the infection-induced inflammation affects neural crest development still remains obscure. In this study, we employed meta-analysis to demonstrate the potential correlation between infection-induced inflammation and craniofacial anomalies, which was usually derived from the problems in neural crest cell development. The correlation was further verified by inflammatory cytokine release and the activation of nuclear factor kappa-light-chain enhancer of activated B cells signaling in lipopolysaccharide-treated HH10 chicken embryos. In such an inflammatory condition, AP-2α- and Pax7-labeled pre-migratory and migratory neural crest cells in HH10 chicken embryos were significantly less than the ones in control. The bioinformatics analysis of RNA-seq data demonstrated that the principal differential gene expression occurred in transforming growth factor-beta (TGF-β) signaling pathway, which was confirmed by the subsequent experimental results of quantitative PCR and immunofluorescent staining. Under this inflammatory circumstance, whole-mount in situ hybridization, immunofluorescence, and quantitative PCR showed the gene expression changes of key EMT-related transcription factors including upregulated Msx1, downregulated Slug, and FoxD3, as well as adhesion molecules and extracellular matrix protein including upregulated Cadherrin6B, E-cadherin, N-cadherin, and Laminin at the dorsal portion of neural tube of HH10 chicken embryos. Meanwhile, the bioinformatics analysis of RNA-seq data also manifested the differential gene expressions relevant to cell proliferation, which was confirmed by proliferating cell nuclear antigen Western blot data and co-immunofluorescence staining of human natural killer-1 and phosphorylated histone H3. In brief, this study revealed for the first time that the double-edged sword role of TGF-β signaling pathway between intrauterine inflammation (protective role) and cranial neural crest development (harmful role).

The lysine methyltransferase SETD2 is a dynamically expressed regulator of early neural crest development

SETD2 is a histone H3 lysine 36 (H3K36) tri-methylase that is upregulated in response to neural crest induction. Because the H3K36 di-methylase NSD3 and cytoplasmic non-histone protein methylation are necessary for neural crest development, we investigated the expression and requirement for SETD2 in the neural crest. SetD2 is expressed throughout the chick blastoderm beginning at gastrulation. Subsequently, SetD2 mRNA becomes restricted to the neural plate, where it is strongly and dynamically expressed as neural tissue is regionalized and cell fate decisions are made. This includes expression in premigratory neural crest cells, which is downregulated prior to migration. Likely due to the early onset of its expression, SETD2 morpholino knockdown does not significantly alter premigratory Sox10 expression or neural crest migration; however, both are disrupted by a methyltransferase mutant SETD2 construct. These results suggest that SETD2 activity is essential for early neural crest development, further demonstrating that lysine methylation is an important mechanism regulating the neural crest.

Single-cell RNA analysis identifies pre-migratory neural crest cells expressing markers of differentiated derivatives.

The neural crest is a migratory population of stem-like cells that contribute to multiple traits including the bones of the skull, peripheral nervous system, and pigment. How neural crest cells differentiate into diverse cell types is a fundamental question in the study of vertebrate biology. Here, we use single-cell RNA sequencing to characterize transcriptional changes associated with neural crest cell development in the zebrafish trunk during the early stages of migration. We show that neural crest cells are transcriptionally diverse and identify pre-migratory populations already expressing genes associated with differentiated derivatives, specifically in the xanthophore lineage. Further, we identify a population of Rohon-Beard neurons in the data. The data presented identify novel genetic markers for multiple trunk neural crest cell populations and Rohon-Beard neurons providing insight into previously uncharacterized genes critical for vertebrate development.

Notch signaling is a critical initiator of roof plate formation as revealed by the use of RNA profiling of the dorsal neural tube

BackgroundThe dorsal domain of the neural tube is an excellent model to investigate the generation of complexity during embryonic development. It is a highly dynamic and multifaceted region being first transiently populated by prospective neural crest (NC) cells that sequentially emigrate to generate most of the peripheral nervous system. Subsequently, it becomes the definitive roof plate (RP) of the central nervous system. The RP, in turn, constitutes a patterning center for dorsal interneuron development. The factors underlying establishment of the definitive RP and its segregation from NC and dorsal interneurons are currently unknown.ResultsWe performed a transcriptome analysis at trunk levels of quail embryos comparing the dorsal neural tube at premigratory NC and RP stages. This unraveled molecular heterogeneity between NC and RP stages, and within the RP itself. By implementing these genes, we asked whether Notch signaling is involved in RP development. First, we observed that Notch is active at the RP-interneuron interface. Furthermore, gain and loss of Notch function in quail and mouse embryos, respectively, revealed no effect on early NC behavior. Constitutive Notch activation caused a local downregulation of RP markers with a concomitant development of dI1 interneurons, as well as an ectopic upregulation of RP markers in the interneuron domain. Reciprocally, in mice lacking Notch activity, both the RP and dI1 interneurons failed to form and this was associated with expansion of the dI2 population.ConclusionsCollectively, our results offer a new resource for defining specific cell types, and provide evidence that Notch is required to establish the definitive RP, and to determine the choice between RP and interneuron fates, but not the segregation of RP from NC.

Neural crest multipotency and specification: power and limits of single cell transcriptomic approaches.

The neural crest is a unique population of multipotent cells forming in vertebrate embryos. Their vast cell fate potential enables the generation of a diverse array of differentiated cell types in vivo. These include, among others, connective tissue, cartilage and bone of the face and skull, neurons and glia of the peripheral nervous system (including enteric nervous system), and melanocytes. Following migration, these derivatives extensively populate multiple germ layers. Within the competent neural border ectoderm, an area located at the junction between the neural and non-neural ectoderm during embryonic development, neural crest cells form in response to a series of inductive secreted cues including BMP, Wnt, and FGF signals. As cells become progressively specified, they express transcriptional modules conducive with their stage of fate determination or cell state. Those sequential states include the neural border state, the premigratory neural crest state, the epithelium-to-mesenchyme transitional state, and the migratory state to end with post-migratory and differentiation states. However, despite the extensive knowledge accumulated over 150 years of neural crest biology, many key questions remain open, in particular the timing of neural crest lineage determination, the control of potency during early developmental stages, and the lineage relationships between different subpopulations of neural crest cells. In this review, we discuss the recent advances in understanding early neural crest formation using cutting-edge high-throughput single cell sequencing approaches. We will discuss how this new transcriptomic data, from 2017 to 2021, has advanced our knowledge of the steps in neural crest cell lineage commitment and specification, the mechanisms driving multipotency, and diversification. We will then discuss the questions that remain to be resolved and how these approaches may continue to unveil the biology of these fascinating cells.

Profiling NSD3-dependent neural crest gene expression reveals known and novel candidate regulatory factors

The lysine methyltransferase NSD3 is required for the expression of key neural crest transcription factors and the migration of neural crest cells. Nevertheless, a complete view of the genes dependent upon NSD3 for expression and the developmental processes impacted by NSD3 in the neural crest was lacking. We used RNA sequencing (RNA-seq) to profile transcripts differentially expressed after NSD3 knockdown in chick premigratory neural crest cells, identifying 674 genes. Gene Ontology and gene set enrichment analyses further support a requirement for NSD3 during neural crest development and show that NSD3 knockdown also upregulates ribosome biogenesis. To validate our results, we selected three genes not previously associated with neural crest development, Astrotactin 1 (Astn1), Dispatched 3 (Disp3), and Tropomyosin 1 (Tpm1). Using whole mount in situ hybridization, we show that premigratory neural crest cells express these genes and that NSD3 knockdown downregulates (Astn1 and Disp3) and upregulates (Tpm1) their expression, consistent with RNA-seq results. Altogether, this study identifies novel putative regulators of neural crest development and provides insight into the transcriptional consequences of NSD3 in the neural crest, with implications for cancer.

Premigratory neural crest stem cells generate enteric neurons populating the mouse colon and regulating peristalsis in tissue-engineered intestine.

Hirschsprung's disease (HSCR) is a common congenital defect. It occurs when bowel colonization by neural crest‐derived enteric nervous system (ENS) precursors is incomplete during the first trimester of pregnancy. Several sources of candidate cells have been previously studied for their capacity to regenerate the ENS, including enteric neural crest stem cells (En‐NCSCs) derived from native intestine or those simulated from human pluripotent stem cells (hPSCs). However, it is not yet known whether the native NCSCs other than En‐NCSCs would have the potential of regenerating functional enteric neurons and producing neuron dependent motility under the intestinal environment. The present study was designed to determine whether premigratory NCSCs (pNCSCs), as a type of the nonenteric NCSCs, could form enteric neurons and mediate the motility. pNCSCs were firstly transplanted into the colon of adult mice, and were found to survive, migrate, differentiate into enteric neurons, and successfully integrate into the adult mouse colon. When the mixture of pNCSCs and human intestinal organoids was implanted into the subrenal capsule of nude mice and grown into the mature tissue‐engineered intestine (TEI), the pNCSCs‐derived neurons mediated neuron‐dependent peristalsis of TEI. These results show that the pNCSCs that were previously assumed to not be induced by intestinal environment or cues can innervate the intestine and establish neuron‐dependent motility. Future cell candidates for ENS regeneration may include nonenteric NCSCs.

Mcrs1 interacts with Six1 to influence early craniofacial and otic development

The Six1 transcription factor plays a major role in craniofacial development. Mutations in SIX1 and its co-factor, EYA1, are causative for about 50% of Branchio-otic/Branchio-oto-renal syndrome (BOR) patients, who are characterized by variable craniofacial, otic and renal malformations. We previously screened for other proteins that might interact with Six1 to identify additional genes that may play a role in BOR, and herein characterize the developmental role of one of them, Microspherule protein 1 (Mcrs1). We found that in cultured cells, Mcrs1 bound to Six1 and in both cultured cells and embryonic ectoderm reduced Six1-Eya1 transcriptional activation. Knock-down of Mcrs1 in embryos caused an expansion of the domains of neural plate genes and two genes expressed in both the neural plate and neural crest (zic1, zic2). In contrast, two other genes expressed in pre-migratory neural crest (foxd3, sox9) were primarily reduced. Cranial placode genes showed a mixture of expanded and diminished expression domains. At larval stages, loss of Mcrs1 resulted in a significant reduction of otic vesicle gene expression concomitant with a smaller otic vesicle volume. Experimentally increasing Mcrs1 above endogenous levels favored the expansion of neural border and neural crest gene domains over cranial placode genes; it also reduced otic vesicle gene expression but not otic vesicle volume. Co-expression of Mcrs1 and Six1 as well as double knock-down and rescue experiments establish a functional interaction between Mcrs1 and Six1 in the embryo, and demonstrate that this interaction has an important role in the development of craniofacial tissues including the otic vesicle.

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.

The Paf1 complex and P-TEFb have reciprocal and antagonist roles in maintaining multipotent neural crest progenitors.

Multipotent progenitor populations are necessary for generating diverse tissue types during embryogenesis. We show the RNA polymerase-associated factor 1 complex (Paf1C) is required to maintain multipotent progenitors of the neural crest (NC) lineage in zebrafish. Mutations affecting each Paf1C component result in near-identical NC phenotypes; alyron mutant embryos carrying a null mutation in paf1 were analyzed in detail. In the absence of zygotic paf1 function, definitive premigratory NC progenitors arise but fail to maintain expression of the sox10 specification gene. The mutant NC progenitors migrate aberrantly and fail to differentiate appropriately. Blood and germ cell progenitor development is affected similarly. Development of mutant NC could be rescued by additional loss of positive transcription elongation factor b (P-TEFb) activity, a key factor in promoting transcription elongation. Consistent with the interpretation that inhibiting/delaying expression of some genes is essential for maintaining progenitors, mutant embryos lacking the CDK9 kinase component of P-TEFb exhibit a surfeit of NC progenitors and their derivatives. We propose Paf1C and P-TEFb act antagonistically to regulate the timing of the expression of genes needed for NC development.

Adult tissue-derived neural crest-like stem cells: Sources, regulatory networks, and translational potential.

Neural crest (NC) cells are a multipotent stem cell population that give rise to a diverse array of cell types in the body, including peripheral neurons, Schwann cells (SC), craniofacial cartilage and bone, smooth muscle cells, and melanocytes. NC formation and differentiation into specific lineages takes place in response to a set of highly regulated signaling and transcriptional events within the neural plate border. Premigratory NC cells initially are contained within the dorsal neural tube from which they subsequently emigrate, migrating to often distant sites in the periphery. Following their migration and differentiation, some NC‐like cells persist in adult tissues in a nascent multipotent state, making them potential candidates for autologous cell therapy. This review discusses the gene regulatory network responsible for NC development and maintenance of multipotency. We summarize the genes and signaling pathways that have been implicated in the differentiation of a postmigratory NC into mature myelinating SC. We elaborate on the signals and transcription factors involved in the acquisition of immature SC fate, axonal sorting of unmyelinated neuronal axons, and finally the path toward mature myelinating SC, which envelope axons within myelin sheaths, facilitating electrical signal propagation. The gene regulatory events guiding development of SC in vivo provides insights into means for differentiating NC‐like cells from adult human tissues into functional SC, which have the potential to provide autologous cell sources for the treatment of demyelinating and neurodegenerative disorders.

Early expression of Tubulin Beta-III in avian cranial neural crest cells

Neural crest cells are a transient stem-like cell population that forms in the dorsal neural tube of vertebrate embryos and then migrates to various locations to differentiate into diverse derivatives such as craniofacial bone, cartilage, and the enteric and peripheral nervous systems. The current dogma of neural crest cell development suggests that there is a specific hierarchical gene regulatory network (GRN) that controls the induction, specification, and differentiation of these cells at specific developmental times. Our lab has identified that a marker of differentiated neurons, Tubulin Beta-III (TUBB3), is expressed in premigratory neural crest cells. TUBB3 has previously been identified as a major constituent of microtubules and is required for the proper guidance and maintenance of axons during development. Using the model organism, Gallus gallus, we have characterized the spatiotemporal localization of TUBB3 in early stages of development. Here we show TUBB3 is expressed in the developing neural plate, is upregulated in the pre-migratory cranial neural crest prior to cell delamination and migration, and it is maintained or upregulated in neurons in later developmental stages. We believe that TUBB3 likely has a role in early neural crest formation and migration separate from its role in neurogenesis.

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.