Draxin alters laminin organization during basement membrane remodeling to control cranial neural crest EMT

Premigratory neural crest cells arise within the dorsal neural tube and subsequently undergo an epithelial-to-mesenchymal transition (EMT) to leave the neuroepithelium and initiate migration. Draxin is a Wnt modulator that has been shown to control the timing of cranial neural crest EMT. Here we show that this process is accompanied by three stages of remodeling of the basement membrane protein laminin, from regression to expansion and channel formation. Loss of Draxin results in blocking laminin remodeling at the regression stage, whereas ectopic maintenance of Draxin blocks remodeling at the expansion stage. The latter effect is rescued by addition of Snail2, previously shown to be downstream of Draxin. Our results demonstrate an essential function for the Wnt modulator Draxin in regulating basement membrane remodeling during cranial neural crest EMT

RNA-binding protein Elavl1/HuR is required for maintenance of cranial neural crest specification

Neural crest development is transcriptionally controlled via sequential activation of gene regulatory networks (GRNs). Recent evidence increasingly implicates a role for post-transcriptional regulation in modulating the output of these regulatory circuits. Using RNA-sequencing data from avian embryos to identify potential post-transcriptional regulators, we observed enrichment during early neural crest development of Elavl1, which encodes for the RNA-binding protein HuR. Immunohistochemical analyses revealed expression of HuR following establishment of the neural plate border. Perturbation of HuR resulted in premature neural crest delamination from the neural tube as well as significant reduction in transcripts associated with the neural crest specification GRN (Axud1 and FoxD3), phenotypes also observed with downregulation of the canonical Wnt inhibitor Draxin. RNA pulldown further shows that Draxin is a specific target of HuR. Importantly, overexpression of exogenous Draxin was able to rescue the cranial neural crest specification defects observed with HuR knockdown. Thus, HuR plays a critical a role in the maintenance of cranial neural crest specification, at least partially via Draxin mRNA stabilization. Together, these data highlight an important intersection of post-transcriptional regulation with modulation of the neural crest specification GRN

RNA-binding protein Elavl1/HuR is required for maintenance of cranial neural crest specification

Neural crest development is transcriptionally controlled via sequential activation of gene regulatory networks (GRNs). Recent evidence increasingly implicates a role for post-transcriptional regulation in modulating the output of these regulatory circuits. Using RNA-sequencing data from avian embryos to identify potential post-transcriptional regulators, we observed enrichment during early neural crest development of Elavl1, which encodes for the RNA-binding protein HuR. Immunohistochemical analyses revealed expression of HuR following establishment of the neural plate border. Perturbation of HuR resulted in premature neural crest delamination from the neural tube as well as significant reduction in transcripts associated with the neural crest specification GRN (Axud1 and FoxD3), phenotypes also observed with downregulation of the canonical Wnt inhibitor Draxin. RNA pulldown further shows that Draxin is a specific target of HuR. Importantly, overexpression of exogenous Draxin was able to rescue the cranial neural crest specification defects observed with HuR knockdown. Thus, HuR plays a critical a role in the maintenance of cranial neural crest specification, at least partially via Draxin mRNA stabilization. Together, these data highlight an important intersection of post-transcriptional regulation with modulation of the neural crest specification GRN

P-bodies are sites of rapid RNA decay during the neural crest epithelial-mesenchymal transition

The epithelial-mesenchymal transition (EMT) drives cellular movements during development to create specialized tissues and structures in metazoans, using mechanisms often coopted during metastasis. Neural crest cells are a multipotent stem cell population that undergo a developmentally regulated EMT and are prone to metastasis in the adult, providing an excellent model to study cell state changes and mechanisms underlying EMT. A hallmark of neural crest EMT during avian development is temporally restricted expression followed by rapid down-regulation of the Wnt antagonist Draxin. Using live RNA imaging, here we demonstrate that rapid clearance of Draxin transcripts is mediated post-transcriptionally via localization to processing bodies (P-bodies), small cytoplasmic granules which are established sites of RNA processing. Contrasting with recent work in immortalized cell lines suggesting that P-bodies are sites of storage rather than degradation, we show that targeted decay of Draxin occurs within P-bodies during neural crest migration. Furthermore, P-body disruption via DDX6 knockdown inhibits not only endogenous Draxin down-regulation but also neural crest EMT in vivo. Together, our data highlight a novel and important role for P-bodies in an intact organismal context−controlling a developmental EMT program via post-transcriptional target degradation

Temporal changes in plasma membrane lipid content induce endocytosis to regulate developmental epithelial-to-mesenchymal transition

Epithelial-to-mesenchymal transition (EMT) is a dramatic change in cellular physiology during development and metastasis which involves coordination between cell signaling, adhesion, and membrane protrusions. These processes all involve dynamic changes in the plasma membrane, yet how membrane lipid content regulates membrane function during developmental EMT remains incompletely understood. By screening for differential expression of lipid-modifying genes over the course of EMT in avian neural crest, we have identified the ceramide-producing enzyme neutral sphingomyelinase 2 (nSMase2) as a critical regulator of a developmental EMT. nSMase2 expression begins at the onset of EMT, and in vivo knockdown experiments demonstrate that nSMase2 is necessary for neural crest migration. Further, we find that nSMase2 promotes Wnt and BMP signaling, and is required to activate the mesenchymal gene expression program. Mechanistically, we show that nSMase2 is sufficient to induce endocytosis, and that inhibition of endocytosis mimics nSMase2 knockdown. Our results support a model in which nSMase2 is expressed at the onset of neural crest EMT to produce ceramide and induce membrane curvature, thus increasing endocytosis of Wnt and BMP signaling complexes and activating pro-migratory gene expression. These results highlight the critical role of plasma membrane lipid metabolism in regulating transcriptional changes during developmental EMT programs

Draxin acts as a molecular rheostat of canonical Wnt signaling to control cranial neural crest EMT

Neural crest cells undergo a spatiotemporally regulated epithelial-to-mesenchymal transition (EMT) that proceeds head to tailward to exit from the neural tube. In this study, we show that the secreted molecule Draxin is expressed in a transient rostrocaudal wave that mirrors this emigration pattern, initiating after neural crest specification and being down-regulated just before delamination. Functional experiments reveal that Draxin regulates the timing of cranial neural crest EMT by transiently inhibiting canonical Wnt signaling. Ectopic maintenance of Draxin in the cranial neural tube blocks full EMT; while cells delaminate, they fail to become mesenchymal and migratory. Loss of Draxin results in premature delamination but also in failure to mesenchymalize. These results suggest that a pulse of intermediate Wnt signaling triggers EMT and is necessary for its completion. Taken together, these data show that transient secreted Draxin mediates proper levels of canonical Wnt signaling required to regulate the precise timing of initiation and completion of cranial neural crest EMT

Draxin alters laminin expression during basement membrane reorganization to control cranial neural crest EMT

Premigratory neural crest cells arise within the dorsal neural tube and subsequently undergo an epithelial-to-mesenchymal transition (EMT) to leave the neuroepithelium and initiate migration. Draxin is a Wnt modulator that has been shown to control the timing of cranial neural crest EMT. Here we show that this process is accompanied by three stages of remodeling of the basement membrane protein laminin, from regression to expansion and channel formation. Loss of Draxin results in blocking laminin remodeling at the regression stage, whereas ectopic maintenance of Draxin blocks remodeling at the expansion stage. The latter effect is rescued by addition of Snail2, previously shown to be downstream of Draxin. Our results demonstrate an essential function for the Wnt modulator Draxin in regulating basement membrane remodeling during cranial neural crest EMT

Bimodal function of chromatin remodeler Hmga1 in neural crest induction and Wnt-dependent emigration

During gastrulation, neural crest cells are specified at the neural plate border, as characterized by Pax7 expression. Using single-cell RNA sequencing coupled with high resolution in situ hybridization to identify novel transcriptional regulators, we show that chromatin remodeler Hmga1 is highly expressed prior to specification and maintained in migrating chick neural crest cells. Temporally-controlled CRISPR-Cas9-mediated knockouts uncovered two distinct functions of Hmga1 in neural crest development. At the neural plate border, Hmga1 regulates Pax7-dependent neural crest lineage specification. At premigratory stages, a second role manifests where Hmga1 loss reduces cranial crest emigration from the dorsal neural tube independent of Pax7. Interestingly, this is rescued by stabilized ß-catenin, thus implicating Hmga1 as a canonical Wnt activator. Together, our results show that Hmga1 functions in a bimodal manner during neural crest development to regulate specification at the neural plate border, and subsequent emigration from the neural tube via canonical Wnt signaling

Migration and Diversification of the Vagal Neural Crest

Arising within the neural tube between the cranial and trunk regions of the body axis, the vagal neural crest shares interesting similarities in its migratory routes and derivatives with other neural crest populations. However, the vagal neural crest is also unique in its ability to contribute to diverse organs including the heart and enteric nervous system. This review highlights the migratory routes of the vagal neural crest and compares them across multiple vertebrates. We also summarize recent advances in understanding vagal neural crest ontogeny and discuss the contribution of this important neural crest population to the cardiovascular system and endoderm-derived organs, including the thymus, lungs and pancreas