Cadherin-6B is proteolytically processed during epithelial-to-mesenchymal transitions of the cranial neural crest.
ABSTRACT: The epithelial-to-mesenchymal transition (EMT) is a highly coordinated process underlying both development and disease. Premigratory neural crest cells undergo EMT, migrate away from the neural tube, and differentiate into diverse cell types during vertebrate embryogenesis. Adherens junction disassembly within premigratory neural crest cells is one component of EMT and, in chick cranial neural crest cells, involves cadherin-6B (Cad6B) down-regulation. Whereas Cad6B transcription is repressed by Snail2, the rapid loss of Cad6B protein during EMT is suggestive of posttranslational mechanisms that promote Cad6B turnover. For the first time in vivo, we demonstrate Cad6B proteolysis during neural crest cell EMT, which generates a Cad6B N-terminal fragment (NTF) and two C-terminal fragments (CTF1/2). Coexpression of relevant proteases with Cad6B in vitro shows that a disintegrin and metalloproteinases (ADAMs) ADAM10 and ADAM19, together with ?-secretase, cleave Cad6B to produce the NTF and CTFs previously observed in vivo. Of importance, both ADAMs and ?-secretase are expressed in the appropriate spatiotemporal pattern in vivo to proteolytically process Cad6B. Overexpression or depletion of either ADAM within premigratory neural crest cells prematurely reduces or maintains Cad6B, respectively. Collectively these results suggest a dual mechanism for Cad6B proteolysis involving two ADAMs, along with ?-secretase, during cranial neural crest cell EMT.
Project description: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.
Project description: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 ?-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 ?-catenin, which stabilizes and redistributes both proteins to the cytosol and nucleus, leading to up-regulation of ?-catenin, CyclinD1, Snail2, and Snail2 promoter-based GFP expression in vivo. A CTF2 ?-catenin-binding mutant, however, fails to alter gene expression, indicating that CTF2 modulates ?-catenin-responsive EMT effector genes. Notably, CTF2 association with the endogenous Snail2 promoter in the neural crest is ?-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.
Project description: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.
Project description: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.
Project description:Neural crest cells form within the neural tube and then undergo an epithelial to mesenchymal transition (EMT) to initiate migration to distant locations. The transcriptional repressor Snail2 has been implicated in neural crest EMT via an as of yet unknown mechanism. We report that the adaptor protein PHD12 is highly expressed before neural crest EMT. At cranial levels, loss of PHD12 phenocopies Snail2 knockdown, preventing transcriptional shutdown of the adhesion molecule Cad6b (Cadherin6b), thereby inhibiting neural crest emigration. Although not directly binding to each other, PHD12 and Snail2 both directly interact with Sin3A in vivo, which in turn complexes with histone deacetylase (HDAC). Chromatin immunoprecipitation revealed that PHD12 is recruited to the Cad6b promoter during neural crest EMT. Consistent with this, lysines on histone 3 at the Cad6b promoter are hyperacetylated before neural crest emigration, correlating with active transcription, but deacetylated during EMT, reflecting the repressive state. Knockdown of either PHD12 or Snail2 prevents Cad6b promoter deacetylation. Collectively, the results show that PHD12 interacts directly with Sin3A/HDAC, which in turn interacts with Snail2, forming a complex at the Cad6b promoter and thus revealing the nature of the in vivo Snail repressive complex that regulates neural crest EMT.
Project description:Neural crest cells are an embryonic cell population that is crucial for proper vertebrate development. Initially localized to the dorsal neural folds, premigratory neural crest cells undergo an epithelial-to-mesenchymal transition (EMT) and migrate to their final destinations in the developing embryo. Together with epidermally-derived placode cells, neural crest cells then form the cranial sensory ganglia of the peripheral nervous system. Our prior work has shown that ?N-catenin, the neural subtype of the adherens junction ?-catenin protein, regulates cranial neural crest cell EMT by controlling premigratory neural crest cell cadherin levels. Although ?N-catenin down-regulation is critical for initial neural crest cell EMT, a potential role for ?N-catenin in later neural crest cell migration, and formation of the cranial ganglia, has not been examined. In this study, we show for the first time that migratory neural crest cells that will give rise to the cranial trigeminal ganglia express ?N-catenin and Cadherin-7. ?N-catenin loss- and gain-of-function experiments reveal effects on the migratory neural crest cell population that include subsequent defects in trigeminal ganglia assembly. Moreover, ?N-catenin perturbation in neural crest cells impacts the placode cell contribution to the trigeminal ganglia and also changes neural crest cell Cadherin-7 levels and localization. Together, these results highlight a novel function for ?N-catenin in migratory neural crest cells that form the trigeminal ganglia.
Project description: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.
Project description:ADAMs are cell surface metalloproteases that control multiple biological processes by cleaving signaling and adhesion molecules. ADAM13 controls cranial neural crest (CNC) cell migration both by cleaving cadherin-11 to release a promigratory extracellular fragment and by controlling expression of multiple genes via its cytoplasmic domain. The latter activity is regulated by ?-secretase cleavage and the translocation of the cytoplasmic domain into the nucleus. One of the genes regulated by ADAM13, the protease calpain8, is essential for CNC migration. Although the nuclear function of ADAM13 is evolutionarily conserved, it is unclear whether the transcriptional regulation is also performed by other ADAMs and how this process may be regulated. We show that ADAM13 function to promote CNC migration is regulated by two phosphorylation events involving GSK3 and Polo-like kinase (Plk). We further show that inhibition of either kinase blocks CNC migration and that the respective phosphomimetic forms of ADAM13 can rescue these inhibitions. However, these phosphorylations are not required for ADAM13 proteolysis of its substrates, ?-secretase cleavage, or nuclear translocation of its cytoplasmic domain. Of significance, migration of the CNC can be restored in the absence of Plk phosphorylation by expression of calpain-8a, pointing to impaired nuclear activity of ADAM13.
Project description:The neural crest is a transient population of migratory cells that differentiates to form a variety of cell types in the vertebrate embryo, including melanocytes, the craniofacial skeleton, and portions of the peripheral nervous system. These cells initially exist as adherent epithelial cells in the dorsal aspect of the neural tube and only later become migratory after an epithelial-to-mesenchymal transition (EMT). Snail2 plays a critical role in mediating chick neural crest cell EMT and migration due to its expression by both premigratory and migratory cranial neural crest cells and its ability to down-regulate intercellular junctions components. In an attempt to delineate the role of cellular junction components in the neural crest, we have identified the adherens junction molecule neural alpha-catenin (alphaN-catenin) as a Snail2 target gene whose repression is critical for chick neural crest cell migration. Knock-down and overexpression of alphaN-catenin enhances and inhibits neural crest cell migration, respectively. Furthermore, our results reveal that alphaN-catenin regulates the appropriate movement of neural crest cells away from the neural tube into the embryo. Collectively, our data point to a novel function of an adherens junction protein in facilitating the proper migration of neural crest cells during the development of the vertebrate embryo.
Project description:Although a network of transcription factors that specifies neural crest identity in the ectoderm has been defined, expression of neural crest transcription factors does not guarantee eventual migration as a neural crest cell. While much work has gone into determining regulatory relationships within the transcription factor network, the ability of protein modifications like phosphorylation to modulate the function of neural crest regulatory factors and determine when and where they are active also has crucial implications. Paladin, which was previously classified as a phosphatase based on sequence similarity, is expressed in chick neural crest precursors and is maintained throughout their epithelial to mesenchymal transition and migration. Loss of Paladin delays the expression of transcription factors Snail2 and Sox10 in premigratory neural crest cells, but does not affect accumulation of FoxD3, Cad6B or RhoB, indicating that Paladin differentially modulates the expression of genes previously thought to be coregulated within the neural crest gene regulatory network. Both gain and loss of Paladin function result in disrupted neural crest migration, reinforcing the importance of precisely regulated phosphorylation for neural crest migration. Mutation of critical, catalytic cysteine residues within Paladin's predicted phosphatase active site motifs did not abolish the function of Paladin in the neural crest. Collectively, these data indicate that Paladin is an antiphosphatase that modulates the activity of specific neural crest regulatory factors during neural crest development. Our work identifies a novel regulator of phosphorylation status that provides an additional layer of regulation in the neural crest.