87 - Identifying the Role of the Cadherin-6B Long N-terminal Fragments in Cranial Neural Crest Cell Development

Poster Board Number: 87
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Abstract Body : Cranial neural crest cells (NCCs) undergo a hallmark epithelial-to-mesenchymal transition (EMT) early in embryonic development. During this process, stationary premigratory NCCs delaminate from the neural folds, adopt a mesenchymal morphology, and begin to migrate. Cadherin-based cell junctions play a critical role in the regulation of EMT by initially maintaining epithelial structural integrity within the neural folds. However, premigratory cranial NCCs must disassemble these junctions during EMT to facilitate migration and ultimately differentiation into diverse cell types such as cartilage, bone, sensory neurons, and melanocytes. As such, the generation of migratory NCCs is crucial to build various tissues within the vertebrate head, and defects in these developmental processes can lead to craniofacial abnormalities and disease. In our prior work, we demonstrated that chick premigratory NCCs express and later downregulate Cadherin-6B (Cad6B) through proteolytic processing and endocytosis to promote delamination and EMT. Cad6B proteolysis generates two extracellular N-terminal fragments (long NTF (L-NTF) and short NTF (S-NTF)) and an intracellular C-terminal fragment (CTF2) with functions that remain to be explored. To this end, we discovered that the Cad6B L-NTF and S-NTF promote NCC delamination and EMT, in part, by increasing matrix metalloproteinase 2 (MMP2) activity and enhancing degradation of the extracellular matrix (ECM) through which premigratory NCCs must traverse to become migratory. Given these findings and those in other systems, we hypothesize that Cad6B NTFs regulate EMT through multiple mechanisms. To address our hypothesis, we performed mass spectrometry to identify Cad6B L-NTF binding proteins. Our initial results uncovered NTF interactors with roles in promoting EMT and migration through regulation of signal transduction, cell junctions and their turnover, and ECM remodeling. Through immunohistochemistry assays, we confirmed expression of putative NTF interactors (e.g., ErbB4, ZO-1, and clathrin) in premigratory NCCs and those undergoing EMT. Further, we showed that MMP16, which converts inactive MMP2 to active MMP2, physically interacts with Cad6B L-NTFs in vivo, providing a potential molecular mechanism for the increased MMP2 activity observed in the presence of Cad6B NTFs. Future experiments will elucidate the function of specific L-NTF binding proteins, and decipher the role of Cad6B L-NTF-MMP16 interactions in MMP2 activation, during NCC EMT. To perform these studies, we will take a multidisciplinary approach in the chick embryo involving biochemistry, molecular perturbation assays, immunohistochemistry, and high-resolution confocal imaging. Collectively, our results will clarify the function of Cad6B L-NTFs in NCC EMT and provide insights into craniofacial development and disease. This work is funded by NSF IOS-1947169 (L.A.T.).