PROJECT SUMMARY This proposal aims to address an outstanding issue in developmental biology: What cellular and molecular mechanisms endow immotile cells with the capacity to migrate and ultimately pattern the vertebrate embryo? Neural crest cells (NCCs) provide an exceptional model in which to address this fundamental question, as they differentiate to form various cell types including cranial bones and cartilage, sensory neurons, and melanocytes. Abnormalities in NCC formation are thus responsible for diverse human diseases, such as cancers, craniofacial anomalies, and disorders of the peripheral nervous system. Initially stationary epithelial cells, premigratory NCCs undergo an epithelial-to-mesenchymal transition (EMT), which is typified by changes in cell adhesion and morphology, along with breakdown of extracellular matrix, to become motile. The en masse nature of cranial NCC EMT requires an extraordinary degree of coordination, but how premigratory NCCs initiate and orchestrate this transition remains obscure. Our published and preliminary data have begun to bridge this knowledge gap, revealing the importance of Cadherin-6B (Cad6B) proteolysis during EMT, which reduces cell-cell adhesion to liberate NCCs but also generates peptides (Cad6B N-terminal fragments, NTFs; Cad6B C-terminal fragment 2, CTF2) with novel pro-EMT functions. Cad6B NTFs augment protease activity to facilitate degradation of extracellular matrix substrates, and Cad6B CTF2 modulates expression of critical EMT genes involved in cellular invasion, as identified by both targeted and global transcriptomics approaches. Herein, we propose to elucidate how Cad6B proteolytic peptides function in concert to control NCC EMT, using chick cranial NCCs as a unique in vivo model for EMT that has the advantage of direct translatability to human development. The aims in this proposal will test the hypothesis that EMT is orchestrated by cadherin peptides with distinct activities and functions within the premigratory cranial NCC population. To define the role of Cad6B NTFs during NCC EMT (Aim 1), we will evaluate the interrelationship among NTFs, proteases, and substrates, and determine the expression and function of NTF binding partners previously obtained via a novel mass spectrometry assay in the embryo. To investigate the role of Cad6B CTF2 as a transcriptional modulator during NCC EMT (Aim 2), we will continue characterizing newly identified CTF2 target genes and determine their mechanism of regulation by CTF2, and use ChIP-seq, followed by CTF2-associated sequence mapping and bioinformatics, to reveal new target genes and DNA binding motifs important for CTF2-mediated gene expression. Our proposed research is innovative as it takes a multidisciplinary approach combining embryology, biochemistry, bioinformatics, and novel microscopic analyses to examine the coordination of EMT in vivo. These studies will have great significance to the field by providing a heuristic paradigm to tackle similar questions for other normal developmental processes involving EMTs and for aberrant EMTs in other cell types that lead to birth defects and developmental disorders.