Craniofacial Developmental Dynamics

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This project is focused primarily on determining mechanisms of morphogenesis and maintenance of salivary glands and other organs. We are addressing the following major questions: 1. How do embryonic glands and other organs generate their characteristic architectures during development? 2. What are the contributions of local regulation of organ-specific gene expression, cell adhesion, extracellular matrix, integrins, signal transduction, and local cell migration to organ development? Branching morphogenesis of developing organs requires coordinated but still relatively incompletely understood changes in gene expression, epithelial cell-cell adhesion, cell-matrix adhesion, and cell motility. We had previously performed single-cell and bulk RNA transcriptomic analyses on embryonic submandibular versus parotid salivary glands to characterize their molecular identities at the very early stage of bud initiation. A key finding was the surprising degree of differences in gene expression patterns between these two types of salivary gland quite early in development, indicating gland specificity at even this early single-bud stage. In ongoing studies, recombination experiments in which the mesenchymal tissues and epithelial tissues of these two types of gland are being swapped to characterize alterations in gene expression patterns. Our laboratory has recently developed and refined methods for efficient CRISPR-mediated knockout of specific genes in embryonic mouse salivary gland epithelial ex vivo explants. Such salivary epithelial explants can be cultured successfully under serum-free conditions plus exogenous FGF7 and 5% Matrigel in 96-well plates, which can permit the facile manipulation and recovery of cultured epithelial buds. An efficient workflow for the production of high-titer lentivirus within one week was described, including the use of a fluorescence reporter associated with the guide RNA to highlight the nuclei of transduced cells. Transduction was 80% efficient, and three different genes were readily ablated, including a key integrin gene essential for branching morphogenesis. These procedures should accelerate studies of the roles of specific genes in salivary gland epithelial development. In another protocol paper, highly detailed methods for guide RNA design using CRISPOR, Golden Gate cloning, and production of lentiviruses was described and successfully applied to embryonic salivary epithelial explants. A research collaboration with the laboratories of Achim Werner and Laura Kerosuo applied microscopy methods to contribute to studies identifying roles for a novel tissue-specific ubiquitin switch of functions important for craniofacial, brain, and skin early development. A second study has involved collaboration with the Kerosuo laboratory on neural crest stem cells in development. A third paper contributed to a study implicating the proteoglycan biglycan in bone development. These studies are beginning to elucidate the complex mechanisms that underlie the cell and tissue dynamics involved in craniofacial organ development and maintenance. Understanding these underlying morphogenetic mechanisms during embryonic development should promote more effective tissue engineering for restoration of damaged organ function.