In this project, we are focusing primarily on determining the mechanisms of morphogenesis of salivary glands and other organs. We are addressing the following major questions: 1. How do embryonic salivary glands and other branched organs generate their characteristic branched architectures during the process of branching morphogenesis? Specifically, how is the formation of clefts, buds, and ducts mediated and coordinated at molecular and biophysical levels? How can we facilitate bioengineering for organ replacement, particularly of salivary glands, by understanding branching morphogenesis and by promoting specific steps? 2. What are the contributions of the selective regulation of extracellular matrix, integrins, signal transduction, specific gene expression, and cell migration in branching morphogenesis, as well as in other major tissue rearrangements such as cranial neural crest development? Branching morphogenesis of developing organs requires coordinated but still relatively poorly understood changes in epithelial cell-cell adhesion and cell motility. Our previous studies had identified a step-wise regulatory cascade involving fibronectin, the novel regulator Btbd7, and the transcription factor Snail2 (Slug) affecting cell adhesion involving E-cadherin and cell migration. We have extended these initial findings to more in-depth analyses of this complex process in vitro and in vivo. We developed a Btbd7 knockout mouse model to test our hypothesis that Btbd7 is a crucial regulator of branching morphogenesis in vivo. In normal mice, Btbd7 levels are elevated in the peripheral cells of branching epithelial end buds, a location at which it could enhance cell motility, reduce cell-cell adhesion, and promote dynamics of cleft formation. Genetic ablation of Btbd7 in mice severely disrupts branching morphogenesis of embryonic salivary gland, lung, and kidney. Loss of Btbd7 results in more tightly packed and elongated outer bud cells, which display stronger E-cadherin localization, reduced cell motility, and decreased dynamic, transient cell-cell separations associated with cleft formation. In striking contrast, inner bud cells remain unaffected. Mechanistic analyses using cultured MDCK cells to mimic outer vs. inner bud cell behavior established that Btbd7 promotes the loss of E-cadherin from cell-cell junctions with enhanced migration and transient cell separation. Mechanistically, Btbd7 serves to enhance E-cadherin ubiquitination, internalization, and degradation via proteasomal activity in MDCK cells and in intact peripheral epithelial bud cells for regulating cell dynamics. Consequently, these studies show how the new regulatory molecule, Btbd7, can function at the periphery of developing organs to regulate the local dynamics of cell adhesion and motility during epithelial branching morphogenesis. We previously established that during salivary gland morphogenesis, a single post-translational alteration in microtubules affecting acetylation can disrupt branching morphogenesis. This molecular alteration in salivary mesenchyme cells alters the mesenchymal microenvironment and promotes the maintenance and differentiation of a subset of epithelial progenitor cells, which can account for the impairment of branching morphogenesis. Specifically, hyperacetylation of microtubules in salivary mesenchymal cells increases cytokeratin 14-positive (K14+) progenitors. This effect on K14+ progenitors and their differentiated progeny, myoepithelial cells, occurs in epithelial basal and suprabasal layers of epithelial cells in the distal endbud region of developing salivary glands. Mechanistically, this process involves alterations in signaling via transforming growth factor beta-1 plus Notch signaling pathways. Thus, a single post-translational alteration in the cytoskeletal system of mesenchyme cells can dictate the maintenance and differentiation of adjacent epithelial progenitor cells to alter epithelial branching morphogenesis. We demonstrated previously that neural crest formation can be regulated by the extracellular matrix protein anosmin by altering cellular signaling. Although anosmin was reported to bind heparan sulfate, FGF receptor, and UPA, we examined for roles of integrins, which play major roles in cell adhesion and migration. We identified three beta-1 integrins as anosmin adhesion receptors. These studies link integrin-matrix interactions to the growth factor regulatory system in neural crest development. These studies are beginning to elucidate the complex regulatory systems important for the cell and tissue dynamics involved in craniofacial organ development. Understanding these underlying morphogenetic mechanisms should promote more effective tissue engineering for restoration of damaged organ function.
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