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 processes? 2. What are the contributions of the selective, local regulation of extracellular matrix, integrins, signal transduction, specific gene expression, and cell migration in organ 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 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. 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 proteolytic 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. Ongoing research is focused on determining the molecular mechanisms of action of Btbd7. Because antibody reagents to various candidate downstream target molecules that are available against human proteins often fail to cross-react adequately with mouse or canine cells, we are focusing on trying to develop a robust human cell line system for in-depth characterizations of the molecular interactions and regulatory mechanisms used by Btbd7. We are also continuing to explore the roles of ubiquitination in Btbd7 functions. Another approach being applied is to perform gene expression analyses of the early development of salivary glands using both bulk and single-cell RNA-sequencing. Preliminary studies indicate the practicality of analyzing early embryonic salivary gland epithelia versus mesenchyme with a goal of identifying new molecules necessary for salivary gland development and organ specificity. These studies are beginning to elucidate the complex regulatory systems important for the cell and tissue dynamics involved in craniofacial organ development, particularly of salivary glands. Understanding these underlying morphogenetic mechanisms should promote more effective tissue engineering for restoration of damaged organ function.
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