We are focusing 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 physical 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? We are applying a variety of approaches to begin answering these complex questions. These approaches include: microdissection, RNA interference, 3D organ explants and cell culture, confocal immunofluorescence and brightfield time-lapse microscopy, genetic ablation, and a variety of other functional inhibition and reconstitution approaches. Our previous research implicated the extracellular matrix protein anosmin in neural crest craniofacial morphogenesis. Genetic defects in anosmin result in human Kallmann syndrome. Its functions include cell adhesion, neuronal migration, and neural crest formation. Anosmin consists of multiple domains, and it has been reported to bind heparan sulfate, FGF receptor, and UPA. We established cell adhesion and spreading assays for anosmin and used them for antibody inhibition analyses to search for anosmin's integrin adhesion receptor(s). Using both anti-functional antibody analyses with a panel of anti-integrin antibodies and specific adhesive peptide competitive inhibition assays, we have identified three integrins that serve to mediate cell adhesion to anosmin. The alpha5-beta1 fibronectin receptor, alpha4-beta1, and alpha9-beta1 integrins are each needed for effective adhesive receptor function in cell adhesion and cell spreading on anosmin; adhesion is also inhibited competitively by both RGD- and CS1-based peptides known to disrupt specific integrin adhesive functions. This identification of anosmin-integrin adhesion receptors should facilitate studies of anosmin function in cell and developmental biology. During salivary gland morphogenesis, we find that a single post-translational change in microtubules affecting acetylation in mesenchymal cells alters the mesenchymal microenvironment and promotes the maintenance and differentiation of a subset of epithelial progenitor cells, which impairs branching morphogenesis. Specifically, hyperacetylation of microtubules in mesenchymal cells increased cytokeratin 14-positive (K14+) progenitors in epithelial cells at the distal endbud region of developing salivary glands. Mechanistically, this process engages the transforming growth factor and Notch signaling pathways. We conclude that a simple post-translational alteration in mesenchyme microtubules dictates the maintenance and differentiation of adjacent epithelial progenitor cells to alter branching morphogenesis of the epithelium. In other descriptive and mechanistic studies of branching morphogenesis, we had recently characterized the overall patterns of both individual cell movement and basement membrane translocation during salivary gland branching morphogenesis. We identified a regulatory process involving fibronectin, the novel regulator Btbd7, and the transcription factor Snail2 (Slug) affecting cell adhesion involving E-cadherin and cell migration. The next phase will be to integrate these findings into an in-depth understanding of this complex process in vitro and in vivo. We will perform more in-depth confocal descriptive analyses at higher resolution combined with further molecular analyses of the roles of regulatory molecules such as Btbd7. For example, we have generated a Btbd7 knockout mouse model that we are characterizing in depth to evaluate its roles in various types of branching morphogenesis. We will also investigate further how matrix proteins signal from the plasma membrane to the nucleus and then to the cytoskeleton to promote dynamic epithelial cell behavior in various epithelial systems in vitro and in vivo.
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