In this project, 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 tissues expand rapidly and generate their characteristic branched architecture during the process of branching morphogenesis? Specifically, how is the formation of clefts, buds, and ducts mediated and coordinated, and in this process, how do epithelial tissues expand rapidly while remaining constrained by the basement membrane? 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 roles of the regulation of extracellular matrix, signal transduction, selective gene expression, and cell migration in branching morphogenesis and 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 explant and cell culture; confocal immunofluorescence and brightfield time-lapse microscopy; and a variety of functional inhibition and reconstitution approaches. A general problem in embryonic organ morphogenesis is how epithelial tissues can expand so rapidly during branching while still remaining confined by a basement membrane. Since organs can display transient high rates of cell motility during branching, keeping the motile epithelial cells confined is necessary to avoid tissue mixing. Remodeling of the basement membrane by proteolysis was assumed to occur during branching morphogenesis to accommodate expanding epithelia, but how such remodeling occurs was not clear. We discovered that the basement membrane is highly dynamic during branching of the salivary gland, exhibiting both local and global remodeling. At the tip of the epithelial end bud, the basement membrane becomes perforated by hundreds of well-defined microscopic holes at regions of the bud undergoing rapid expansion; similar perforations at the expanding tip regions were also found during lung and kidney morphogenesis. Further characterization of the dynamics of morphogenesis in developing salivary glands revealed that epithelial cells at the expanding tips of buds often actively extend blebs and protrusions but not the whole cell body through these perforations. At these sites of numerous micro-perforations, the basement membrane appears more distensible and mesh-like, which would permit controlled epithelial expansion while maintaining tissue integrity. In addition, however, during branching morphogenesis the entire basement membrane itself translocates rearward away from the bud tip and towards the main duct. Basement membrane then accumulates and thickens around the forming secondary ducts, apparently stabilizing them during branching. Both local and global dynamics of the basement membrane depend on protease and myosin II activity, and these latter activities synergize. Our findings suggest that the basement membrane is rendered distensible by proteolytic degradation to allow it to be translocated by cells through actomyosin contractility to support branching morphogenesis. Now that we have characterized the overall patterns of cell movement and basement membrane translocation during salivary gland branching morphogenesis, including recently identifying a regulatory process involving fibronectin, the novel regulator Btbd7, and the transcription factor Snail2 (Slug), the next phase will be to integrate these findings into an in-depth understanding of this complex process. We will perform more in-depth confocal descriptive analyses at higher resolution combined with further molecular analyses of the roles of molecules such as Btbd7. We will investigate further how matrix proteins signal from the plasma membrane to the nucleus to promote dynamic epithelial cell behavior in various epithelial systems in vitro and in vivo.
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