In this project, we are focusing on determining the mechanisms of salivary gland and neural crest formation. 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;whole-embryo, organ, and cell culture;confocal immunofluorescence and brightfield time-lapse microscopy;and a variety of functional inhibition and reconstitution approaches. During branching morphogenesis, epithelial cells of salivary glands and other organs become transiently motile. However, the local patterns of migration and region-specific differences in cell motility throughout developing glands are poorly understood. We developed a photo-convertible fluorescent transgenic mouse system to quantify the migration dynamics of individual salivary gland epithelial cells at specific locations. Local groups of migrating cells in organs of a mouse line expressing the fluorescent protein KikGR (Kikumi Green-Red) could be photo-converted from green to red fluorescence using a narrow beam of laser light. The red-fluorescing cells could then be tracked to characterize their movements in 3D. We determined the motility pattern and role of integrin interactions of these cells with the basement membrane compared to cell-cell interactions mediated by E-cadherin. We discovered that salivary gland epithelial cells are most highly motile in peripheral bud regions associated with the basement membrane. These cells often move laterally, repetitively bumping along this 2D surface during branching morphogenesis. Inhibiting interactions of these cells with the basement membrane using anti-integrin antibodies slowed rates of cell migration and disrupted both tissue organization and overall morphogenesis. This study points to an unexpected function of the basement membrane in stimulating local cell motility at this stage in embryonic development. We also established that the basement membrane-associated motility of these outer bud cells depends on myosin II, but not E-cadherin. In striking contrast, cell motility of inner bud cells was restrained by E-cadherin, and inhibition of this cell-cell adhesion molecule accelerated the rates of cell migration by inner, but not outer, bud cells. These findings identify the importance of integrin-dependent basement membrane association for the tissue organization and lateral motility of morphogenetic outer bud epithelial cells, which is complemented by E-cadherin mediated cell-cell adhesion to inhibit inner bud cell motility. Besides this new role in embryonic cell motility, basement membranes are particularly well known for their function in maintaining tissue boundaries between epithelia and mesenchymal tissues. However, during organ morphogenesis, how do epithelial tissues expand rapidly while still remaining confined by the basement membrane? Defining this type of mechanism will help elucidate normal organ morphogenesis, but it may also illuminate how this basement membrane barrier is breached aberrantly by tumor cell proteolysis. We now describe the stage-dependent appearance of hundreds of microscopic, well-defined, protease-dependent perforations in the basement membrane surrounding the tips of rapidly expanding end buds in the embryonic salivary gland;they are not present in cleft or duct regions. Formation and maintenance of these micro-perforations also depends on myosin II-based actomyosin contractility;in fact, proteases and contractility function cooperatively to form and maintain perforations. These tiny perforations increase the pliability of the basement at the expanding tip of the end bud. We also discovered that the basement membrane translocates rearward towards the duct at 8 micrometers per hour during salivary gland branching morphogenesis;this rate is faster and opposite in direction to the outward expansion of epithelial buds. We established that the basement membrane accumulates near the center of the end bud and the duct in both salivary gland and lung. Immunostaining and live-organ imaging revealed that collagen IV increases in fluorescence intensity as the micro-perforations disappear, and it becomes more fibrillar and compacted. This basement membrane accumulation most likely supports secondary duct structure, since previous work showed that collagenase disruption of the basement membrane results in cleft and duct regression. In mesenchyme-free epithelial rudiments, the epithelium alone could translocate the basement membrane rearward, indicating an epithelial source of the motive force. This dynamic basement membrane translocation also depends on both myosin II and protease activity. Inhibiting contractility with blebbistatin inhibited the translocation almost immediately, whereas the glands required overnight treatment with a broad protease inhibitor to halt the translocation. This delay suggests that the contractile translocation machinery itself is not directly inhibited by protease activity. Instead, the 2-fold buildup and stiffening of the basement membrane may itself restrict the ability of epithelial cells to pull the matrix rearward. Overall, both micro-perforations and translocation of the basement membrane each require contractility and proteases during development in this unique, dynamic matrix remodeling process that appears to support both focal, rapid expansion of epithelia and subsequent stabilization of secondary ducts. Because the alpha5 integrin subunit had never been cloned from chicken and was absent from chicken genomic sequences, we cloned and sequenced it. Besides identifying conserved sequences, we were able to show that a small 1.5-fold experimental increase in levels of this integrin by transfection produced a highly selective increase in expression of just 1 out of 11 growth and transcription factors that was not mimicked by another integrin. The target was the newly identified neural crest regulator BMP-5. This finding suggests that various previously reported changes of integrin levels in development may be important for regulating selective gene expression, beyond roles in cell adhesion and kinase-mediated signaling. We had previously identified Btbd7 as a novel regulator of epithelial cell dynamics in salivary gland development. Btbd7 is focally induced by fibronectin, and increases epithelial cell dynamics via the down-regulation of E-cadherin and up-regulation of the transcription factor Snail2;this process resembled a partial local EMT. We are investigating further how this matrix protein signals 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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