The overall goal of the Matrix and Morphogenesis Unit is to identify the mechanisms of growth factor/extracellular matrix interactions that regulate branching morphogenesis of salivary glands. In addition, we are interested in discovering novel genes required for submandibular gland initiation and early branching morphogenesis.
We aim to understand basic developmental mechanisms during salivary gland organogenesis to design therapeutic approaches for the functional regeneration of diseased or damaged adult salivary tissue. Loss of salivary gland function in humans often occurs after removal of salivary tumors, after therapeutic radiation of head and neck tumors, as a result of Sjogrens Syndrome, and as a side effect of medications. In diseases such as Sjogrens Syndrome or after radiation treatment there is a significant loss of the salivary gland tissue that secretes saliva, while the ductal tissue remains intact. Understanding the biological processes that result in the formation salivary glands during development will provide targets for gland regeneration and/or tissue engineering approaches to restore secretory function. Our primary model system to study salivary gland organogenesis is the developing mouse submandibular gland. We focus on the role of fibroblast growth factors and the extracellular matrix during gland morphogenesis. The fibroblast growth factors are essential for mouse submandibular gland development. Mice lacking FGF10 or FGFR2b do not develop salivary glands, but the role of FGFs during branching morphogenesis and at discrete stages of gland development are not known. We profiled gene expression during mouse salivary gland development and found that the expression of multiple FGFs and their receptor isoforms are developmentally regulated. We hypothesize that individual FGFs and their receptors regulate discrete developmental events during branching morphogenesis, and may play different roles at different times during development. The major questions we address include: 1. What are the distinct roles of FGFR1b and FGFR2b within the salivary epithelium and how do their signaling pathways regulate cell proliferation, survival, or apoptosis during morphogenesis? Both FGFR1 and FGFR2 are important for ex vivo branching morphogenesis. We hypothesized that the epithelial splice isoforms FGFR1b and FGFR2b are necessary for epithelial morphogenesis. We have used molecular biology approaches to study the role of both FGFR1b and FGFR2b signaling in the epithelium. In the past year we have developed techniques to knockdown FGFR gene expression with siRNA, we have used soluble FGFRs to compete the endogenous FGF ligands, and chemical inhibitors of FGF signaling to define the isoform-specific signaling events and downstream effects on gene expression. We have identified a major role for both FGFR1 and FGFR2 in the regulation of epithelial cell proliferation during development of the salivary epithelium. Apoptosis does not appear to play a major role in regulating epithelial morphogenesis. The knockdown of FGFR1 in epithelial culture decreases gene expression of FGFR2, Lama5 (Laminin alpha 5 is a major Laminin isoform in the basement membrane of SMGs), and Fgf1. The knockdown of FGFR2 expression decreases FGF10-mediated epithelial morphogenesis and also decreases gene expression of FGFR1, Lama5 and Fgf1. The gene expression of other FGFs and Laminin ?-chains did not change. Our findings suggest that FGFR signaling regulates both FGF/FGFR gene expression in an autocrine manner, and that it also regulates expression of extracellular matrix molecules during morphogenesis. Modulation of FGFR signaling or a downstream target of FGFR signaling may be a useful to stimulate salivary gland regeneration. 2. How do FGF7 (which binds FGFR2b) and FGF10, (which both binds both FGFR2b and FGFR1b) regulate distinct epithelial morphologies? Using salivary gland epithelial rudiments cultuured without mesenchyme, we have discovered that both FGF7 and FGF10 promote epithelial proliferation but FGF7 induces epithelial budding, whereas FGF10 also induces duct elongation, suggesting they use different receptors and downstream signaling pathways. We have used FGFR isoform immunolocalization to show that FGFR1 is at the tips of the elongating ducts when treated with FGF10, whereas FGFR1b is localized around the entire epithelial bud with FGF7 treatment. After mesenchyme-free epithelial organ culture with individual FGFs we used subsequent real time PCR analysis of gene expression to analyze changes in gene expression. We discovered that FGF7- but not FGF10-dependent morphogenesis is PI3K-dependent, and that FGF7 increases expression of FGFR1b and Fgf1. Our findings suggest that the FGF7 induced increase FGFR1 and FGF1 may result in increased proliferation resulting in epithelial bud formation. 3. What role do proteases play in regulating matrix remodeling during branching morphogenesis? Matrix Metalloproteinases (MMP) and other protease activities are important for branching morphogenesis. We hypothesized that FGFs regulate MMP expression during branching morphogenesis. The morphogenesis of salivary epithelium cultured in Matrigel requires both FGF and protease activity. FGF7 increases epithelial MMP2 expression, whereas, FGF10 increases mesenchyme MMP9 expression compared to FGF7-treated glands. We have used gelatin and casein zymography, and real time PCR analysis of FGF and MMP expression to analyze the role of MMPs in gland development. We are currently using time-lapse confocal analysis of salivary epithelial rudiments to localize protease function during epithelial morphogenesis. Protease activity during FGF induced morphogenesis was localized using quenched fluorescent collagen added to the Laminin matrix. The sites of most active matrix remodeling are the epithelial rudiment surface particularly at cleft sites, and along the ductal structures. In conclusion, our studies on the basic biologic mechanisms that result in branching morphogenesis provide a rationale for a biologically-based therapeutic approach for regeneration of salivary gland tissue. Understanding the cellular processes involved in tissue morphogenesis including proliferation, differentiation, apoptosis, and migration is critical to regenerating tissue. The regeneration of salivary glands could be a potential therapeutic option in cases
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