This project focuses on identifying and characterizing novel molecules and new mechanisms underlying craniofacial development and their relevance to tissue engineering, with particular focus on salivary and neural crest development. We are addressing the following major questions: 1. How do embryonic salivary glands and other tissues generate large epithelial surface areas during the process of branching morphogenesis? Specifically, how is cleft formation to delineate buds mediated and regulated? How can we facilitate bioengineering for organ replacement, particularly of salivary glands, by understanding branching morphogenesis and by developing reconstitution approaches? 2. What are the roles of regulation of extracellular matrix, signaling, and selective gene expression versus cell movements in branching morphogenesis and in other major tissue rearrangements such as cranial neural crest development? We are applying a variety of approaches to begin to answer these complex questions, including laser microdissection, gene expression profiling, RNA interference, whole-embryo culture, organ and cell culture, confocal immunofluorescence and video time-lapse microscopy, and a variety of functional inhibition and reconstitution approaches. Potential future clinical replacement of salivary gland function destroyed by radiation therapy for oral cancer or by Sjogrens syndrome will be challenging, because it will require restoration of enough secretory epithelium to produce adequate volumes of salivary fluid to alleviate xerostomia (salivary hypofunction). This general biological problem of how to obtain sufficient surface area in compact organs for secretion is normally solved during embryonic development by the process of branching morphogenesis. During development, a single embryonic bud first develops clefts and buds. It then undergoes repetitive branching to provide the large surface areas needed for effective secretory output. Regardless of whether eventual clinical replacement will involve salivary regeneration or an artificial salivary gland, a major challenge is how to create numerous branched epithelial structures. We have been applying a variety of approaches to identify novel mechanisms, with a particular focus on extracellular matrix-cell interactions and dynamic movements of both cell and extracellular matrix that drive branching. We had previously established essential roles for fibronectin, its integrin receptor, and random cell motility in salivary branching morphogenesis. Our unpublished studies also implicated actomyosin contractility in branching. We will continue to examine the underlying mechanisms and roles of cell migration and local tissue remodeling in branching morphogenesis. Our current working model is that there are direct causal links between fibronectin expression/local matrix accumulation and the process of cleft extension to delineate buds, e.g. fibronectin-induced local expression of specific regulatory molecules. In order to search for such novel regulators of morphogenesis, we applied two approaches after laser microdissection to compare gene expression patterns of epithelial cells adjacent to clefts with those in buds. One approach used our previously developed technique of T7-SAGE. The other used T7 amplification and Agilent mouse whole-genome microarrays. The goal has been to identify genes that are differentially activated at each site, not only comparing expression patterns in cells adjacent to clefts with those at the outer surface of end buds, but also with the expression patterns of cells at the center of buds, in the primary duct, or in secondary ducts. Besides fibronectin and TIMP3, each of which are now known to be needed for branching morphogenesis, we found a variety of additional changes in a number of known and prevoiusly uncharacterized genes. We are testing whether any of these genes may play a role in branching morphogenesis by qPCR analyses, RNA interference to inhibit function, and attempted gene transfer approaches. Craniofacial bones, teeth, and surrounding connective tissues are derived primarily from specific cell populations in cranial neural folds. During embryonic development, the neural folds are formed at the boundary of epidermal ectoderm and neural ectoderm, and they produce the cranial neural crest and olfactory epithelium. Although well-known regulatory factors such as FGF, WNT, and BMP are involved in neural crest and olfactory epithelium formation, little is known about the roles of extracellular matrix proteins in these important in vivo processes. We hypothesized that one or more extracellular matrix proteins play a regulatory role in these developmental processes. Using cDNA microarray analysis, we compared forming cranial neural folds and ventral neural ectoderm to identify two dozen genes expressed 10-fold or higher in newly forming chick cranial neural crest compared to neural tube. We are focusing on strongly differentially expressed extracellular matrix proteins and are attempting to characterize a role for one of them in neural crest formation, as well as its potential modulatory effects on growth factors involved in these processes such as FGFs and BMPs. Our goal is to identify potential roles for novel growth factor-extracellular matrix collaborations in normal and defective embryonic development.
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