Our studies in this project have focused 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 generate their large epithelial surface area during the process of branching morphogenesis? Specifically, how is cleft formation that delineates buds and ducts 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 the 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 answering these complex questions. These approaches include: laser microdissection;gene expression profiling;RNA interference;whole-embryo, organ, and cell culture;confocal immunofluorescence and brightfield time-lapse microscopy;and a variety of functional inhibition and reconstitution approaches. Salivary glands are formed from embryonic neural crest and other tissues. Their complex architecture is established by the dramatic process of branching morphogenesis in which a simple bud is transformed rapidly into a complex branched early organ. Branching is also crucial for the formation of many other organs including lungs, kidneys, and mammary glands. Understanding the mechanisms of branching morphogenesis and applying this knowledge to control tissue self-assembly and branching should accelerate tissue engineering approaches to regenerate damaged salivary glands and create an artificial salivary gland. Our central strategy has been to identify novel regulators by first identifying differentially expressed genes between closely adjacent epithelial sites. We have focused particularly on cells adjacent to forming clefts that divide buds into branched structures versus neighboring non-clefting/branching cells. A second major strategy was to develop knockdown and over-expression systems;we previously published novel approaches to using siRNA to analyze organ development. A third key approach has been to use time-lapse confocal microscopy to analyze dynamics of individual cells and tissues during morphogenesis. We have combined these approaches to identify novel mechanisms of branching morphogenesis. Microanatomical atlas of early salivary gland development: We have used laser-microdissection with SAGE (serial analysis of gene expression) or whole-genome microarrays to identify novel candidate regulators. We completed data acquisition and general bioinformatic analyses, and all primary datasets including 84 microarrays of laser-microdissected regions are now available for use by the community through the GEO database and at http://sgmap.nidcr.nih.gov. These microarray studies are now published in J. Dental Research. As an example of the value of this approach, we identified a novel regulatory change: a substantial reduction in GSK3 expression at forming clefts. We confirmed this finding at the protein level, and global inhibition of GSK3 in developing salivary glands enhanced branching morphogenesis. We expect that other new regulators of salivary development will be discovered using this and other databases. We have been studying Btbd7 (cleftin) as a novel regulator of branching morphogenesis. Our previously published studies established that local, transient fibronectin gene expression is crucial for successful branching of multiple organs including salivary glands and lungs, and that salivary branching morphogenesis involves a high level of relatively random cell motility combined with inward progression of fibronectin at forming clefts. However, it was not clear how fibronectin could function to form clefts because it is a non-motor matrix protein. We recently succeeded in establishing a novel regulatory and morphogenetic pathway involving the novel regulator Btbd7 (cleftin), a 126 kilodalton protein with previously unknown functions, which we discovered to be expressed primarily at the base of forming clefts. We have identified the following novel regulatory and morphogenetic pathway: Fibronectin induces Btbd7, which in turn induces Snail2 and decreases E-cadherin protein levels, resulting in cell separation and scattering that promotes intercellular gap formation and cleft progression. Our findings are being clarified through ongoing characterization of epithelial cell morphology and dynamics during salivary gland branching morphogenesis. For example, direct visualization of cleft formation in living glands revealed that clefts advance in a discontinuous manner through the formation of transient gaps between cells near the base of forming clefts. This loss of cell-cell adhesion between the orderly peripheral epithelial cells allows local cleft progression. We are continuing to characterize individual cell movements are a variety of sites throughout developing salivary glands. We are also working collaboratively with John Chiorini to determine the best system using viral gene transfer vectors for epithelium- or mesenchyme-specific transduction, or for microinjection. In ongoing studies, we have been characterizing anosmin as a regulatory matrix protein involved in cranial neural crest formation and craniofacial morphogenesis. Cranial neural crest cells are a stem cell population that is a major contributor to craniofacial bones, teeth, salivary glands, and surrounding connective tissues. During embryonic development, the neural crest is formed at the boundary of epidermal ectoderm and neural ectoderm. Although many growth and transcription factors are known to regulate neural crest formation and dispersal of neural crest cells to target tissues, extracellular matrix molecules were thought to play only downstream roles, e.g., providing fibronectin-rich or basement membrane pathways for neural crest cell emigration. We searched for some matrix molecule(s) that might play a more central regulatory role in modulating the growth factor and transcriptional pathways essential for neural crest formation. Using microarray screening and other approaches, we discovered that anosmin shows strong differential expression in the early chick neural crest, which is a widely used model system for analyzing neural crest function. RNA interference and other approaches suggest that anosmin plays essential roles in neural crest function, with striking effects on the generation of neural crest cells and craniofacial morphogenesis. These studies are being completed.
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