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 understand normal organ morphogenesis, but it may also illuminate how this basement membrane barrier is breached aberrantly by tumor cell proteolysis. We have observed numerous tiny, protease-dependent perforations in the basement membrane surrounding the tips of rapidly expanding end buds in the embryonic salivary gland;they were not present in cleft or duct regions. We hypothesize that these tiny perforations increase the elasticity of the basement membrane to permit rapid expansion of epithelia. We 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 cell model systems. Cranial neural crest cells are stem cell-like cells that generate craniofacial bones, teeth, salivary glands, and surrounding connective tissues. During embryonic development, the neural crest is formed at the boundary of the epidermal ectoderm and the neural ectoderm. We recently identified the extracellular matrix molecule anosmin as a novel regulatory protein necessary for balancing FGF, BMP, and WNT signaling for normal cranial neural crest formation and craniofacial morphogenesis. These studies used RNA interference, overexpression, and protein microinjection studies to establish that anosmin plays essential roles in neural crest function. We also identified BMP5 as a new regulator of neural crest formation. We concluded that this single extracellular matrix protein can play crucial roles in morphogenesis by modulating the balance of multiple growth factor activity-receptor functions. An unanswered question from the latter study was whether integrin matrix receptors could also play a regulatory role beyond the current paradigm in which integrins function only to mediate neural crest cell adhesion and migration. Our newest studies indicate that the integrin alpha5-beta1 can by itself promote selective expression of a growth factor. 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 mere 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 now in press 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.

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National Institute of Dental & Craniofacial Research
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Daley, William P; Yamada, Kenneth M (2013) ECM-modulated cellular dynamics as a driving force for tissue morphogenesis. Curr Opin Genet Dev 23:408-14
Endo, Yukinori; Ishiwata-Endo, Hiroko; Yamada, Kenneth M (2013) Cloning and characterization of chicken *5 integrin: endogenous and experimental expression in early chicken embryos. Matrix Biol 32:381-6
Hsu, Jeff C; Koo, Hyun; Harunaga, Jill S et al. (2013) Region-specific epithelial cell dynamics during branching morphogenesis. Dev Dyn 242:1066-77