Our overall goal is to characterize the mechanisms by which cell-surface and cytoplasmic regulatory molecules control signaling and cytoskeletal processes that mediate craniofacial development and functionally related biological processes in vivo. Cellular processes that they regulate during embryonic development include cell adhesion, migration, and tissue morphogenesis. We are placing particular emphasis on the developing salivary gland, neural crest, and potential tissue engineering applications. We hypothesize that related biological processes are involved in normal morphogenesis and tissue repair and other processes such as AIDS pathogenesis. Common mechanisms are likely to include choreographed changes in cell-cell and cell-matrix adhesion, migration, and signal transduction pathways. Understanding these fundamental processes should help to clarify mechanisms of pathogenesis and to identify novel potential targets for therapeutic intervention, e.g., for regenerative and tissue engineering approaches. Our primary focus has been on determining new mechanisms underlying craniofacial development and their relevance to tissue engineering. We are addressing the following major questions: 1. How do embryonic salivary glands and other branching tissues such as lungs develop? Specifically, how do clefts and branches form, and how is the process mediated and regulated? 2. What basic biological knowledge is needed for bioengineering organ replacement, particularly of salivary glands? For example, what are the most natural and effective approaches to attaching cells to biomaterial scaffolds, what are the cellular responses, and how will it be possible to generate sufficient functional capacity in an artificial organ? 3. Are similar processes and principles involved in other aspects of development and in diseases, such as HIV disease? We have applied a variety of approaches to begin to answer these complex questions, including laser microdissection, gene expression profiling, and RNA interference, organ and cell culture, confocal immunofluorescence, and functional inhibition and reconstitution approaches. Clinical replacement of salivary gland function destroyed by radiation therapy for oral cancer or by Sjogren?s disease will be challenging, because it will require providing enough secretory epithelium to produce enough salivary fluid to alleviate xerostomia. This general biological problem of how to obtain sufficient surface area in compact organs for secretion is solved during embryonic development by a process termed 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 a bioartificial salivary gland, a biological challenge facing us involves how to create numerous branched epithelial structures. We have applied a variety of approaches to identify novel mechanisms, with particular focus on extracellular-to-intracellular signaling pathways. We had previously established methods for generating accurate gene expression data from miniscule samples of embryonic salivary glands, e.g., to compare gene expression in forming cleft regions with expression at the tips of branching lobules or in adjacent mesenchyme. Preliminary differential gene expression data using this T7-SAGE (serial analysis of gene expression) approach unexpectedly implicated fibronectin in branching morphogenesis. We have therefore tested extensively the role of fibronectin and its alpha-5 beta-1 integrin receptor in cleft formation during branching using a variety of experimental inhibition and augmentation approaches. Local, developmentally regulated synthesis and assembly of fibronectin into fibrils was shown to be a key regulator of salivary braanching morphogenesis. Related findings were obtained for vertebrate kidney and lung branching morphogenesis. A collaborative study has extended this work to demonstrate a major role for the Wnt signaling pathway in targeting fibronectin in lung morphogenesis. We are presently focusing on the salivary gland system of branching morphogenesis to initiate a search for novel upstream and parallel regulators of branching morphogenesis using our T7-SAGE analysis, in-depth studies of signaling pathways, and examination for potential roles of cell migration in salivary morphogenesis. Although one approach to replacement of destroyed salivary tissue is regeneration, a complementary approach is to develop an implantable artificial salivary gland. We have collaboratively linked our research on cell adhesion and movement to expertise in the NIDCR Gene Therapy and Therapeutics Branch in a joint program to develop a bioartificial salivary gland. This fiscal year, our collaborative team received U.S. Patent No. 6,743,626 for the ?Artificial Salivary Gland.? For successful tissue engineering of such artificial organs, additional knowledge will be needed about the detailed mechanisms of cellular responses to biomaterials. Consequently, we are analyzing cell interactions with various artificial materials or protein preparations. Because the biophysical properties of the substrate or scaffold will be important for effective tissue engineering, we collaborated with researchers at the National Institute of Standards and Technology (NIST) to examine physical parameters in cell-biomaterial interactions. Using a novel gradient combinatorial polymer system, we were able to identify distinct roles of nanometer-scale surface roughness on cell proliferation and actin cytoskeletal organization. We are currently evaluating cell interactions and the formation of specific cell adhesions to various potential tissue engineering materials such as PuraMatrix. Because interactions at the surfaces of cells are likely to play important roles in disease, we have been involved in a long-term collaboration with FDA researchers to characterize cell-surface and extracellular interactions involved in AIDS pathogenesis. Ongoing studies have explored the roles of adhesion molecules and secreted HIV-Tat protein. A recent study has demonstrated a central role for cell-cell contact in the activation of HIV replication in latently infected cells. Our Branch had initiated a genome project led by the Molecular Biology Section in collaboration with our Section and discovered the enamel protein ameloblastin. This project has recently identified and characterized the novel regulatory protein epiprofin, which is found in developing teeth and limb buds, and which functions to promote cell proliferation. To help accelerate more general progress in the area, we originated an NIDCR contract awarded to Washington University to facilitate the discovery of new human genes and to characterize the patterns of gene regulation to promote the study of human craniofacial development. Expression data and materials from this contract are freely available to the extramural community through the project website: http://hg.wustl.edu/COGENE. While using digital images in our projects, it became clear that there was substantial potential for misuse by researchers of current powerful image analysis software while preparing data for analysis or publication. We collaborated with a professional journal editor to write an educational paper identifying ethical issues in contemporary image processing.
| Daley, William P; Matsumoto, Kazue; Doyle, Andrew D et al. (2017) Btbd7 is essential for region-specific epithelial cell dynamics and branching morphogenesis in vivo. Development 144:2200-2211 |
| Wei, Cindy; Larsen, Melinda; Hoffman, Matthew P et al. (2007) Self-organization and branching morphogenesis of primary salivary epithelial cells. Tissue Eng 13:721-35 |
| Green, J Angelo; Yamada, Kenneth M (2007) Three-dimensional microenvironments modulate fibroblast signaling responses. Adv Drug Deliv Rev 59:1293-8 |
| Stepp, Mary Ann; Liu, Yueyuan; Pal-Ghosh, Sonali et al. (2007) Reduced migration, altered matrix and enhanced TGFbeta1 signaling are signatures of mouse keratinocytes lacking Sdc1. J Cell Sci 120:2851-63 |
| Moon, Hye-Sung; Even-Ram, Sharona; Kleinman, Hynda K et al. (2006) Zyxin is upregulated in the nucleus by thymosin beta4 in SiHa cells. Exp Cell Res 312:3425-31 |
| Devadas, Krishnakumar; Boykins, Robert A; Hardegen, Neil J et al. (2006) Selective side-chain modification of cysteine and arginine residues blocks pathogenic activity of HIV-1-Tat functional peptides. Peptides 27:611-21 |
| Larsen, Melinda; Artym, Vira V; Green, J Angelo et al. (2006) The matrix reorganized: extracellular matrix remodeling and integrin signaling. Curr Opin Cell Biol 18:463-71 |
| Larsen, Melinda; Wei, Cindy; Yamada, Kenneth M (2006) Cell and fibronectin dynamics during branching morphogenesis. J Cell Sci 119:3376-84 |
| Even-Ram, Sharona; Artym, Vira; Yamada, Kenneth M (2006) Matrix control of stem cell fate. Cell 126:645-7 |
| Even-Ram, Sharona; Yamada, Kenneth M (2005) Cell migration in 3D matrix. Curr Opin Cell Biol 17:524-32 |
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