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 their high epithelial surface area in the process of branching morphogenesis? Specifically, how is the formation of clefts and 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 cell motility versus extracellular matrix expression or movement and their regulation in branching morphogenesis and 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, 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 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 major challenge is how to create numerous branched epithelial structures. We have applied 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 and its integrin receptor in salivary branching morphogenesis. Our unpublished studies have also implicated actomyosin contractility in branching. These molecular systems have previously been associated with cell migration, suggesting that branching morphogenesis could involve cell motility. We developed methods to directly visualize individual epithelial cell movements in intact developing organs. Adenoviral labeling by GFP (green fluorescent protein) of individual cells of isolated epithelial rudiments followed by organ culture permitted confocal time-lapse microscopy of cell movements during branching. A surprisingly high level of embryonic mouse epithelial cell movement was documented in early morphogenesis affecting all cells observed. This cell motility disappeared later in development. Digital tracking of the patterns of cell movement showed that they are non-choreographed and relatively random, suggesting that high tissue plasticity is needed transiently to permit rapid cleft formation and budding. ? This observation of active, seemingly chaotic, migration also suggested that reconstitution of glands from separated, individual cells might be possible. Isolated salivary gland epithelia were enzymatically dispersed into single cells. When provided with an appropriate matrix microenvironment, the dispersed epithelial cells initially spontaneously formed amorphous aggregates, which then progressed to reconstitute branching with the successful formation of multiple buds. In these regenerating buds, the molecular organization of adhesion components and actin was very similar to buds of intact salivary rudiments cultured in parallel, and they eventually expressed the key salivary membrane water channel protein aquaporin-5, as well as other salivary gland markers. Interestingly, tracking GFP-labeled cells from buds or ducts revealed no sorting out, and randomly reaggregated and interspersed cells were subsequently able to spontaneously organize bud formation. Co-culturing with salivary mesenchyme cells allowed branching to continue longer, consistent with classical studies postulating roles for epithelial-mesenchymal interactions in morphogenesis. These studies using isolated single salivary cells demonstrate that the system is highly robust in reconstituting branching morphogenesis, providing hope for regenerative approaches starting from cultured cells to generate branched epithelia for restoring salivary gland function.? ? During salivary gland morphogenesis, even in the presence of this chaotic cell migration, there is steady translocation of a 3D wedge comprised of fibronectin moving gradually inward from the surface of the gland to form each cleft. This dynamic but highly focal 3D matrix translocation occurs at the same rate as integrin-driven translocation of fibronectin in a process that we had previously described in fibroblasts and implicated in the formation of fibronectin fibrils. Because we have previously established that salivary gland cleft formation depends on both fibronectin and the integrin alpha5-beta1, this translocation process may provide a novel mechanism for cleft formation in which a steadily moving matrix partition driven by integrin translocation separates chaotically moving, highly plastic epithelial cells to form a cleft. We plan to examine whether this translocation involves large-scale movements of basement membrane into the cleft versus local fibronectin extension and whether other proteins besides fibronectin are involved.? ? In order to identify novel regulators of morphogenesis, we applied our previously developed method termed T7-SAGE with laser microdissection to compare gene expression patterns of epithelial cells adjacent to clefts with those in buds. The goal was to identify genes that are differentially activated at each site. Besides fibronectin and TIMP3, each of which are now known to be needed for branching morphogenesis, we have found two previously uncharacterized genes that are present at >10-fold higher ratios in cleft versus bud epithelial cells. We are characterizing their expression and potential role in morphogenesis using in situ hybridization, RT-PCR, and RNA interference approaches.? ? A process of cell separation that is even more dramatic than cleft formation occurs during the scattering of cells at the initiation of cranial neural crest cell migration; these cells ultimately generate many of the tissues and structures of the face and mouth. We used ultra-micro array analysis to identify two dozen genes expressed 10-fold or higher in newly forming chick cranial neural crest compared to neural tube. We intend to identify strongly differentially expressed extracellular matrix proteins and characterize a role for one of them in neural crest cell specification or migration.
| 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 |
Showing the most recent 10 out of 58 publications
