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 have placed particular emphasis on the developing salivary gland and neural crest, as well as potential tissue engineering approaches. We hypothesize that related biological processes are involved in normal morphogenesis and tissue repair, and also in pathological processes such as AIDS pathogenesis. Common mechanisms are likely to include choreographed changes in cell-matrix and cell-cell adhesion, migration, and associated 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. We are addressing the following major questions:? 1. How do embryonic salivary glands and other tissues generate their high epithelial surface area by 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 in depth? ? 2. What are the roles of cell motility versus extracellular matrix dynamics and regulation in branching morphogenesis and other major tissue rearrangements such as cranial neural crest development? ? 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, RNA interference, organ and cell culture, confocal immunofluorescence and video time-lapse microscopy, and functional inhibition and reconstitution approaches.? The long-term goal of clinical replacement of salivary gland function destroyed by radiation therapy for oral cancer or by Sjogren?s disease is challenging, because it will require restoration of enough secretory epithelia to produce an adequate volume 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 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, the 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 dynamics of cell and matrix movements driving branching. ? We had previously established essential roles for fibronectin, its integrin receptor, and PI 3-kinase in salivary branching morphogenesis, and unpublished studies implicated actomyosin contractility in branching. Because all of these molecular systems had previously been associated with cell migration, we examined whether branching morphogenesis involved 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 high rate of embryonic mouse epithelial cell movement was observed in early morphogenesis that was lost later in development, suggesting that high internal organ plasticity is needed transiently to permit rapid cleft formation and budding. However, these epithelial cell movements appeared non-choreographed and chaotic, suggesting another source of cleft positional information. That source appears to be fibronectin, which we found to translocate steadily inward as a wedge between the motile cells. The rate of translocation is the same as we had previously characterized for integrin and fibronectin translocation in the process of extracellular matrix assembly, suggesting that similar mechanisms underlie these cell surface translocation processes. We are currently characterizing the capacity of such motile epithelial cells to reconstitute early steps of branching morphogenesis after complete dissociation and reassembly into glandular structures within a three-dimensional gel. We are also currently searching for novel genes that help to regulate or mediate the process of branching triggered by fibronectin. ? Nonmuscle cellular myosins and actin are thought to play crucial roles in many developmental and wound repair processes, but the roles of the major myosin IIA gene are not yet clear. We collaborated to characterize a mouse gene knockout of myosin IIA, which was published last year. In the absence of myosin IIA, cell-cell interactions were defective, resulting in major tissue disorganization in vivo. Since then, our laboratory has undertaken cell biological characterization of cells lacking this predominant myosin of nonmuscle cells. Our findings relating to cell contractility are not surprising: knockout or siRNA knockdown cells and cells treated with the myosin II inhibitor blebbistatin show losses of focal adhesions and actin stress fibers, and they become defective in their ability to contract fibrin gels. Unexpectedly, we are finding that myosin IIA-deficient cells show enhanced random cell migration, with hyperactive cell protrusiveness and elaborate leading lamellae involving Rac and its activator Tiam1. We are characterizing the mechanisms of this phenotype. ? Because interactions at the cell surface are likely to play important roles in many diseases, we have been involved in a long-term collaboration to characterize cell-surface and extracellular interactions involved in AIDS pathogenesis. Ongoing studies have explored the roles of adhesion molecules, secreted HIV-Tat protein, and signal transduction in HIV disease, and we have attempted to develop novel methods to generate a therapeutic vaccine. We used a novel method for generating a peptide vaccine using peptides with the same amino acid sequence as biologically active HIV-Tat peptides we had previously identified, but using residues modified to suppress their potent cell biological activity. Specifically, we substituted chemically modified cysteine and arginine residues at key sites of our HIV-Tat peptides. These substitutions resulted in suppression of problematic biological activities such as enhancement of HIV replication, which will be important if these peptides are to be used in a potential therapeutic AIDS vaccine. Even though biologically suppressed, the peptides remained effective immunogens for producing antibodies against HIV-1 Tat protein. ? In summary, a unifying theme in our recent studies has been the central importance of understanding dynamics, whether of local cell surface interactions, of individual cell movements, or most recently of local regions of three-dimensional extracellular matrix during complex developmental or disease processes. In this project, our fundamental research expertise on integrins, signaling, migration, and cell interactions is being applied to understand and modify organ development, reconstitution/regeneration, tissue engineering, and disease.
| 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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