Mechanisms of Development /Related Processes

Dental &Craniofacial Research

Investigators

Linked publications & trials

Abstract

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 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 in pathological processes such as AIDS pathogenesis. Common mechanisms are likely to include choreographed changes in cell-matrix and cell-cell 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 tissues generate sufficient 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 and its 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, and 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 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 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 recently established that a local burst of fibronectin mRNA expression is needed for the formation of clefts during branching morphogenesis of salivary glands, with an analogous requirement for fibronectin in development of lung and kidney. We collaborated with Bellusci and co-workers in studies demonstrating that lung development requires the Wnt pathway and fibronectin. We confirmed that fibronectin is needed for early lung branching in mice, and in fact, adding exogenous fibronectin rescues inhibition of the Wnt signaling pathway. These studies indicate a key role for Wnt signaling and downstream fibronectin expression in lung branching morphogenesis. We are also continuing a long-standing collaboration with the NIDCR Gene Therapy and Therapeutics Branch to develop a bioartificial salivary gland. Current studies are focusing on trying to reconstitute branching morphogenesis using artificial aggregates of individual epithelial cells. Cellular actin and myosin are thought to play crucial roles in many developmental processes, yet the roles of the major myosin II-A gene in developmental processes was not clear. We have been collaborating with the Adelstein laboratory in NHLBI to generate and characterize a gene knockout of myosin II-A. Analyses of mutated mouse embryonic stem cells and embryos with experimentally induced deficiencies in myosin II revealed an unexpected, crucial role in cell adhesion for a particular isoform, myosin II-A. In the absence of myosin II-A, cell-cell interactions were defective, resulting in major tissue disorganization in vivo. In vitro, the cells lacked cohesion and were continually shed from aggregates of embryonic stem cells. These effects in vitro and in vivo were associated with defective localization of components of the major E-cadherin cell-to-cell adhesion system. These findings link myosin to cell adhesion. Knockout mice die in early development with defects in cadherin-mediated cell-to-cell adhesion and failure of tissue formation. Mouse embryonic stem cells deficient in myosin II-A show defects in cell-cell cohesion, and individual cells or aggregates anomalously attach and spread more rapidly. We are currently characterizing the cell biological phenotype of cells lacking this predominant myosin of non-muscle cells. Because interactions at the cell surface are likely to play important roles in many diseases, we have been involved in a long-term collaboration with the Dhawan laboratory in the FDA 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 been attempting to develop novel methods to generate a therapeutic vaccine. This fiscal year, we investigated the roles of cell interactions and inflammatory cytokines such as TNF-alpha in the process by which dormant human cells latently infected with HIV-1 are activated to begin producing infectious virus. This work showed that human macrophages can activate HIV-1 viral replication in latently infected cells by a cell-cell interaction requiring living cells and cytokines that mediate rapid nuclear localization of NF-kB and induction of proinflammatory cytokines. Overall, these approaches are applying our fundamental research expertise on integrins, signaling, migration, and cell interactions to understand and modify organ development, regeneration, tissue engineering, and disease.