Epithelial branching morphogenesis is essential for forming many organs such as exocrine glands, salivary gland, lung, and kidney. Repetitive epithelial cleft and bud formation create the three-dimensional branching structures characteristic of many organs, yet the mechanisms are poorly understood. Objectives: To identify molecules that are necessary for branching morphogenesis, laser microdissection (LMD) and serial analysis of gene expression (SAGE) (http://www.sagenet.org) were performed. Methods: Embryonic 12-day salivary glands were cultured on membranes, and cleft and bud epithelia of frozen-sectioned salivary gland were laser-microdissected (Sakai, T. et al, Nature 423, 876-881, 2003). Profiles of gene expression in salivary epithelium from small amounts of cells were identified by T7-SAGE (Sakai, T. et al. Current Protocols in Cell Biology 19.3.1-19.3.30) Results: Initial T7-SAGE data showed that cleft epithelium expressed an extracellular protein, fibronectin (FN), higher than bud epithelium. We hypothesized that a direct mechanism for branching morphogenesis could be site-specific accumulation of FN, which is involved in morphogenetic processes and is developmentally expressed in embryonic branching organs. Exogenously added FN substantially promoted salivary gland cleft formation and branching, whereas other matrix proteins had no effect. Furthermore, knockdown of FN expression by siRNA or function-blocking antibodies reduced branching. Conclusion: We conclude that FN expression is required for cleft formation in glandular branching morphogenesis, and that T7-SAGE gene expression analysis provides new possibilities for the characterization of region-specific expression profiles in microdissected samples from complex, heterogeneous tissues.

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