7043

TGFB3

epithelial mesenchymal transformation (EMT) of the medial edge epithelial (MEE) seam creates palatal confluence. This work aims to elucidate the molecular mechanisms by which TGFbeta3 brings about palatal seam EMT. We collected mRNA for PCR analysis from individual transforming MEE cells by laser microdissection techniques and demonstrated that TGFbeta3 stimulates lymphoid-enhancing factor 1 (LEF1) mRNA synthesis in MEE cells. We show with antisense beta-catenin oligonucleotides that up-regulated LEF1 is not activated by beta-catenin in palate EMT. We ruled out other TGFbeta3 targets, such as RhoA and MEK1/2 pathways, and we present evidence using dominant-negative Smad4 and dominant-negative LEF1 showing that TGFbeta3 uses Smads both to up-regulate synthesis of LEF1 and to activate LEF1 transcription during induction of palatal EMT. When phospho-Smad2 and Smad4 are present in the nucleus, LEF1 is activated without beta-catenin. Our paper is the first to show that the Smad2,4/LEF1 complex replaces beta-catenin/LEF1 during activation of EMT in vivo by TGFbeta3.

epithelial-mesenchymal transition (EMT), crucial during embryogenesis for new tissue and organ formation, is also considered to be a prerequisite to cancer metastasis. We report here that the protein tyrosine phosphatase Pez is expressed transiently in discrete locations in developing brain, heart, pharyngeal arches, and somites in zebrafish embryos. We also find that Pez knock-down results in defects in these organs, indicating a crucial role in organogenesis. Overexpression of Pez in epithelial MDCK cells causes EMT, with a drastic change in cell morphology and function that is accompanied by changes in gene expression typical of EMT. Transfection of Pez induced TGFbeta signaling, critical in developmental EMT with a likely role also in oncogenic EMT. In zebrafish, TGFbeta3 is co- expressed with Pez in a number of tissues and its expression was lost from these tissues when Pez expression was knocked down. Together, our data suggest Pez plays a crucial role in organogenesis by inducing TGFbeta and EMT.

During early heart development at the looped heart stage, endothelial cells in the outflow tract and atrioventricular (AV) regions transform into mesenchyme to generate endocardial cushion tissue. This endocardial epithelial-mesenchymal transition (EMT) is regulated by several regulatory pathways, including the transforming growth factor-beta (TGFbeta), bone morphogenetic protein (BMP), and Rho-ROCK pathways. Here, we investigated the spatiotemporal expression pattern of ROCK1 mRNA during EMT in chick and examined whether TGFbeta or BMP could induce the expression of ROCK1. At the onset of EMT, ROCK1 expression was up-regulated in endothelial/mesenchymal cells. A three-dimensional collagen gel assay was used to examine the mechanisms regulating the expression of ROCK1. In AV endocardium co-cultured with associated myocardium, ROCK1 expression was inhibited by either anti-TGFbeta3 antibody, anti-ALK2 antibody or noggin, but not SB431542 (ALK5 inhibitor). In cultured preactivated AV endocardium, TGFbeta3 protein induced the expression of ROCK1, but BMP did not. AV endothelial cells that were cultured in medium supplemented with TGFbeta3 plus anti-ALK2 antibody failed to express ROCK1. These results suggest that the expression of ROCK1 is up-regulated at the onset of EMT and that signaling mediated by TGFbeta3/ALK2 together with BMP is involved in the expression of ROCK1.CI - (c) 2008 Wiley-Liss, Inc.

mutation in interferon regulatory factor 6 (IRF6) is known to cause syndromic and non-syndromic cleft lip/palate in human. In this study, we investigated the molecular mechanisms related to IRF6 during palatal fusion using palatal shelves organ culture. The results showed that ablation of Irf6 resulted in a delay in TGFbeta3-regulated palatal fusion. Ectopic expression of IRF6 was able to promote palatal fusion and rescue shTgfbeta3-induced fusion defect. These findings indicate that IRF6 is involved in TGFbeta3-mediated palatal fusion. Molecular analysis revealed that ectopic expression of IRF6 increased the expression of SNAI2, an epithelial mesenchymal transition (EMT) regulator, and diminished the expression of various epithelial markers, such as E-cadherin, Plakophilin and ZO-1. In addition, knockdown of Irf6 expression decreased SNAI2 expression, and restored the expression of ZO-1 and Plakophilin that were diminished by TGFbeta3. Blocking of Snai2 expression delayed palatal fusion and abolished the IRF6 rescuing effect associated with shTgfbeta3-induced fusion defect. These findings indicate that TGFbeta3 increases IRF6 expression and subsequently regulates SNAI2 expression, and IRF6 appears to regulate EMT during palatal fusion via SNAI2. Taken together, this study demonstrates that IRF6 is a mediator of TGFbeta3, which regulates EMT and fusion process during the embryonic palate development.