Unknown,Transcriptomics,Genomics,Proteomics

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Follistatin antagonizes transforming growth factor b3 induced epithelial mesenchymal transition in vitro. Implications for murine palatal development supported by microarray analysis Differentiation 2007

ABSTRACT: Follistatin antagonizes TGF-b3-induced epithelial-mesenchymal transition in vitro: Implications for murine palate development by microarray analysis

Cleft palate is among the most frequent human birth defects. A number of genes and encoded proteins have been shown to play a crucial role in the process of palatogenesis. The central role is attributed to the cytokine TGF-ß3 which is expressed in the medial edge epithelium, (MEE), covering the growing palatal shelves already prior to the fusion process. Gene targeted mice with a palatal cleft phenotype may be divided into factors acting upstream of TGFß-3 in regulating its gene expression or downstream in its signal transduction pathway. The fusion process itself gets explained either by a transition of the epithelial cells into mesenchymal cells (EMT.) or by programmed cell death (apoptosis) of the epithelial cells. No explanation could ever been given why the TGF-ß3 producing epithelial cells do not undergo differentiation or apoptosis prior to the contact of the two shelves. We screened the MEE for expression of known antagonists of TGFß-molecules and detected the gene coding for follistatin to be coexpressed with TGF-ß3. We can show that follistatin directly binds to TGFß3 and that it completely blocks TGF-ß3 induced EMT of an epithelial cell line in vitro. EMT of the cells was analyzed by microarray hybridization.

ORGANISM(S): Mus musculus

SUBMITTER: Joachim Gruen

PROVIDER: E-TABM-395 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Cleft palate results from a mixture of genetic and environmental factors and occurs when the bilateral palatal shelves fail to fuse. The objective of this study was to search for new genes involved in mouse palate formation. Gene expression of murine embryonic palatal tissue was analyzed at the various developmental stages before, during, and after palate fusion using GeneChip? microarrays. Ceacam1 was one of the highly up-regulated genes during and after fusion in palate formation, and this was confirmed by quantitative real-time PCR. Immunohistochemical staining showed that CEACAM1 was expressed at a very low level in palatal epithelium before fusion, but highly expressed in the midline of the palate during and after fusion. To investigate the developmental role of CEACAM1, function-blocking antibody was added to embryonic mouse palate in organ culture. Palatal fusion was inhibited by this function-blocking antibody. To investigate the subsequent developmental role of CEACAM1, we characterized Ceacam1-deficient (Ceacam1-/-) mice. Epithelial cells persisted abnormally at the midline of the embryonic palate even on day E16.0, and palatal fusion was delayed in Ceacam1-/- mice. TGF?3 expression, apoptosis, and cell proliferation in palatal epithelium were not effected in the palate of Ceacam1-/-mice. CEACAM1 expression was down-regulated in Tgfb3-/- palate. However, exogenous TGF?3 did not induce CEACAM1 expression. These results suggest that CEACAM1 has roles in both the initiation of palate formation via epithelial cell adhesion and TGF signaling has some indirect effect on CEACAM1. Global gene expression profiling of palatal processes before, during and after fusion of palatal shelves We used microarray to investigate the gene expression of palatal tissue during palatal development. Palatal processes were microdissected at the stages of palatal development (before, during and after fusion) for RNA extraction and hybridization on Affymetrix microarrays.

Project description:We identify a role for two evolutionarily related, secreted metalloproteases of the ADAMTS family (A disintegrin-like and metalloprotease domain with thrombospondin type-1 motif), ADAMTS20 and ADAMTS9, in palatogenesis. Adamts20 mutations cause the mouse white spotting mutant belted (bt), whereas Adamts9 is essential for survival beyond 7.5 days of gestation (E7.5). Functional overlap of Adamts9 with Adamts20 was established in bt/bt:Adamts9+/- mice, which have increased white spotting relative to bt mice, as previously reported, and a fully penetrant cleft palate. Palatal closure was delayed, although eventually completed, in both bt/+;Adamts9+/- and bt/bt mice, demonstrating a cooperative role of these related genes. Adamts9 and Adamts20 are both expressed in palatal mesenchyme, with Adamts9 expressed exclusively in microvascular endothelial cells. Palatal shelves from bt/bt:Adamts9+/- mice fused in culture, suggesting an intact TGF signaling pathway in palatal epithelium, and indicating a temporally specific delay in palatal shelf elevation and growth toward the midline. Palatal shelf mesenchymal cells showed a statistically significant decrease of cell proliferation at E13.5 and E14.5, as well as decreased processing of versican, an ADAMTS substrate, at these stages. Vcan haploinsufficiency led to a greater penetrance of cleft palate in bt mice, and impaired proliferation was also seen in palatal mesenchymal cells of these mice, suggesting a role for ADAMTS-mediated versican proteolysis in palatal closure. In a parallel with recent work identifying a role for a bioactive ADAMTS-generated versican fragment in regulating apoptosis during interdigital web regression, we propose that versican proteolysis may influence palatal mesenchymal cell proliferation. Palatal shelves were dissected from four E13.75 Adamts9+/-:bt/bt embyos (correspond to the 4 samples: Palate_Adamts9+/-:bt/bt_Rep1, Palate_Adamts9+/-:bt/bt_Rep2, Palate_Adamts9+/-:bt/bt_Rep3 and Palate_Adamts9+/-:bt/bt_Rep4) and age-matched 3 wild-type C57Bl/6 embryos (correspond to the 3 samples: Palate_WT_Rep1, Palate_WT_Rep2, and Palate_WT_Rep3) that were used as the controls

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Follistatin antagonizes transforming growth factor b3 induced epithelial mesenchymal transition in vitro. Implications for murine palatal development supported by microarray analysis Differentiation 2007

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