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Keywords:

  • palatal fusion;
  • medial edge epithelium;
  • transforming growth factor;
  • small interfering RNA

Abstract

The molecular regulation of palatogenesis continues to be an active area of investigation to provide a foundation for understanding the molecular etiology of cleft palate. Transforming growth factor (TGF) -β type III receptor (TβR-III) has been shown to be specifically expressed in the medial edge epithelium at critical stages of palatal shelf adherence during palatogenesis. The aim of this study was to examine TβR-III mRNA localization and expression levels in vivo and to determine the requirement for TβR-III expression during palatal fusion in vitro. TβR-III gene expression was analyzed by in situ hybridization in tissue specimens and real-time reverse transcriptase-polymerase chain reaction using specific cells in the palatal shelf isolated by laser capture microdissection. TβR-III was knocked down in embryonic day (E) 13 palatal shelves in organ culture. Palatal shelf organ cultures were treated with small interfering RNA (siRNA) at final concentrations of 300, 400, and 500 nM, respectively. The treatment with siRNA specific for TβR-III decreased the amount of protein by approximately 75%. The reduction in TβR-III resulted in a delay in the process of palatal fusion compared with control. The protein expression of phospho-Smad2 was decreased in the TβR-III siRNA group. In addition, palatal organ cultures treated with TβR-III siRNA + rhTGF-β3 completely fused by 72 hr in vitro. These results support our hypothesis that TβR-III has a critical role in the process of palatal fusion. Developmental Dynamics 236:791–801, 2007. © 2007 Wiley-Liss, Inc.