Cleft palate is one of the most common birth defects in human. Mammalian palatogenesis is a complex and multiple step process, largely depending on the sequential and reciprocal interactions between apposed epithelial and mesenchymal tissues involving diffusible growth factors and homeobox genes. Genetic or environmental interruption at any step of this complex process can result in cleft palate. Bone Morphogenetic proteins (BMPs), Sonic hedgehog (Shh), and Fibroblast Growth Factors (FGFs) have been implicated in the formation of many vertebrate organs. However, their potential role in mammalian palatogenesis remains unknown. Our previous studies demonstrated that Msx1 homeobox gene, mutations, which are associated with non-syndromic cleft palate and tooth agenesis in human, control a genetic hierarchy involving BMPs and Shh in the regulation of outgrowth of the anterior portion of the secondary palatal shelves. In the anterior portion of developing palatal shelves, the expression of Bmp4, which is regulated by Msx1 in the mesenchyme, maintains Shh expression in the medial edge epithelium (MEE). Expression of Shh in the MEE is required for the growth of palatal shelves via activation of Bmp2 in palatal mesenchyme. Our preliminary studies also demonstrated a restricted expression of FGFR2 in the posterior palatal mesenchyme, which can respond to FGF signal in terms of gene expression, suggesting that FGF signaling may regulate the development of the posterior portion of the palate. In this application, three specific aims are proposed to further test the regulation and function of the BMP-Shh pathway in anterior palatal development, and to establish a role for FGF signaling in the regulation of posterior palate development.
Aim 1 is designed to examine the regulatory mechanism of Shh expression in the MEE by mesenchymal BMP4. The specific type I BMP receptor that is involved in this regulation will also be determined. Transgenic mice will be generated that carry either a dominant-negative BMP receptor-IA transgene driven by the K14 promoter or a constitutively active BMP receptor-IA transgene driven by the Msx1 promoter.
In Aim 2, the role of Shh in palate formation will be defined by analyzing Shh conditional knockout mice and transgenic mice overexpressing Shh in the palatal epithelium. It will also test whether ectopic Shh expression in the palatal epithelium of the Msx1 mutants can bypass the requirement for both Msx1 and BMP4 in palatal mesenchyme to restore cell proliferation and rescue cleft palate. Lastly, in Aim 3, the role for FGF signaling in palatogenesis will be established by testing FGF's function in the regulation of cell proliferation and survival in vitro by organ culture, and in vivo by analyzing Fgf10 knockout mice that exhibit a cleft palate. Our long-term goal is to unveil the molecular mechanisms of mammalian palatogenesis and cleft palate formation, which would provide insight for genetic prevention and therapy for human cleft palate.
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