Mechanisms of FGFR2 Signaling in Salivary Gland Branching Morphogenesis

描述（由申请人提供）：成纤维细胞生长因子（FGF）及其受体（FGFR）在唾液腺分支形态发生中起重要作用。在小鼠中，Fgf 10或其受体Fgfr 2b的靶向破坏导致唾液腺发育不全，证明了FGF 10-FGFR 2b信号传导途径在唾液腺发育中的重要作用。然而，Fgf 10或Fgfr 2b基因敲除小鼠中唾液腺的缺乏排除了在这些小鼠模型中对分支形态发生的进一步研究。因此，有必要开发一种替代方法来深入了解Fgfr 2b细胞内信号通路在唾液腺分支形态发生中的作用。我们已经开发了一种新的小鼠模型系统，其中Fgfr 2下游的特定信号传导途径已被敲入突变废除，而不损害受体的酪氨酸激酶活性以激活其他信号传导途径。我们的假设是，Fgfr 2b信号通过Frs 21是必不可少的唾液腺分支形态发生。目的1：（1）通过组织学和离体器官培养分析不同突变品系小鼠下颌下腺（SMG）的分支形态发生：（2）使用无间充质SMG培养来研究上皮形态发生;以及（3）使用表达突变型和野生型受体的细胞系来评估Fgfr 1b和Fgfr 2b在分枝期间对Frs 21的上皮转磷酸化的机制形态发生在目标2中，我们将确定Frs 21介导的Grb 2和Shp 2信号通路在SMG分支形态发生中的作用。我们的假设是Shp 2是Fgfr 2b信号通过Frs 21的SMG分支形态发生的关键介质。我们将使用两种基因工程突变小鼠来验证这一假设。在第一个突变小鼠模型中，四个Grb 2结合位点突变为苯丙氨酸（4F），因此不能招募Grb 2。在第二种突变小鼠模型中，两个Shp 2结合位点突变为苯丙氨酸（2F），因此不能募集Shp 2。我们将进行（1）组织学和离体SMG器官培养，以确定Grb 2和Shp 2在分支形态发生中的作用;和（2）重组SMG培养，以通过培养野生型上皮细胞与Grb 2或Shp 2突变间充质来确定突变是细胞自主还是非细胞自主，反之亦然。此外，我们提出了两种方法来确定新的遗传途径：a）全基因组的突变SMG的上皮和间充质中的mRNA表达分析，和B）SMG中靶基因的基于表达的途径分析。总的来说，这项工作将提供一个详细的分子图片FGF信号，介导的Fgfr 2b亚型和对接蛋白Frs 21，调节唾液腺分支形态发生。这些研究的结果可能使得能够设计使用FGFR 2b介导的途径来调节祖细胞分化和形态发生的用于唾液腺再生的新方法。

DESCRIPTION (provided by applicant): Fibroblast growth factors (FGF) and their receptors (FGFR) play a central role in salivary gland branching morphogenesis. In mice, targeted disruption of Fgf10 or its receptor, Fgfr2b, causes salivary gland agenesis, demonstrating the essential role of FGF10-FGFR2b signaling pathways in salivary gland development. However, the lack of salivary glands in Fgf10 or Fgfr2b knockout mice precludes further investigation of branching morphogenesis in these mouse models. Therefore, it is necessary to develop an alternative approach to gain insights about the role of Fgfr2b intracellular signaling pathways in salivary gland branching morphogenesis. We have developed a novel mouse model system in which specific signaling pathways downstream of Fgfr2 have been abrogated by a knock-in mutation, without compromising the tyrosine kinase activity of the receptor to activate other signaling pathways. Our hypothesis is that Fgfr2b signaling via Frs21 is essential for salivary gland branching morphogenesis. In Aim 1, we will (1) analyze the branching morphogenesis of submandibular glands (SMGs) from different mutant mouse strains by histology and ex vivo organ culture; (2) use mesenchyme-free SMG cultures to investigate epithelial morphogenesis; and (3) use cell lines expressing the mutant and wild type receptors to evaluate the mechanism of epithelial transphosphorylation of Frs21 by Fgfr1b and Fgfr2b during branching morphogenesis. In Aim 2, we will determine the role of Frs21-mediated Grb2 and Shp2 signaling pathways in SMG branching morphogenesis. Our hypothesis is that Shp2 is the critical mediator of Fgfr2b signaling via Frs21 for SMG branching morphogenesis. We will test this hypothesis using two strains of genetically engineered mutant mice. In the first mutant mouse model, the four Grb2 binding sites are mutated to phenylalanine (4F), and thus cannot recruit Grb2. In the second mutant mouse model, the two Shp2 binding sites are mutated to phenylalanine (2F), and thus cannot recruit Shp2. We will perform (1) histology and ex vivo SMG organ culture to determine the role of Grb2 and Shp2 in branching morphogenesis; and (2) recombinant SMG culture to determine whether the mutations are cell autonomous or non-cell autonomous by culturing wild type epithelium with Grb2 or Shp2 mutant mesenchyme and vice versa. In addition, we propose two approaches to identify novel genetic pathways: a) genome-wide mRNA expression analysis in the epithelium and mesenchyme of the mutant SMGs, and b) expression-based pathway analysis of target genes in SMG. Collectively, this work will provide a detailed molecular picture of how FGF signaling, mediated by the Fgfr2b isoform and the docking protein Frs21, regulates salivary gland branching morphogenesis. The results of these studies may enable the design of novel methods for salivary gland regeneration using FGFR2b mediated pathways to regulate progenitor cell differentiation and morphogenesis.