Mechanisms of FGFR2 Signaling in Salivary Gland Branching Morphogenesis

描述(申请人提供)：成纤维细胞生长因子及其受体(FGFR)在唾液腺分支形态发生中起核心作用。在小鼠中，靶向干扰Fgf10或其受体FGFR2b导致唾液腺发育不全，证明了FGF10-FGFR2b信号通路在唾液腺发育中的重要作用。然而，Fgf10或FGFR2b基因敲除小鼠唾液腺的缺乏阻碍了对这些小鼠模型中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FGFR2b细胞内信号通路在唾液腺分支形态发生中的作用。我们开发了一种新的小鼠模型系统，在该系统中，FGFR2下游的特定信号通路已被敲入突变取消，而不影响受体的酪氨酸激酶活性来激活其他信号通路。我们的假设是，FGFR2b信号通过Frs21对唾液腺分支形态发生至关重要。在目标1中，我们将(1)通过组织学和体外器官培养分析不同突变小鼠下颌下腺(SMGs)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；(3)使用表达突变和野生型受体的细胞系来评估Fgfr1b和FGFR2b在分支形态发生过程中Fgfr1b和FGFR2b对Frs21上皮转磷酸化的机制。在目标2中，我们将确定Frs21介导的Grb2和Shp2信号通路在SMG分支形态发生中的作用。我们的假设是Shp2是FGFR2b信号通过Frs21在SMG分支形态发生中的关键中介。我们将使用两个品系的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Grb2结合位点突变为苯丙氨酸(4F)，因此无法招募Grb2。在第二个突变小鼠模型中，Shp2的两个结合位点突变为苯丙氨酸(2F)，因此无法招募Shp2。我们将进行(1)组织学和体外SMG器官培养，以确定Grb2和Shp2在分支形态发生中的作用；(2)重组SMG培养，通过用Grb2或Shp2突变的间质培养野生型上皮来确定突变是细胞自主的还是非细胞自主的，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)在突变的SMG的上皮和间质中进行全基因组的mRNA表达分析，以及b)在SMG中基于表达的靶基因的途径分析。总而言之，这项工作将提供一幅详细的分子图像，说明FGFR2b异构体和对接蛋白Frs21介导的成纤维细胞生长因子信号如何调节唾液腺分支形态发生。这些研究结果可能有助于设计新的唾液腺再生方法，使用FGFR2b介导的途径来调节前体细胞的分化和形态发生。

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.