Growth Factor Signaling in Mouse Palatogensis

描述（申请人提供）：非综合征性唇腭裂，是人类主要的先天性出生缺陷之一。尽管经常发生，相对较少的了解发育和遗传基础，导致唇裂和/或腭裂形成。哺乳动物的腭发育是一个复杂的多步骤过程，很大程度上取决于顺序和互惠的组织相互作用。几个生长因子家族的成员已被证明在腭发育过程中介导这种组织相互作用，包括BMP、FGF、TGF¿和Shh。最近的研究已经开始暗示Wnt信号在腭发育中的作用。人类腭裂表型与WNT3和WNT5A突变有关。我们发现wnt5a缺乏导致二次腭裂的完全性，在前后腭的组织学、细胞和分子水平上表现出明显的表型改变。我们进一步证明，Ror2是Wnt5a非典型信号的孤儿酪氨酸激酶受体，与Wnt5a基因相互作用，并介导Wnt5a非典型信号在发育中的腭调节定向细胞迁移和细胞增殖。然而，与Ror2-/-腭或Wnt5a+/-腭相比，Wnt5a突变体在组织学上表现出更严重的腭缺损；Ror2+/-腭，表明有其他受体参与。基于这些观察结果以及Ryk基因缺失小鼠表现出腭裂表型和发育中缺乏典型Wnt信号活动的事实，我们假设Wnt5a信号通过Ror2-和Ryk-介导的非典型途径调节腭裂发生。为了验证这一假设，我们提出了三个目标：1)确定Wnt5a不通过典型途径发出信号来调节腭发育；2)确定Wnt5a信号通过ror2介导的非规范途径调节腭发育过程中上皮-间质相互作用；3)确定Ryk在腭发育过程中介导Wnt5a信号通路的作用。结合遗传杂交，组织学，细胞和分子分析，以及体外器官培养和细胞迁移分析将被用来实现这些目标。所获得的结果将为更好地理解人类腭裂形成的机制提供见解。公共卫生相关性：腭裂是最常见的人类出生缺陷之一。这种复杂的先天缺陷是由多基因和环境因素共同造成的。尽管经常发生，但对导致腭裂形成的发育和遗传基础知之甚少。生长因子在胚胎发生中起着至关重要的作用。几种WNT生长因子的突变与人类腭裂缺陷有关。本研究旨在研究Wnt信号在腭发育调控中的作用，并试图建立Wnt5a及其受体在腭发育过程中的分子和细胞机制。本研究结果将为我们了解人类腭裂的形成提供基础信息，并为人类腭裂的遗传预防和治疗提供见解。

DESCRIPTION (provided by applicant): Non-syndromic cleft lip and cleft palate, one of the major groups of congenital birth defects in human beings. Despite the frequent occurrence, relatively little is known about the developmental and genetic basis that leads to a cleft lip and/or cleft palate formation. Mammalian palatogenesis is a complex and multiple step process, largely depending on the sequential and reciprocal tissue interactions. Members of several families of growth factors have been demonstrated to play a role in mediating such tissue interaction during palatogenesis, including BMP, FGF, TGF¿, and Shh. Recent studies have begun to implicate a role of Wnt signaling in the palate development. The cleft palate phenotype has been linked to mutations in WNT3 and WNT5A in humans. We have found that Wnt5a-deficiency leads to a complete cleft of the secondary palate, which exhibits distinct phenotypic alterations at histology, cellular and molecular levels in the anterior and posterior palate. We further demonstrated that Ror2, a orphan tyrosine kinase receptor for Wnt5a noncanonical signaling, genetically interacts with Wnt5a and mediates Wnt5a noncanonical signaling in regulating directional cell migration and cell proliferation in the developing palate. However, Wnt5a mutants exhibit much severer palatal defects histologically as compared to that in Ror2-/- palate or Wnt5a+/-;Ror2+/- palate, indicating an involvement of another receptor(s). Based on these observations and the fact that mice deficient in Ryk exhibit a cleft palate phenotype and the absence of the canonical Wnt signaling activity in the developing palate, we hypothesized that Wnt5a signals through Ror2- and Ryk- mediated noncanonical pathway to regulate palatogenesis. Three aims are proposed to test this hypothesis: 1) to establish that Wnt5a does not signal through the canonical pathway to regulate palatogenesis; 2) to determine that Wnt5a signals through Ror2-mediated noncanonical pathway to regulate epithelial-mesenchymal interaction during palatogenesis; and 3) to define the role of Ryk in mediating Wnt5a signaling in palate development. A combination of genetic crosses, histological, cellular and molecular assays, as well as in vitro organ culture and cell migration assays will be utilized to accomplish these goals. The results obtained will provide an insight for a better understanding of the mechanisms of cleft palate formation in humans. PUBLIC HEALTH RELEVANCE: Cleft palate represents one of the most frequent human birth defects. This complex birth defect is caused by both multigenic and environmental contributions. Despite the frequent occurrence, relatively little is known about the developmental and genetic basis that leads to a cleft palate formation. The growth factor plays vital roles in embryogenesis. Mutations in several WNT growth factors have been associated with human cleft palate defect. This proposal studies the role of Wnt signaling in the regulation of the palate development and attempts to establish molecular and cellular mechanisms underlying Wnt5a and its receptors during palatogenesis. The results obtained from this study will provide fundamental information for our understanding of cleft palate formation in humans, and provide an insight for the genetic prevention and therapy of the human cleft palate.