Growth Factor Signaling in Mouse Palatogensis

描述（由申请人提供）：非综合征性唇腭裂，是人类先天性出生缺陷的主要类型之一。尽管经常发生，但对导致唇腭裂形成的发育和遗传基础知之甚少。哺乳动物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。已经证明几个生长因子家族的成员在腭发育期间介导这种组织相互作用中起作用，包括BMP、FGF、TGF β和Shh。最近的研究已经开始暗示Wnt信号在腭发育中的作用。腭裂表型与人类WNT 3和WNT 5A的突变有关。我们已经发现，Wnt 5a缺陷导致完全裂的继发腭，表现出不同的表型改变在组织学，细胞和分子水平的前，后腭。我们进一步证明，Ror 2，一个孤儿酪氨酸激酶受体的Wnt 5a非经典信号，遗传相互作用与Wnt 5a和介导的Wnt 5a非经典信号在调节定向细胞迁移和细胞增殖发育腭。然而，与Ror 2-/-腭或Wnt 5a +/-; Ror 2 +/-腭相比，Wnt 5a突变体在组织学上表现出更严重的腭缺陷，表明另一种受体参与。基于这些观察结果以及Ryk缺陷小鼠表现出腭裂表型和发育中的腭中缺乏经典Wnt信号传导活性的事实，我们假设Wnt 5a信号通过Ror 2和Ryk介导的非经典途径来调节腭发生。本研究拟通过三个方面来验证这一假说：1）确定Wnt 5a不通过经典途径来调控腭发育; 2）确定Wnt 5a通过Ror 2介导的非经典途径来调控腭发育过程中上皮-间充质相互作用; 3）确定Ryk在腭发育中介导Wnt 5a信号传导中的作用。将利用遗传杂交、组织学、细胞和分子测定以及体外器官培养和细胞迁移测定的组合来实现这些目标。所获得的结果将为更好地理解人类腭裂形成的机制提供一种见解。公共卫生相关性：腭裂是最常见的人类出生缺陷之一。这种复杂的出生缺陷是由多基因和环境的贡献。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。本研究旨在探讨Wnt信号在腭发育调控中的作用，并试图建立Wnt 5a及其受体在腭发育中的分子和细胞机制。本研究的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.