Roles of the Fat pathway in cartilage patterning and polarity

描述(申请人提供)：一个有功能的骨骼系统依赖于胚胎发育过程中软骨和关节的协调发展。然而，人们对控制软骨大小、形状或连通性的细胞和分子机制知之甚少。解开引导间充质细胞凝聚并排列成软骨形成前堆叠的信号的进展，对于阐明塑造骨骼组织和生长的早期事件尤为重要。了解这些过程将有助于更好地诊断和治疗骨骼畸形和出生缺陷。此外，控制软骨形态发生和分化的分子可能对改善软骨和关节损伤的治疗以及从干细胞诱导软骨形成的生物工程努力具有相当重要的临床意义。我们最近发现，Rere是调节果蝇Dachsous(DCHS)-Fat信号的转录因子Atroin的近亲，是斑马鱼头面部发育所必需的 骨骼发育过程中平面细胞极性(PCP)中的脂肪通路。我们实验室的戏剧性结果表明，与PCP一致，Fat3在软骨分化中发挥了更深远的作用。缺乏Rere或Fat3的胚胎发育出软骨，在这些软骨中，咽弓中的堆积和分化是分离的，这导致了关节融合。此外，在颅面发育过程中，Rere和Fat3在协调骨骼祖细胞对BMP和成纤维细胞生长因子信号的反应中起着关键作用。我们建议通过三组实验来探索脂肪通路和PCP在软骨和关节形成中的作用。第一个目标将解决脂肪途径结合软骨堆积和分化的假设。软骨和关节表型将在Rere和Fat3突变体中使用实时成像进行评估。我们将确定软骨中脂肪的DCHS配体(S)，并创建异位源来研究信号的传播和调制。我们还将比较脂肪途径和Wnt/PCP的活性以及它们在软骨发育中的相互依赖。第二个目标将解决这样的假设，即脂肪途径协调对BMPS和其他生长因子的极化反应，从而将早期的构图事件与后来的器官形成联系起来。为此，我们有了新的细胞骨架极性和纤毛的标记，揭示了骨骼祖细胞中意想不到的极性边界。最后，第三个目标将集中在关节形成上，并测试脂肪途径通过诱导消除或过度表达Rere或Fat3来防止关节间区软骨堆积和分化的假设。总之，这些研究将导致对脊椎动物脂肪途径在细胞-细胞交流中相对未被探索的功能的机械性见解，并为尚未解决的人类先天性骨骼畸形的原因确定候选基因。

DESCRIPTION (provided by applicant): A functional skeletal system depends upon the coordinated development of cartilages and joints during embryogenesis. However, little is known about the cellular and molecular mechanisms that control cartilage size, shape or connectivity. Progress unraveling the signals that direct mesenchymal cells to condense and align into prechondrogenic stacks is of particular importance in elucidation of the early events that shape the organization and growth of the skeleton. Understanding these processes will allow better diagnostic approaches and treatments for skeletal malformations and birth defects. Moreover, molecules that control cartilage morphogenesis and differentiation may be of considerable clinical importance both for improvements in the treatment of cartilage and joint injuries and in bioengineering efforts to induce cartilage formation from stem cells. Our recent finding that Rere, a close relative of the transcription factor Atrophin which regulates Dachsous (Dchs)-Fat signaling in flies, is required for craniofacial development in zebrafish has implicated the Fat pathway in planar cell polarity (PCP) during skeletogenesis. Dramatic results from our laboratory now demonstrate an even more profound role for a Fat3 in cartilage differentiation, consistent with PCP. Embryos deficient in Rere or Fat3 develop cartilages in which stacking and differentiation are uncoupled in the pharyngeal arches and this leads to joint fusion. Moreover, Rere and Fat3 are critical for coordinating responses of skeletal progenitors to Bmp and Fgf signaling during craniofacial development. We propose to explore the roles of the Fat pathway and PCP in cartilage and joint formation in three sets of experiments. The first aim will address the hypothesis that the Fat pathway couples cartilage stacking and differentiation. Cartilage and joint phenotypes will be evaluated in Rere and Fat3 mutants using live imaging. We will identify the Dchs ligand(s) for Fat in cartilage and create ectopic sources to study signal propagation and modulation. We will also compare the activities of the Fat pathway and Wnt/PCP and their interdependence in cartilage development. The second aim will address the hypothesis that the Fat pathway coordinates polarized responses to Bmps and other growth factors, thereby linking early patterning events with later organ formation. For this we have new markers of cytoskeletal polarity and cilia that reveal unexpected boundaries of polarity in skeletal progenitors. Finally, the third aim will focus on joint formation and test the hypothesis that the Fat pathway prevents cartilage stacking and differentiation in the joint interzone with inducible elimination or overexpression of Rere or Fat3. Together these studies will lead to mechanistic insights into the relatively unexplored functions of the vertebrate Fat pathway in cell-cell communication and identify candidate genes for as yet unresolved causes of human congenital skeletal malformations.