Notch Signaling and Left-Right Asymmetry Determination

描述（由申请人提供）：在器官发生期间，胚胎的内部器官显示出显著的左右不对称（L-R）。建立这种不对称性的一系列事件包括：a）对称性的最初破坏，这导致在胚胎组织者内部和周围建立特定的基因表达模式; B）将L-R位置信息从组织器传递到侧板中胚层，以及c）L-R信息向器官原基的转移，其导致不对称形态发生的特定程序的详细阐述。近年来，参与这三个过程的各种基因已经被鉴定出来。我们的初步结果表明，此外，Notch途径，一个主要的发育信号通路，可能有一个重要的作用，在L-R的决定。该建议的主要重点将是研究Notch信号通路在上述三个事件中的每一个事件中的作用，以及它与其他已知的参与L-R决定的基因的相互作用。我们计划在体内使用现代可视化技术来解决这些问题，同时在模型动物如小鸡，斑马鱼和小鼠中进行遗传干预。具体而言，我们建议：1）在斑马鱼、鸡和小鼠胚胎中研究纤毛在破坏L-R对称性中的作用以及RFX家族转录因子对其的调节; 2）进行Notch途径各种组分的功能获得和丧失实验（鸡、小鼠和斑马鱼），并建立了其与小鼠结纤毛运动和流动以及斑马鱼体内Ca 2+的关系; 3）分析已知参与斑马鱼野生型和突变型胚胎L-R不对称性的基因的时空表达，试图获得斑马鱼胚胎LR不对称性建立的数学模型; 4）通过转基因工程斑马鱼胚胎的大规模诱变筛选鉴定Nodal、Notch和Pitx 2上游的新基因。到目前为止，很少有基因与人类偏侧性障碍的病因学有关。对L-R决定的早期阶段的研究和在各种脊椎动物模式生物中涉及的新基因的鉴定无疑将为导致人类侧化缺陷的遗传因素提供新的线索。

DESCRIPTION (provided by applicant): During organogenesis the internal organs of the embryo display significant left-right asymmetry (L-R). A sequence of events for the establishment of this asymmetry includes: a) the initial breaking of symmetry, which leads to the establishment of specific patterns of gene expression in and around the embryonic organizer; b) the relay of L-R positional information from the organizer to the lateral plate mesoderm, and c) the transfer of L-R information to the organ primordia that leads to the elaboration of specific programs of asymmetric morphogenesis. A variety of genes involved in these three processes have been identified in recent years. Our preliminary results indicate that, additionally, the Notch pathway, a major developmental signaling pathway, may have an important role in L-R determination. The main focus of this proposal will be to study the role of the Notch signaling pathway during each one of the three events described above, and its interaction with other known genes implicated in L-R determination. We plan to tackle these questions using modem visualizing techniques in vivo, in parallel with genetic interventions in model animals such as chick, zebrafish and mouse. Specifically, we propose to: 1) study the role of cilia in breaking L-R symmetry and their regulation by the RFX family of transcription factors in zebrafish, chick and mouse embryos; 2) Perform gain- and loss-of-function experiments of various components of the Notch pathway (chick, mouse and zebrafish) and establish its relationship with nodal cilia movement and flow in mice and Ca 2+ in zebrafish; 3) analyze the spatio-temporal expression of genes known to be involved in L-R asymmetry in zebrafish wild-type and mutant embryos, in an effort to obtain a mathematical model of the establishment of LR asymmetry in the zebrafish embryo; and 4) identify novel genes upstream of Nodal, Notch and Pitx2 by means nf large-scale mutagenesis screens of transgenically engineered zebrafish embryos. Until now, very few genes have been implicated in the etiology of human laterality disorders. The study of the early stages of L-R determination and the identification of new genes involved in various vertebrate model organisms would undoubtedly shed new light on the genetic factors contributing to laterality defects in humans.