Notch Signaling and Left-Right Asymmetry Determination

描述(申请人提供)：在器官发生过程中，胚胎的内脏表现出明显的左右不对称(L-R)。建立这种不对称性的一系列事件包括：a)最初对称性的破坏，导致在胚胎组织器内和周围建立特定的基因表达模式；b)L-R的位置信息从组织器传递到侧板中胚层；以及c)L-R信息转移到器官原基，导致不对称形态发生的特定程序的制定。近年来，人们发现了参与这三个过程的各种基因。我们的初步结果表明，Notch通路作为一条主要的发育信号通路，可能在L-R的测定中发挥重要作用。这项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，以及它与其他与L-R测定有关的已知基因的相互作用。我们计划在活体内使用现代可视化技术来解决这些问题，同时对雏鸡、斑马鱼和老鼠等模型动物进行遗传干预。具体地说，我们建议：1)研究纤毛在斑马鱼、鸡和小鼠胚胎中打破L-R对称性中的作用及其转录因子Rfx家族对其的调节；2)对Notch途径的各个组成部分(鸡、小鼠和斑马鱼)进行功能得失实验，并建立其与小鼠纤毛运动和流动以及斑马鱼体内钙离子的关系；3)分析已知的参与L-R不对称性的基因在斑马鱼野生型和突变型胚胎中的时空表达，以期获得斑马鱼胚胎中LR不对称性建立的数学模型；4)通过对转基因斑马鱼胚胎进行大规模诱变筛选，发现Nodal、Notch和Pitx2上游的新基因。到目前为止，很少有基因与人类偏侧疾病的病因学有关。对L-R测定的早期阶段的研究，以及在各种脊椎动物模式生物中发现新的基因，无疑将为揭示导致人类偏侧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.