Biological Roles of Nodal Related Genes in Embryogenesis

描述（申请人提供）：该建议描述了一个系统的努力来理解脊椎动物胚胎中左右（L-R）不对称解剖学的发展，包括（a）控制“左”与“右”的分子，（B）它们在胚胎组织内和之间的表达，运动和解释，（c）在Nodal“左指定”信号通过期间组织的状态，以及细胞对该信号的生物学反应，（d）确保向左信号一次产生并处于正确水平的机制，以及（e）L-R形态发生如何在整个胚胎中整合。我们确定了该领域的主要差距，我们可以利用我们的专业知识和模型系统的力量来获得重大的新生物学和机械学见解。在非洲爪蟾中的发现将与包括哺乳动物在内的所有脊椎动物物种相关，非洲爪蟾是研究的绝佳选择，因为胚胎的大小，对操作的适应性以及易于获得的外部发育时间点。我们的研究包括理解Xnr 1的表达模式是如何转化为“反应活性图”内L侧板中胚层（LPM）在核内磷酸-Smad 2（P-Smad 2）的水平，以及如何这张地图与细胞和组织的不对称形态发生重排。与在早期胚胎发生中起作用的L-R特化的非保守机制相反，所有脊椎动物显示Nodal-Lefty-Pitx 2“左性特化”基因盒的保守L LPM表达。我们将：（1）通过Nodal/Xnr 1表达波解决L与R的特化机制;确定Xnrf表达的LPM能力图及其前向传播机制;测试分隔L和R隔室的腹侧中线。我们将Xnr 1表达转化为P-Smad 2组织反应图。(2)在细胞骨架和细胞/组织水平上，在其对Xnr 1信号传导的响应之前、期间和之后创建LPM架构图谱（LPM何时是上皮性的和极化的？组织层间空间是否允许Xnr 1/Xlefty更自由地移动？Xnr 1信号对不对称形态发生的细胞结构和组织反应是什么？它们是如何与我们的P-Smad 2地图精确关联的？）。除了填补一个主要的空白，并普遍有用的领域，这些结果是了解Xnr 1/Xlefty运动的限制的基础。(3)评估Xnr 1/Xlefty在LPM内和整个胚胎中的运动路线、方向和速度（及其稳定性），从而了解LPM中Xnr 1表达的动态变化，以及我们最近检测到的胚胎两侧之间的远程通信。单侧Nodal表达是导致内脏和心血管系统解剖不对称的主要L-R决定因素。失调的Nodal信号传导可能发生在癌症中。该提案对人类健康的意义很高：（1）它探索了控制复杂组织中细胞间信号传导的强度，持续时间和范围的因素。(2)脊椎动物之间相似的L-R解剖结构提高了人类先天性L-R异常的重要性，这是常见的（1/8500出生）。心血管系统的形成涉及协调的前后和L-R形态发生;缺陷导致严重的异常，而人类的位置缺陷与L-R途径基因（例如，nodal、lefty、Pitx 2、Cryptic和Zic 3。(3)节点信号传导可能是环境毒性剂的靶点。

DESCRIPTION (provided by applicant): This proposal describes a systematic effort to understand the development of left-right (L-R) asymmetric anatomy in vertebrate embryos in terms of (a) the molecules controlling "leftness" vs. 'rightness", (b) their expression, movement, and interpretation within and between embryonic tissues, (c) the state of tissues during passage of the Nodal "leftness-specifying" signal, and cell biological responses to this signal, (d) the mechanisms ensuring that a leftness signal is generated once and at the correct level, and (e) how L-R morphogenesis is integrated across the entire embryo. We identify major gaps in the field where we can leverage our expertise and the power of the model system to gain major new biological and mechanistic insights. Discoveries in Xenopus will be relevant to all vertebrate species including mammals, and Xenopus is an excellent choice for the studies because of the embryo's size, resilience to manipulation, and easily accessed time points of external development. Our studies include an understanding of how the Xnr1 expression pattern is transformed into a "response-activity map" within L lateral plate mesoderm (LPM) at the level of intranuclear phospho-Smad2 (P-Smad2), and how this map correlates with the cell and tissue rearrangements of asymmetric morphogenesis. In contrast to the non-conserved mechanisms of L-R specification that operate in earlier embryogenesis, all vertebrates show conserved L LPM expression of the Nodal-Lefty-Pitx2 "leftness-specifying" gene cassette. We will: (1) Address the mechanism of specification of L vs. R via the Nodal/Xnr1 expression wave; determine an LPM competence map for Xnrf expression and its mechanism of anterior propagation; test for a ventral midline separating the L & R compartments. We will convert Xnr1 expression into a P-Smad2 tissue-response map. (2) Create an LPM architecture atlas before, during and after its response to Xnr1 signaling, at the cytoskeletal and cell/tissue level (when is LPM epithelial and polarized? Does inter-tissue layer space allow freer movement of Xnr1/Xlefty? What are the cytoarchitectural and tissue responses to Xnr1 signaling for asymmetric morphogenesis? How do they precisely correlate with our P-Smad2 map?). As well as filling a major gap and being generally useful to the field, these results are the base for understanding constraints on Xnr1/Xlefty movement. (3) Assess the route, direction, and speed of movement of Xnr1/Xlefty (and their stabilities) within LPM and across the embryo, providing insight on the dynamic shift of Xnr1 expression in LPM, and our recently-detected long-range communication between both sides of the embryo. Unilateral Nodal expression is the prime L-R determinant leading to asymmetric anatomy of the viscera and cardiovascular system. Dysregulated Nodal signaling may occur in cancer. The proposal's significance to human health is high: (1) It explores the factors controlling strength, duration, and range of intercellular signaling in complex tissues. (2) The similar L-R anatomy amongst vertebrates raises the significance to human congenital L-R abnormalities, which are common (1 in 8500 births). Cardiovascular system formation involves coordinated anterior-posterior and L-R morphogenesis; defects cause severe anomalies, and human situs defects link to L-R pathway genes (e.g., nodal, lefty, Pitx2, Cryptic, and Zic3. (3) Nodal signaling may be a target for environmental toxic agents.