Ahead of the Curve: Mechanisms of Left-Right Asymmetric Stomach Morphogenesis

项目摘要 出生缺陷是婴儿死亡的主要原因，但在大多数情况下，其病因不明。一些 最常见和最复杂的畸形是在具有异常左右（LR）不对称的家族中发现的， 这表明许多先天性缺陷可能是由扰动的偏侧性引起的。最初的胚胎活动， 确定LR体轴，包括双侧对称性的早期破坏，以及随后的左侧 决定因素的表达，如节点和Pitx 2，现在已经很好地理解。然而，基因和 在器官水平上，LR发育的最后阶段所涉及的形态发生事件， 未知长期的目标是确定LR不对称器官发生的机制。客观 在这个应用程序是确定分子和细胞的过程中，产生LR不对称性（曲率） 在一个单独的器官里，胃。初步分析确定了放射状细胞中的LR不对称性 在发育中的胃的重排作为其弯曲的驱动力。为了确认 一种新的模式生物（Lepidobatrachus laevis） 被雇用了。这个物种的超大胚胎促进了基因发现的方法， 在大多数模型中难以处理：从左半部分与右半部分解剖的组织的转录组谱分析（RNASeq） 胚胎胃。先导数据集包括具有LR不对称表达模式和功能的基因 在胃弯曲的时候。中心假设是胃弯曲是由不同的左， 正确的调节网络，差异调节控制放射状细胞重排的细胞事件。 青蛙胚胎独特的实验顺从性将被用来通过三个特定的实验来验证这一假设。 目的：1）产生正常和异常胃弯曲的分子特征。全面 LR胃基因的时空分布将在两个正常的背景下产生和比较， LR不对称性和实验诱导的LR轴缺陷。2)确定胃的细胞功能- 特定LR基因。选择基因将在功能丧失和获得测定中进行测试，以确定它们的功能。 影响发育中胃的放射状细胞重排。3)确定监管层级 控制胃弯曲的神经系统实验扰动与时空分布相结合， 揭示了控制不对称形态发生的核心基因调控相互作用。总体方法是 创新，因为它利用了独特物种的独特属性来解决一个关键问题， LR发育中未回答的问题：发育器官的近似机制是什么？ 成为LR非对称实体？这项拟议中的研究意义重大，因为它将立即推动 通过定义基因、形态发生学，我们对正常偏侧性和与偏侧性相关的出生缺陷的理解 过程和监管逻辑，管理LR不对称的出现在器官水平。

Project Summary Birth defects are a leading cause of infant mortality, yet in most cases, their etiology is unknown. Some of the most common and complex malformations are found in families with abnormal left-right (LR) asymmetry, suggesting that many congenital defects may result from perturbed laterality. The initial embryonic events that determine the LR body axis, including the early breaking of bilateral symmetry, and subsequent left-sided expression of determinants such as nodal and Pitx2, are now well understood. However, the genetic and morphogenetic events involved in the final phases of LR development, at the organ level, remain largely unknown. The long term goal is to ascertain the mechanisms of LR asymmetric organogenesis. The objective in this application is to identify the molecular and cellular processes that generate LR asymmetry (curvature) within an individual organ, the stomach. Preliminary analyses identified LR asymmetries in radial cell rearrangements in the developing stomach as the driving force for its curvature. To identify the proximate effectors of this novel asymmetric morphogenetic program, a new model organism (Lepidobatrachus laevis) was employed. The extra-large embryos of this species facilitated a gene-discovery approach that would be intractable in most models: transcriptome profiling (RNASeq) of tissues dissected from left vs. right halves of the embryonic stomach. Pilot datasets include genes with LR asymmetric expression patterns and functions during stomach curvature. The central hypothesis is that stomach curvature is determined by distinct left and right regulatory networks which differentially modulate the cellular events controlling radial cell rearrangement. The unique experimental amenability of frog embryos will be used to test this hypothesis via three specific aims: 1) Generate molecular signatures of normal and abnormal stomach curvature. Comprehensive spatiotemporal profiles of LR stomach genes will be generated and compared in the context of both normal LR asymmetry and experimentally-induced LR axis defects. 2) Determine the cellular function of stomach- specific LR genes. Select genes will be tested in loss- and gain-of-function assays to determine their influence on radial cell rearrangement in the developing stomach. 3) Determine the regulatory hierarchy that controls stomach curvature. Experimental perturbations combined with spatiotemporal profiling will reveal core gene regulatory interactions governing asymmetric morphogenesis. The overall approach is innovative because it takes advantage of distinctive attributes of a unique species to address one of the key unanswered questions in LR development: what are the proximate mechanisms by which developing organs become LR asymmetric entities? The proposed research is significant because it will immediately advance our understanding of normal laterality and laterality-related birth defects by defining the genes, morphogenetic processes and regulatory logic that govern the emergence of LR asymmetry at the organ level.