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，其病因尚不清楚。一些人 最常见和最复杂的畸形是在左-右不对称(LR)异常的家族中发现的， 这表明许多先天缺陷可能是由偏侧紊乱引起的。最初的胚胎事件 确定LR体轴线，包括早期两侧对称性的破坏，以及随后的左侧 决定因素的表达，如结点和PITX2，现在已经很好地理解了。然而，基因和 在器官水平上，在LR发育的最后阶段涉及的形态发生事件在很大程度上仍然存在 未知。长期目标是确定LR不对称器官发生的机制。目标是 在这个应用中是为了识别产生LR不对称性(曲率)的分子和细胞过程 在一个单独的器官中，胃。初步分析确定了放射状细胞中的LR不对称性 发育中的胃的重排是其弯曲的驱动力。以识别最接近的 这一新的不对称形态发生程序的效应器--一种新的模式生物(Lep Obatrachus Laevis) 被雇佣了。这个物种的超大胚胎促进了一种基因发现的方法，这将是 在大多数模型中难以处理：从左半部与右半部解剖的组织的转录组图谱(RNAseq) 胚胎的胃。试点数据集包括具有LR不对称表达模式和功能的基因 在胃曲期间。中心假设是胃曲度由明显的左侧和左侧决定。 正确的调控网络，对控制放射状细胞重排的细胞事件进行不同的调制。 青蛙胚胎独特的实验适应性将被用来通过三个特定的 目的：1)生成正常和异常胃曲的分子特征。全面 将生成LR胃基因的时空分布，并在两种正常情况下进行比较 LR不对称性和实验诱导的LR轴缺陷。2)测定胃的细胞功能- 特定的LR基因。选定的基因将在功能丧失和功能获得分析中进行测试，以确定它们的 对发育期胃径向细胞重排的影响。3)确定监管层级 来控制胃曲度。实验扰动与时空剖析相结合将 揭示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 创新是因为它利用了独特物种的独特属性来解决关键问题之一 LR发育中未回答的问题：发育器官的最近机制是什么 成为LR非对称实体？这项拟议的研究意义重大，因为它将立即推进 通过定义基因、形态发生学对正常偏侧和偏侧相关出生缺陷的认识 在器官水平上控制LR不对称性出现的过程和调控逻辑。

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.