Seq-ing the etiology of birth defects in a new frog model, Lepidobatrachus laevis

描述（由申请人提供）：大多数人类出生缺陷的原因是未知的。心脏和消化道的结构缺陷经常被发现与其他器官系统的异常左右不对称有关，这表明这种畸形可能是由扰动的偏侧性造成的，但在单个器官内形成不对称的发育过程仍然难以捉摸。长期目标是确定控制解剖学左右不对称发展的形态发生机制。这种探索性的R21应用的目的是获得一个全面的看法左右不对称的分子差异内的胚胎心脏和肠道管，因为它们经历不对称的“循环”，一个基本的器官发生事件，定向最关键的解剖不对称。为了实现这一目标，独特的胚胎学特征的一种新的模式两栖动物，Lepidobatrachus laevis，将开发和利用。Lepidobatrachus有大量的胚胎，有助于在循环过程中精确切除早期心脏和肠道的左半部分和右半部分，从而在不对称形态发生的关键阶段实现迄今为止不可行的左右转录组分析方法。中心假设是，识别转录本之间的差异表达的对侧的一半循环器官将确定控制左右不对称形态发生的新分子。该假设将通过两个特定的目的进行测试：1）鉴定在循环心脏和肠管的左侧和右侧之间差异表达的转录物;和2）验证左或右富集转录物对于不对称器官形态发生的生物学相关性。根据目标1，RNAseq方法（由PI已经构建的Lepidobatrachus转录组草案支持）将用于完成全基因组表达分析，这将揭示与关键解剖不对称形成相关的单侧富集转录本。在目标2下，已证实的将外源性试剂精确靶向两栖动物发育器官左侧或右侧的能力，以及由相当大的Lepidobatrachus提供的发育器官不对称性的前所未有的亚细胞分辨率，将用于验证选择的单侧富集的转录物在不对称哺乳动物中的体内功能。 形态发生该方法是创新的，因为它利用独特的非模型生物体的独特属性来理解左右发育领域中的关键未回答的问题之一：发育器官获得关键的左右不对称解剖特征的机制是什么？这项拟议的研究意义重大，因为它有望通过识别新的分子类别和新的细胞过程来立即加速我们对一些最常见的出生缺陷病因的理解，这些分子和细胞过程塑造了心脏和肠道的基本左右不对称性。

DESCRIPTION (provided by applicant): The causes of most human birth defects are unknown. Structural defects in the heart and digestive tract are frequently found in association with abnormal left-right asymmetries in other organ systems, suggesting that such deformities may result from perturbed laterality, yet the developmental processes that form asymmetries within individual organs remain elusive. The long term goal is to determine the morphogenetic mechanisms that control the development of anatomical left-right asymmetry. The objective of this exploratory R21 application is to gain a comprehensive view of the left-right asymmetric molecular differences within the embryonic heart and gut tubes as they undergo asymmetric "looping", a fundamental organogenesis event that orients the most crucial anatomical asymmetries. To accomplish this objective, the unique embryological features of a novel model amphibian, Lepidobatrachus laevis, will be developed and exploited. Lepidobatrachus has massive embryos that facilitate precise excision of the left and right halves of the early heart an gut during looping, enabling the heretofore infeasible approach of left-right transcriptome profiling during a key phase of asymmetric morphogenesis. The central hypothesis is that identifying transcripts that are differentially expressed between the contralateral halves of looping organs will identify new molecules that control left-right asymmetric morphogenesis. This hypothesis will be tested via two specific aims: 1) Identify transcripts that are differentialy expressed between the left and right sides of the looping heart and gut tubes; and 2) Validate the biological relevance of left- or right-enriched transcripts for asymmetric organ morphogenesis. Under Aim 1, an RNAseq approach (supported by a draft Lepidobatrachus transcriptome already constructed by the PI) will be used to complete genome-wide expression analyses that will reveal unilaterally-enriched transcripts associated with the formation of key anatomical asymmetries. Under Aim 2, the proven ability to precisely target exogenous reagents to the left or right side of developing organs in amphibians, and the unprecedented subcellular resolution of developing organ asymmetries provided by the sizeable Lepidobatrachus, will be used to authenticate the in vivo function of select unilaterally-enriched transcripts in asymmetric morphogenesis. The approach is innovative because it takes advantage of the distinctive attributes of a unique non-model organism to understand one of the key unanswered questions in the field of left-right development: what are the mechanism(s) by which developing organs acquire critical left- right asymmetric anatomical features? The proposed research is significant because it is expected to immediately accelerate our understanding of the etiology of some of the most common birth defects by identifying new classes of molecules, and new cellular processes, which shape the fundamental left-right asymmetry of the heart and gut.