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

描述(申请人提供)：大多数人类出生缺陷的原因尚不清楚。心脏和消化道的结构缺陷经常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引起的，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左-右不对称发展的形态发生机制。R21的这一探索性应用的目的是全面了解胚胎心脏和肠管在经历不对称“循环”时的左右不对称分子差异，不对称“环”是一种基本的器官发生事件，定位最关键的解剖不对称。为了实现这一目标，将开发和利用一种新的模式两栖动物--鳞翅目两栖动物独特的胚胎学特征。鳞翅目昆虫拥有巨大的胚胎，有助于在循环过程中精确切除早期心脏的左右半部分和肠道，使迄今无法在不对称形态发生的关键阶段进行左右转录组分析的方法成为可能。中心假设是，识别在环状器官的对侧一半之间差异表达的转录本，将识别控制左右不对称形态发生的新分子。这一假说将通过两个特定的目的来检验：1)确定在心脏和肠管的左右两侧差异表达的转录本；2)验证左或右浓缩转录本与不对称器官形态发生的生物学相关性。在目标1下，将使用RNAseq方法(由PI已经构建的LEpobatrachus转录组草案支持)完成全基因组表达分析，揭示与关键解剖不对称的形成有关的单方面浓缩的转录本。在目标2下，已证实的将外源试剂精确地靶向两栖动物发育器官的左侧或右侧的能力，以及由大型鳞毛虫提供的对发育器官不对称性的史无前例的亚细胞分辨率，将被用来验证选择的单边不对称富集型转录本的体内功能。 形态发生。这种方法之所以具有创新性，是因为它利用了一种独特的非模式生物的独特属性来理解左右发育领域中一个关键的悬而未决的问题：发育中的器官获得关键的左右不对称解剖特征的机制(S)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，立即加快我们对一些最常见出生缺陷的病因学的理解，这些分子和新的细胞过程塑造了心脏和肠道的基本左右不对称。

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.