CARDIAC DEVELOPMENT DURING SITUS INVERSUS EMBRYOGENESIS

早期开发中的关键事件包括启动循环 胚胎心管，以及伴随而来的左右关系的建立 不对称。尽管决定分子运动方向的分子事件 环路仍未确定，一些基因最近已被 属性之前显示的左右不对称表达式 出现心脏跳动。然而，这些不对称表达的基因确实 似乎没有对最初的左右规范负责 不对称。携带INV的转基因小鼠的发育研究 (胚胎旋转倒置)插入突变表明 Inv基因在左右轴的早期发育中是必不可少的 规格。纯合子inv胚胎表现出一致的逆转 形态和分子左右不对称。为了开始…… 对inv基因座进行鉴定，克隆了转基因整合位点并 特色化的。图谱研究表明，转基因的整合 伴随着显著的基因组缺失和染色体内染色体 小鼠4号染色体上的复制.单拷贝基因组序列侧翼 整合站点用于识别跨越的YAC克隆(GO571 已删除的区域。该YAC被提纯并用于微量注射 产生转基因小鼠。六只整合了两只手臂的创始小鼠 被确认为YAC的成员。育种研究表明，G0571YAC 能治愈inv突变表型。因此，这个YAC包含编码 和inv基因的调控元件。为了进一步刻画 该基因及其在建立左右两极中的作用如下 提出了具体的目标：1.确定编码序列 Inv基因(外显子捕获和cDNA文库筛选)。2.确定 候选inv基因表达的胚胎模式 蛋白。3.表征不对称基因表达的变化 YAC腌制中表达的基因(Nodal、Lefty、Snail、Flectin和Dhand) 胚胎，嵌合胚胎和双突变(iv/iv；inv/inv) 胚胎。4.构建inv微型基因，并利用这些微型基因 鉴定和表征蛋白质中的功能结构域。这些 实验应该提供最早的步骤的分子细节 左右轴规范与心脏形态发生的控制。

Critical events in early development include the initiation of looping in the embryonic heart tube, and the concomitant establishment of left-right asymmetry. Although the molecular events that determine the directions of looping remain unidentified, a number of genes have recently been identified that exhibit left-right asymmetric expression prior to the onset of heart looping. However, these asymmetrically expressed genes do not appear to be responsible for the initial specification of left-right asymmetry. Developmental studies of transgenic mice that carry the inv (inversion of embryonic rotation) insertional mutation suggest that the inv gene is essential for the earliest stages of left-right axis specification. Homozygous inv embryos exhibit a consistent reversal of morphological and molecular left-right asymmetry. In order to begin to characterize the inv locus, the transgenic integration site was cloned and characterized. Mapping studies revealed that integration of the transgene was accompanied by a significant genomic deletion and an intrachromosomal duplication on mouse chromosome 4. Single-copy genomic sequences flanking the integration site was used to identify a YAC clone (GO571) that spanned the deleted region. This YAC was purified and used for microinjection to generate transgenic mice. Six founder mice that had integrated both arms of the YAC were identified. Breeding studies revealed that the G0571 YAC can cure the inv mutant phenotype. Therefore this YAC contains the coding and regulatory elements of the inv gene. In order to further characterize this gene and its role in establishing left-right polarity, the following specific aims are proposed: 1. To identify the coding sequences for the inv gene (by exon trapping and cDNA library screening). 2. To determine the embryonic patterns of expression of the candidate inv mRNA and protein. 3. To characterize changes in expression of asymmetrically expressed genes (Nodal, lefty, Snail, flectin and dHAND) in YAC-cured embryos, in chimeric embryos, and in double mutant (iv/iv;inv/inv) embryos. 4. To construct an inv minigene and to use the minigenes to identify and characterize the functional domains in the protein. These experiments should provide molecular details about the earliest steps in left-right axis specification and the control of cardiac morphogenesis.