CARDIAC DEVELOPMENT DURING SITUS INVERSUS EMBRYOGENESIS

早期开发中的关键事件包括： 胚胎的心管，以及伴随的左-右 不对称虽然决定宇宙方向的分子事件 循环仍然不明，一些基因最近被 鉴定出在免疫前表现出左右不对称表达的细胞， 心脏循环的开始然而，这些不对称表达的基因 似乎并不负责最初的规范左右 不对称转基因小鼠的发育研究 （胚胎旋转反转）插入突变表明， inv基因对左右轴的最早期是必需的 规范.纯合子inv胚胎表现出一致的逆转， 形态和分子左右不对称性。为了开始 为了表征inv基因座，克隆了转基因整合位点， 表征了作图研究表明，转基因的整合 伴随着一个显着的基因组缺失和染色体内 小鼠4号染色体上的重复。单拷贝基因组序列侧翼 整合位点用于鉴定跨越 删除的区域。将该YAC纯化并用于显微注射， 产生转基因小鼠。六只将两只手臂 的YAC被识别。育种研究表明，G 0571 YAC 可以治愈inv突变表型。因此，此YAC包含编码 和inv基因的调节元件。为了进一步表征 该基因及其在建立左右极性中的作用， 具体目标是：1。为了识别 inv基因（通过外显子捕获和cDNA文库筛选）。2.以确定 候选inv mRNA的胚胎表达模式， 蛋白3.为了描述不对称表达的变化， 在YAC固化的中表达的基因（Nodal、lefty、Snail、flectin和dHAND） 胚胎、嵌合体胚胎和双突变体（iv/iv;inv/inv） 胚胎4.为了构建inv小基因并使用该小基因来 鉴定和表征蛋白质中的功能结构域。这些 实验应该提供关于最早阶段的分子细节， 左-右轴的规范和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.