Bioelectrical controls of left-right asymmetry

描述(申请人提供)：这项研究的目标是在分子水平上阐明导致脊椎动物身体一致的左-右不对称的新机制。我们将测试当数千个细胞存在时，离子通量机制在建立LR不对称性中所起的作用，并确定细胞骨架在离子转运体的不对称分布中所起的作用。青蛙最近的工作表明，离子通量机制参与了早期的LR图案化。具体地说，在前两次细胞分裂中，四个离子转运体不对称地分布在胚胎的中线上，是正确的LR不对称所必需的。这些数据清楚地描绘了离子转运蛋白的亚细胞定位和下游不对称图案化之间的联系。然而，这一机制依赖于这样一个事实，即在非洲爪哇，第一个大卵裂球的卵裂面通常与整个胚胎的预期中线重合。令人惊讶的是，当在囊胚晚期(~1000个细胞)的青蛙胚胎中诱导出第二个组织者时，异位双胞胎具有正常的LR不对称。与早期的大的卵裂球不同，在这个阶段，单个细胞不能通过细胞内定位将成分分布在胚胎中线上；尽管如此，可以将正确的侧向施加到晚期囊胚的小细胞上--这一背景类似于哺乳动物LR的启动。因此，当存在数以千计的小细胞时，必须有某种机制来正确地对胚胎进行模式识别。我们将测试当存在具有数千个细胞的组织时，在早期青蛙胚胎中操作的机制是否被重复使用。研究的目的有两个：(1)确定在胚胎发育后期是否需要离子通量机制来定向LR轴的正常不对称。(2)了解细胞骨架运动蛋白如何在早期和晚期胚胎中建立生物电极性。在第一个目标中，我们将在一个阶段诱导异位双胞胎，此时青蛙胚胎中存在数千个细胞，并且没有发生大规模的中线定向卵裂。然后，我们将测试四种不同的离子通量机制，这些机制与第一次卵裂期间LR轴的建立有关，以确定是否有任何机制在随后的阶段也发挥作用。在第二个目标中，我们将在早期和晚期干扰单个马达蛋白的功能，并评估由此产生的对离子转运体定位不对称性的影响，以阐明早期和晚期LR启动时的细胞内转运。相关性：偏侧性是胚胎发育的一个重要方面，尤其是心血管系统、内脏器官和大脑的形态发生。了解不对称性是如何建立的，将有助于解决出生缺陷问题，并推动心脏和其他器官的再生医学。

DESCRIPTION (provided by applicant): The goal of this research is to elucidate, at the molecular level, novel mechanisms responsible for generating the consistent left-right (LR) asymmetry of the vertebrate body. We will test what role ion flux mechanisms play in establishing LR asymmetry when thousands of cells are present and identify what role the cytoskeleton plays in the asymmetric distribution of ion transporters. Recent work from the frog indicates that ion flux mechanisms are involved in early LR patterning. Specifically, four ion transporters are asymmetrically distributed across the embryo's midline during the first two cell cleavages and are required for proper LR asymmetry. These data present a clear picture of steps linking subcellular localization of ion transporters and downstream asymmetric patterning. However, this mechanism relies on the fact that in Xenopus, the cleavage plane of the first large blastomeres normally coincides with the prospective midline of the whole embryo. Surprisingly, when a second organizer is induced in frog embryos during the late blastula stage (~1000 cells), the ectopic twin has normal LR asymmetry. Unlike the large early blastomeres, individual cells at this stage cannot distribute components across the embryonic midline by intracellular localization; nonetheless correct laterally can be imposed upon the small cells of the late blastula - a context similar to mammalian LR initiation. Therefore, some mechanism must properly pattern embryos when thousands of small cells are present. We will test whether the mechanisms that operate in early frog embryos are re-used when tissues with thousands of cells are present. Two aims will be addressed: (1) To determine whether ion flux mechanisms are required to orient the normal asymmetry of the LR axis during late stage embryogenesis. (2) To understand how cytoskeletal motor proteins establish bioelectrical polarity in early and late embryos. In the first aim, we will induce ectopic twins at a stage when thousands of cells are present and large-scale, midline-oriented cleavages are not occurring in the frog embryo. We will then test four different ion flux mechanisms that have been implicated in the establishment of the LR axis during the first cleavages to determine whether any also function in later stages. In the second aim, we will disrupt individual motor proteins' function at early and late stages and assess the resulting effect on asymmetry of ion transporter localization, to shed light on intracellular transport on early and late LR initiation. Relevance: Laterality is a crucial aspect of embryonic development, particularly of morphogenesis of the cardiovascular system, visceral organs, and the brain. Understanding how asymmetry is established will help address birth defects and advance regenerative medicine of the heart and other organs.