Bioelectrical controls of left-right asymmetry

描述（由申请人提供）：本研究的目的是在分子水平上阐明导致脊椎动物身体一致性左右（LR）不对称的新机制。我们将测试离子通量机制在建立LR不对称性时发挥的作用，数千个细胞存在，并确定细胞骨架在离子转运蛋白的不对称分布中发挥的作用。青蛙最近的工作表明，离子通量机制参与了早期LR模式。具体而言，四个离子转运蛋白在前两个细胞分裂期间不对称地分布在胚胎的中线上，并且是适当的LR不对称所需的。这些数据提供了连接离子转运蛋白亚细胞定位和下游不对称模式的步骤的清晰图像。然而，这种机制依赖于这样一个事实，即在非洲爪蟾中，第一个大卵裂球的卵裂平面通常与整个胚胎的预期中线重合。令人惊讶的是，当第二个组织者在囊胚后期（约1000个细胞）的青蛙胚胎中诱导时，异位双胞胎具有正常的LR不对称性。与大的早期卵裂球不同，在这个阶段的单个细胞不能通过细胞内定位将组分分布在胚胎中线上;然而，可以将正确的横向施加在晚期囊胚的小细胞上-这与哺乳动物LR启动类似。因此，当存在数千个小细胞时，某些机制必须正确地使胚胎定型。我们将测试当存在具有数千个细胞的组织时，在早期青蛙胚胎中运作的机制是否被重新使用。将处理两个目标：（1）确定在胚胎发生后期是否需要离子流机制来定向LR轴的正常不对称性。(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，我们将在青蛙胚胎中存在数千个细胞并且没有发生大规模中线定向分裂的阶段诱导异位双胞胎。然后，我们将测试四种不同的离子通量机制，这些机制在第一次分裂期间与LR轴的建立有关，以确定是否有任何机制在以后的阶段也起作用。在第二个目标中，我们将在早期和晚期破坏单个马达蛋白的功能，并评估对离子转运蛋白定位不对称性的影响，以阐明早期和晚期LR启动的细胞内转运。相关性：偏侧性是胚胎发育的一个重要方面，特别是心血管系统、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，并促进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.