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产生脊椎动物身体一致的左右（LR）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。最近对青蛙的研究表明，离子通量机制参与了早期的LR模式。具体来说，在前两次细胞分裂期间，四个离子转运体不对称地分布在胚胎中线，这是适当的LR不对称所必需的。这些数据提供了连接亚细胞定位离子转运体和下游不对称模式的步骤的清晰图片。然而，这种机制依赖于这样一个事实，即非洲爪蟾的第一个大卵裂球的卵裂面通常与整个胚胎的未来中线一致。令人惊讶的是，当在囊胚后期（约1000个细胞）的青蛙胚胎中诱导第二个组织者时，异位双胞胎具有正常的LR不对称性。与早期的大卵裂球不同，这一阶段的单个细胞不能通过细胞内定位将成分分布到胚胎中线；尽管如此，侧向正确可以强加于囊胚后期的小细胞-类似于哺乳动物LR起始的背景。因此，当数千个小细胞存在时，某些机制必须正确地塑造胚胎。我们将测试在具有数千个细胞的组织存在时，在早期青蛙胚胎中运作的机制是否可以重复使用。两个目标将被解决：(1)确定是否需要离子通量机制来定向晚期胚胎发育中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，我们将在数千个细胞存在的阶段诱导异位双胞胎，并且在青蛙胚胎中没有发生大规模的中线定向分裂。然后，我们将测试四种不同的离子通量机制，这些机制在第一次裂解过程中与LR轴的建立有关，以确定是否有任何离子通量机制在后期阶段也起作用。在第二个目标中，我们将在早期和晚期破坏单个运动蛋白的功能，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起始的细胞内转运。相关性：侧边性是胚胎发育的一个重要方面，特别是在心血管系统、内脏器官和大脑的形态发生中。了解不对称是如何形成的将有助于解决出生缺陷和推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.