Bioelectrical Controls of Morphogenesis

描述(申请人提供)：脊椎动物身体平面一致的左右不对称是一个令人着迷的发育和进化生物学难题，对生物医学具有深远的意义。每6000个足月出生的婴儿中就有1个会受到偏侧缺陷的影响，分子细节对于理解、检测、预防和修复与LR模式错误相关的出生缺陷至关重要。该领域目前处于混乱状态，缺乏概念模型，可以将小鼠和斑马鱼胚胎的LR模式发现的机制与在非洲爪哇中发现的更早的机制联系起来。我们最近的工作发现了全新的离子流依赖事件，这些事件在早期的LR模式中发挥作用，并在青蛙、雏鸟和斑马鱼中保守。然而，现有的模型不能解释在鸡和哺乳动物的身体平面中，电压梯度如何相对于LR轴可靠地定向。激动人心的数据表明，细胞和组织中的离子通道和泵依赖的电流产生的内源性电压和pH梯度调节着胚胎发育和再生中的一些重要的形态发生事件。最近集中于基因转录网络和分泌信号因子的工作在很大程度上忽视了这些引人入胜的表观遗传生物物理现象。我们的实验室结合了强大的现代分子和细胞生物学、生物物理学、生理学和计算机建模工具；因此，我们的工作不仅将有助于理解左右模式，还将揭示内源离子流动如何控制细胞行为的新的分子细节。我们的方法和试剂为解决主要难题提供了一个宝贵而独特的机会，即在与胚胎中线一致的大卵裂球中建立不对称的细胞质事件在存在许多小细胞的其他动物(如哺乳动物)中如何发挥作用。我们将通过解决以下主要悬而未决的问题来解决这个难题：(1)确定3个特定的离子转运体如何参与自主组织者的焦点将LR不对称强加于大细胞场，以及(2)表征极性和细胞骨架蛋白在控制细胞内离子通量的定位和/或行为方面的相互作用。总之，这些实验利用了一套强大的方法，在一个既适用于分子遗传学又适用于功能生物物理学的模型系统(非洲爪哇)中。通过利用我们已获得的令人兴奋的初步和已发表的数据，我们的工作将揭示可用于洞察形态发生的细胞和进化生物学的新机制，将该领域与主流分子发育生物学相结合，并最终将作为针对出生缺陷、再生和肿瘤生物学许多领域的新方法的基础。

DESCRIPTION (provided by applicant): The consistent left-right (LR) asymmetry of the vertebrate bodyplan is a fascinating puzzle of developmental and evolutionary biology, and has profound implications for biomedicine. Laterality defects affect more than 1 in 6000 babies born to term, and the molecular details are crucial to understanding, detection, prevention, and repair of birth defects related to errors of LR patterning. The field is currently in disarray, lacking conceptual models that can link mechanisms uncovered for the LR patterning of mouse and zebrafish embryos to the much earlier mechanisms that have been discovered in Xenopus. Our recent work has identified completely novel ion flow-dependent events that function in early LR patterning and are conserved to frog, chick, and zebrafish. However, the existing models do not explain how voltage gradients can be reliably oriented with respect to the LR axis in chick and mammalian body-plans. Exciting data indicate that endogenous voltage and pH gradients produced by ion channel and pump-dependent current flows in cells and tissues regulate a number of important morphogenetic events in embryonic development and regeneration. The recent explosion of work focused on gene transcription networks and secreted signaling factors have largely neglected these fascinating epigenetic biophysical phenomena. Our lab combines the powerful modern tools of molecular and cell biology, biophysics, physiology, and computer modeling; thus, our work will not only contribute to understanding left-right patterning but also reveal new molecular details of how endogenous ion flows control cell behavior. Our approaches and reagents present a valuable and unique opportunity to resolve the major puzzle of how early, cytoplasmic events that set up asymmetry in large blastomeres aligned with the embryo's midline may function in other animals (such as mammals) where many small cells exist instead. We will resolve this puzzle by addressing the major outstanding questions through (1) determining how 3 specific ion transporters are involved in the imposition of LR asymmetry onto large cell fields by foci of autonomous "organizers", and (2) characterizing the interaction of polarity and cytoskeleton proteins in controlling the localization and/or behavior of ion fluxes in cells. Together, these experiments take advantage of a powerful set of approaches in a model system (Xenopus) amenable to both molecular genetics and functional biophysics. By capitalizing on the exciting base of preliminary and published data that we have obtained, our work will reveal novel mechanisms that can be used to gain insight into the cell and evolutionary biology of morphogenesis, will integrate this field with mainstream molecular developmental biology, and will ultimately serve as the basis for novel approaches targeting many areas of birth defects, regeneration, and tumor biology.