Induction of limb development in Xenopus

描述(申请人提供)：复杂形态发生过程中细胞行为的协调不仅涉及已被充分研究的生化途径，而且还涉及一个重要而有趣的生物物理系统：离子通道和泵产生的离子流动、跨膜梯度和电场的内源模式。这种生物电信号来自所有细胞(不仅仅是神经和肌肉)，并调节细胞的增殖、迁移、 和差异化。生物电梯度是发育中图案化的关键决定因素， 再生，以及对肿瘤的抵抗。我们的实验室率先开发了用于生物电信号分子水平表征的新技术，以及用于在体内合理调节生物电信号的遗传功能获得和丧失方法。在过去的十年里，我们发现了跨膜电位梯度在干细胞调节、左右模式形成、尾巴再生、眼睛诱导和脊椎动物前后极决定中的几个新角色。此外，我们还确定了将电压变化转换为转录反应的分子步骤，从而充实了生长和形态的生理控制与调节细胞行为的下游遗传成分之间的机制联系。现在强有力的数据表明，细胞生物电特性的调节有可能彻底改变面部、眼睛、脊髓和四肢的再生医学方法，以及癌症的检测和正常化。在这个R03项目中，我们计划克服当今该领域面临的最大障碍：以非常高的时空分辨率改变和监测体内跨膜电位的困难。初步数据表明，特定离子通道的错误表达可以在青蛙中诱导出完整、完整、有功能的前肢；这是理解这种新型发育信号的绝佳机会。阻碍社区完全破解生物电密码的是一个概念验证应用程序，它允许在VIV中调节生物电梯度的空间模式。我们的基本想法是将光遗传学技术，迄今为止仅应用于可兴奋细胞(神经和肌肉)，应用于所有类型的细胞以进行发育调节。我们建议：1)生产表达电压荧光报告蛋白的转基因青蛙，促进体内任何感兴趣器官/组织的生物电学性质的研究；2)生产表达光遗传结构(通道视紫红质和古紫质)的转基因青蛙，通过图案光暴露的方式允许体内感兴趣细胞的去极化或超极化；以及3)使用这项技术通过对肢体诱导和形态发生特有的电压梯度的机械理解来诱导青蛙的异位肢体。通过创造强大和广泛适用的资源，并将它们应用于具有根本重要性和生物医学相关性的问题(肢体损伤)，我们的工作将为推进光遗传学、脊椎动物肢体再生医学领域以及理解发育中的生物物理因素提供关键的原则证明。

DESCRIPTION (provided by applicant): The orchestration of cell behavior during complex morphogenesis involves not only the well-studied biochemical pathways but also an important and fascinating biophysical system: the endogenous patterns of ion flows, transmembrane gradients, and electric fields produced by ion channels and pumps. Such bioelectrical signals are produced from all cells (not just nerve and muscle) and regulate cell proliferation, migration, and differentiation. Bioelectrical gradients are crucial determinants of patterning in development, regeneration, and resistance to neoplasm. Our laboratory has pioneered the development of novel techniques for the molecular-level characterization of bioelectrical signals and genetic gain- and loss-of-function approaches for their rational modulation in vivo. Over the last decade, we have uncovered several new roles for transmembrane potential gradients in stem cell regulation, left-right patterning, tail regeneration, eye induction, and planarian anterior-posterir polarity determination. Moreover, we have identified the molecular steps that transduce voltage changes into transcriptional responses, thus fleshing out the mechanistic links between physiological controls of growth and form and downstream genetic components that regulate cell behavior. Strong data now indicate that modulation of bioelectric properties of cells has the potential to revolutionize approaches in regenerative medicine of the face, eye, spinal cord, and limb, as well as detection and normalization of cancer. In this R03 project, we propose to surmount the biggest barrier facing this field today: the difficulty of changing and monitoring transmembrane potential in vivo with very high spatio-temporal resolution. Preliminary data indicate that misexpression of specific ion channels can induce whole, complete, functional forelimbs in frogs; this is a superb opportunity to understand this new type of developmental signaling. What is holding back the community from fully cracking the bioelectric code is a proof-of-concept application that allows spatial patterns of bioelectric gradients to be regulated in viv. Our basic idea is to move the technology of optogenetics, heretofore applied only to excitable cells (nerve and muscle), to all cell types for developmental regulation. We propose to: 1) produce transgenic frogs expressing voltage fluorescent reporter proteins, facilitating the investigation of bioelectrical properties in any organ/tissue of interest in vivo; 2) produce transgenic frogs expressing optogenetic constructs (Channelrhodopsin and Archeorhodopsin), allowing depolarization or hyperpolarization of cells of interest in vivo by means of patterned light exposure; and 3) use this technology to induce ectopic limbs in froglets by developing a mechanistic understanding of the voltage gradients specific to limb induction and morphogenesis. By creating powerful and widely-applicable resources and applying them to a problem of fundamental importance and biomedical relevance (limb damage), our work will provide a key proof-of-principle to stepping stone to advance the field of optogenetics, the regenerative medicine of the vertebrate limb, and the understanding of biophysical factors in development.