Induction of limb development in Xenopus

描述（由申请人提供）：复杂形态发生期间细胞行为的协调不仅涉及充分研究的生化途径，而且涉及重要且迷人的生物物理系统：离子流的内源性模式、跨膜梯度以及由离子通道和泵产生的电场。这种生物电信号由所有细胞（不仅仅是神经和肌肉）产生，并调节细胞增殖，迁移， 和差异化。生物电梯度是发育模式的关键决定因素， 再生和抗肿瘤。我们的实验室率先开发了生物电信号分子水平表征的新技术，以及用于体内合理调制的遗传获得和功能丧失方法。在过去的十年中，我们已经发现了几个新的作用，跨膜电位梯度在干细胞调控，左右图案，尾巴再生，眼诱导，和Planarian前后极性的决定。此外，我们已经确定了将电压变化转化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。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、脊髓和肢体再生医学的方法，以及癌症的检测和正常化。在这个R 03项目中，我们提出要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诱导青蛙的完整，完整，功能性前肢;这是了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，该应用程序允许在体内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，迄今为止只应用于兴奋细胞（神经和肌肉），用于所有细胞类型的发育调控。我们建议：1）产生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的器官/组织中的生物电特性的研究;（视紫红质和古视紫红质），允许通过图案化光暴露在体内对感兴趣的细胞进行去极化或超极化;和3）通过发展对肢体诱导和形态发生的特定电压梯度的机械理解，使用该技术在小蛙中诱导异位肢体。通过创建强大且广泛适用的资源并将其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领域，脊椎动物肢体再生医学以及对发育中生物物理因素的理解提供关键的原理证明。

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.