Biophysical controls of vertebrate organ regeneration

描述（由申请人提供）：因损伤或疾病而丧失的组织和器官的再生是生物医学的一个关键目标。在临床环境中诱导再生将需要在能够再生的动物中操作的相关模式信号的分子解剖。该领域一直以关注化学信号为主，并准备采用新的方法来解决这个问题。我们的实验室将功能生理学与分子遗传学相结合，以了解图案的新型生物物理控制，并使用它们来控制组织生长。当被切断时，爪蟾的尾巴会形成一个再生芽，迅速产生一个完整的原始尾巴的复制品，包括神经，血管和肌肉。使用这个强大的脊椎动物系统，我们发现内源性离子通量和膜电压梯度在再生中起着至关重要的作用。我们的药物筛选涉及再生所需的H+泵、K+通道和Na+通道，但不是伤口愈合或初级尾生长所需的;这些转运蛋白的活性在芽中建立了一个中度去极化区，这对再生至关重要。我们使用突变体转运蛋白构建体来抑制或拯救再生，证明H+通量是诱导再生所必需的且足够的。这些生物物理事件的上游功能和控制：已知的再生标志物的表达，在芽中的细胞增殖的上调，和轴突图案。我们建议开始了解离子通量在再生中的作用，通过表征：（1）芽基电流的时间过程和性质，（2）牵连的产电基因的表达，（3）下游步骤连接膜电压的分子和再生过程中的形态发生事件。我们的数据提供了第一个诱导再生的离子流的分子调制，和拟议的工作将回答最重要的开放问题，在这个新的领域。这项建议包含了高度的新奇，因为它集中在一个范例，以前没有解决使用分子遗传工具：再生的电控制。这是高回报的，因为它将为复杂的脊椎动物结构（包括脊髓）的再生提供一套新的控制参数。这将对理解基本的形态发生机制以及为有前途的医学方法建立基础以增强或诱导非再生组织的再生具有重要意义。再生组织和器官的能力对于损伤、衰老、感染或手术切除癌症的医疗管理至关重要。我们的工作将提供一种全新的模式，有一天可能使人类再生重要的组织和器官（包括肌肉和脊髓）。

DESCRIPTION (provided by applicant): The regeneration of tissues and organs lost to injury or disease is a key goal of biomedicine. Induction of regeneration in clinical contexts will require a molecular dissection of the relevant patterning signals operating in animals that are able to regenerate. This field has been dominated by a focus on chemical signals and is ready for fresh approaches to the problem. Our lab merges functional physiology with molecular genetics to understand novel biophysical controls of patterning and use them to control tissue growth. When amputated, the Xenopus tail forms a regeneration bud that rapidly produces a perfect duplicate of the original tail, including nerves, blood vessels, and muscle. Using this powerful vertebrate system, we discovered that endogenous ion fluxes and membrane voltage gradients play a crucial role in regeneration. Our drug screen implicated a H+ pump, a K+ channel, and a Na+ channel as required for regeneration but not for wound healing or primary tail growth; the activity of these transporters establishes a moderate zone of depolarization in the bud that is crucial for regeneration. We used mutant transporter constructs to inhibit or rescue regeneration, demonstrating that H+ flux is necessary and sufficient for inducing regeneration. These biophysical events function upstream of and control: known regeneration marker expression, up-regulation of cell proliferation in the bud, and axon patterning. We propose to begin to understand the role of ion flux in regeneration by characterizing: (1) the time-course and properties of blastema currents, (2) the expression of implicated electrogenic genes, (3) the downstream steps linking membrane voltage to molecular and morphogenetic events during regeneration. Our data provide the first induction of regeneration by molecular modulation of ion flows, and the proposed work will answer the most important open questions in this new field. This proposal incorporates a high degree of novelty because it is focused on a paradigm that has not been previously addressed using molecular genetic tools: electrical controls of regeneration. It is high-reward because it would lay bare a new set of control parameters for the regeneration of a complex vertebrate structure (including spinal cord). This will have important implications for understanding basic morphogenetic mechanisms as well as establishing a foundation for promising medical approaches to augment or induce regeneration in non-regenerating tissues. The ability to regenerate tissues and organs is crucial to the medical management of injury, aging, infection, or surgical removal of cancer. Our work will provide an entirely new modality that may, one day, allow human beings to regenerate important tissues and organs (including muscle and spinal cord).