Biophysical controls of vertebrate organ regeneration

描述(申请人提供)：再生因损伤或疾病而失去的组织和器官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对能够再生的动物中操作的相关图案信号进行分子解剖。这一领域一直被对化学信号的关注所主导，并准备采取新的方法来解决这一问题。我们的实验室将功能生理学与分子遗传学相结合，以了解图案化的新生物物理控制，并使用它们来控制组织生长。当被截断时，非洲爪哇尾巴形成一个再生芽，迅速产生与原始尾巴完美复制的尾巴，包括神经、血管和肌肉。利用这个强大的脊椎动物系统，我们发现内源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到再生所需的H泵、K通道和Na通道，而不是伤口愈合或初级尾巴生长所需的；这些转运蛋白的活性在芽中建立了一个对再生至关重要的适度去极化区域。我们使用突变的转运蛋白结构来抑制或挽救再生，证明了H通量是诱导再生的必要条件和充分条件。这些生物物理事件的作用是上游和控制：已知的再生标记表达，芽中细胞增殖的上调，以及轴突模式。我们建议从以下几个方面开始了解离子通量在再生中的作用：(1)胚泡电流的时间过程和特性，(2)相关电基因的表达，(3)在再生过程中将膜电压与分子和形态发生事件联系起来的下游步骤。我们的数据首次提供了通过分子调制离子流来诱导再生的方法，所提出的工作将回答这一新领域中最重要的开放问题。这一提议包含了高度的新颖性，因为它关注的是一种以前没有使用分子遗传工具解决的范例：再生的电子控制。这是一个高回报，因为它将为复杂的脊椎动物结构(包括脊髓)的再生暴露出一套新的控制参数。这将对理解基本的形态发生机制以及为在非再生组织中增强或诱导再生的有前途的医学方法奠定基础。再生组织和器官的能力对于损伤、衰老、感染或癌症手术切除的医疗管理至关重要。我们的工作将提供一种全新的模式，有朝一日可能会允许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).