Chemical Genetics to Define Regenerative Pathways

翻译后摘要：损伤，疾病和老化都导致组织的损失和生活质量降低。如果有促进组织再生的疗法，许多人类疾病都可以得到显著改善。与哺乳动物不同，斑马鱼具有完全再生丢失或受损组织的非凡能力。几十年来，这种再生能力刺激了科学研究，希望找到允许人体组织再生的方法。已经开发了许多实验模型来识别控制再生过程的基因。这些研究的共识是，基本的再生途径是保守的，但我们才刚刚开始确定允许组织再生的网络。仍然存在巨大的信息差距。该提案的重点是定义再生所需的信号通路和基因。在初步研究中，我们建立了两个斑马鱼鳍再生模型，并利用微阵列分析鉴定了一些再生反应基因。一个直接的目标是确定这些基因在再生过程中的作用。此外，该提案将首次使用化学遗传学的力量来探测组织再生。主要目标是确定干扰正常再生的化学物质。假设是，如果一种化学物质专门阻止组织再生，它必须干扰或阻止关键大分子的活动，这些大分子对这一非凡的过程至关重要。作为该提议的初步数据的一部分，筛选了市售化学文库，并鉴定了许多特异性抑制组织再生的小分子。本研究的目的是明确斑马鱼早期鳍再生的分子和细胞机制。潜在的假设是，存在在再生组织中诱导的共同再生途径。这一假设的一个自然延伸是，一旦在斑马鱼中确定了关键基因和途径，它们就可以在其他再生模型中得到验证。两个具体的目标已经开发，以确定新的调节剂的早期生命阶段的鳍再生1）确定和定义的作用，在鳍再生过程中表达的基因; 2）以确定生物活性物质，调节早期生命阶段的再生，并开始确定其生化目标。这些研究将开始，以确定控制再生的分子机制。除了识别组织再生的基因和途径外，这些研究还将产生许多分子标记，可以在其他再生模型（如哺乳动物）中进行评估。在哺乳动物中进行化学遗传筛选是不切实际的，因此啮齿动物不能用于药物发现。这些斑马鱼发现研究的结果是快速的，可以迅速转化为其他模型。在更广泛的范围内，对化学物质的结构和功能关系的分析可能会导致新的药物开发，可用于促进人体组织再生。如果能在损伤后促进组织再生的治疗方法，将有巨大的临床效益。这些研究的成功完成也将突出化学遗传学方法与传统基因敲除或化学诱导遗传学筛选相比在研究复杂过程方面的巨大优势。从培训的角度来看，一些本科生、研究生和高中生将参与拟议的研究。无论他们的职业发展阶段，每个人都将获得在假设制定，实验设计，实验室组织和数据呈现方面的宝贵经验。Tanguay实验室致力于K-12教育和推广，这表明了教师和学生参与研究的长期记录。因此，预计该项目的研究将提供许多机会，使重大的科学发现，但同样重要的是，一个机制，以培训学生，并将结果转化为公众。

Abstract:Injury, disease and aging all result in a loss of tissue and reduced quality of life. Numerous human conditions could be significantly improved if therapies that encourage tissue regeneration were available. Zebrafish, unlike mammals, have the remarkable ability to fully regenerate lost or damaged tissues. This regenerative ability has stimulated scientific investigations for decades with hopes of finding ways to permit human tissue regeneration. A number of experimental models have been developed to identify the genes that control the regeneration process. The consensus from these studies is that the basic regeneration pathways are well-conserved, yet we are just beginning to identify the networks that permit tissue regeneration. There remain enormous information gaps. The focus of this proposal is on defining the signaling pathways and the genes required for regeneration. In preliminary studies, two zebrafish fin regeneration models were developed and a number of regeneration-responsive genes were identified using microarray analysis. An immediate goal is to determine the role of these genes in the regeneration process. In addition, this proposal will, for the first time use the power of chemical genetics to probe tissue regeneration. The main goal is to identify chemicals that perturb normal regeneration. The hypothesis is that if a chemical specifically block tissue regeneration, it must interfere or block the activity of critical macromolecules that are essential for this remarkable process. As part of the preliminary data for this proposal, a commercially available chemical library was screened and a number of small molecules were identified that specifically inhibited tissue regeneration. The goal of this proposal is to define the molecular and cellular mechanisms of early life stage fin regeneration in zebrafish. The underlying hypothesis is that there are common regenerative pathways that are induced in regenerating tissues. A natural extension of this hypothesis is that once critical genes and pathways are identified in zebrafish; they can be validated in other regenerative models. Two specific aims have been developed to identify novel regulators of early life stage fin regeneration 1) Identify and define the role of genes expressed during fin regeneration; 2) To identify bioactives that modulate early life stage regeneration, and begin to define their biochemical targets Together these studies will begin to define the molecular machinery that controls regeneration.The broader impacts for this project are many. In addition to identifying genes and pathways that choreograph tissue regeneration, these studies will generate a number of molecular markers that can be evaluated in other regenerative models such as mammals. It would be impractical to conduct a chemical genetic screen in mammals, so rodent cannot be used for drug discovery. The results these zebrafish discovery studies are rapid and can be rapidly translated to other models. On a broader scale, the analysis the structure and function relationships of the chemicals may lead to novel drug development that may be used to promote tissue regeneration in humans. There would be a tremendous clinical benefit if therapies that encourage tissue regeneration following injury were available. Successful completion of these studies will also highlight the enormous advantages of chemical genetic approaches compared to traditional gene knockout or chemical induced-genetic screens to study complex processes. From a training standpoint, a number of undergraduate, graduate and high school students will be involved with the proposed studies. Regardless of their stage in career development, each will gain valuable experience in hypothesis formulation, experimental design, laboratory organization, and data presentation. The Tanguay laboratory is committed to K-12 education and outreach as demonstrated a long track record in involving teachers and students in research. Thus, it is expected that research from this project will provide numerous opportunities to make significant scientific discoveries, but as importantly, a mechanism to train students and to translate the results to the general public.