Small Molecule Screens to Identify Probes for Studies of Repair and Regeneration

描述(申请人提供)：斑马鱼是研究胚胎模式和器官发生的重要遗传模型。虽然基因工具可以用来探索胚胎发育过程中的基因功能和信号通路，但它们的应用受到时间上严格控制的过程或导致胚胎致死的基因的限制。小分子探针由于其快速和可逆的作用可以克服这些障碍，从而加强遗传学研究，并为揭示信号通路在幼虫和成虫生理中的作用提供了独特的机会。目前，研究成年斑马鱼基因功能的方法包括建立转基因热休克驱动系，使用Gal4-UAS系统等二元基因激活，或使用Cre重组酶等遗传重组来激活基因表达。复杂的组织特异性基因敲除目前在斑马鱼中是不可行的，因此将信号通路的研究限制在 早期开发，当基因产物可以被反义寡核苷酸击倒时。本研究的目的是寻找新的小分子调节因子作为工具来研究这些信号通路在斑马鱼幼体和成体修复和再生中的作用。成纤维细胞生长因子和成纤维细胞生长因子b信号通路在再生、修复和伤口愈合过程中至关重要，但它们作为潜在的药理靶点的开发还有待于阐明它们在这些事件中的确切分子机制。超激活这些通路的小分子将是研究这些通路的作用的有用工具，并代表着开发新的再生疗法的起点。最终，我们将为斑马鱼群体提供一套独特的工具，以研究斑马鱼发育的后期阶段和成年斑马鱼的疾病模型。这些研究将为在组织修复和再生模型中以明确的特异性和体内活性增强成纤维细胞生长因子和转化生长因子b信号提供有效的探针。拟议的工作分为三个具体目标，这三个目标利用了调查人员在这一多PI提案上的互补专业知识。目的1：我们将鉴定激活成纤维细胞生长因子信号通路的化合物。目的2：我们将鉴定激活TGFb信号通路的化合物。目的3：我们将在实验室常用的再生模型中测试新化合物的效果。 公共卫生相关性：这项建议的目标是确定新型的成纤维细胞生长因子和转化生长因子b通路的小分子调节剂，作为分析这些信号通路的作用的工具。研究斑马鱼在幼体和成体中再生的一个主要障碍是在这一过程中功能上抑制或增强这些途径的手段有限。为了克服这些缺点，用抑制或增强这些信号通路的小分子进行治疗是一种理想的方法。最终目标是为斑马鱼群落提供一套独特的工具来研究成纤维细胞生长因子和转化生长因子b信号通路。

DESCRIPTION (provided by applicant): The zebrafish is an important genetic model for studying embryonic patterning and organogenesis. Although genetic tools are available to probe gene function and signaling pathways during embryonic development, their utility is limited with temporally tightly controlled processes or with genes whose perturbation results in embryo lethality. Small molecule probes can overcome these obstacles due to their rapid and reversible actions, thereby enhancing genetic studies and offering a unique opportunity to uncover the roles of signaling pathways in larval and adult physiology. Currently, methods to study gene function in adult zebrafish involves the generation of transgenic heat shock driver lines, the use of binary gene activation such as the Gal4-UAS system, or the use of genetic recombination such as Cre recombinase to activate gene expression. Sophisticated tissue specific gene knockouts are not currently feasible in zebrafish, thus limiting the study of signaling pathways to early development, when gene products can be knocked-down with antisense oligonucleotides. The objective of this proposal is to identify novel small molecule modulators of the FGF and TGFb pathways as tools to dissect the role of these signaling pathways in zebrafish larval and adult repair and regeneration. The FGF and TGFb signaling pathways are critical in regeneration, repair, and wound healing but their exploitation as potential pharmacological targets awaits elucidation of their precise molecular mechanisms during these events. Small molecules that hyper-activate these pathways would be useful tools to study the roles of these pathways and represent starting points for the development of novel regenerative therapies. Ultimately, we will provide the zebrafish community with a unique set of tools to study later stages of development and adult zebrafish models of disease. These studies will provide validated probes for enhancing FGF and TGFb signaling with defined specificity and in vivo activity in models of tissue repair and regeneration. The proposed work is divided into three specific aims, which take advantage of the complementary expertise of investigators on this multi-PI proposal. Aim 1: We will identify compounds that activate the FGF signaling pathway. Aim 2: We will identify compounds that activate the TGFb signaling pathway. Aim 3: We will test the efficacy of the new compounds in regeneration models that are commonly used in our laboratories. PUBLIC HEALTH RELEVANCE: The objective of this proposal is to identify novel small molecule modulators of the FGF and TGFb pathways as tools to dissect the role of these signaling pathways. One major obstacle associated with studying zebrafish regeneration in larvae and adults is the limited number of means to functionally suppress or enhance these pathways in this process. To overcome these shortcomings, treatment with small molecules that suppress or enhance these signaling pathways is a desired approach. The ultimate goal is to provide the zebrafish community with unique set of instruments for studying the FGF and TGFb signaling pathways.