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

描述（由申请人提供）：斑马鱼是研究胚胎模式和器官发生的重要遗传模型。虽然遗传工具可用于探测胚胎发育过程中的基因功能和信号传导途径，但它们的实用性受到时间上严格控制的过程或其扰动导致胚胎致死的基因的限制。小分子探针可以克服这些障碍，由于其快速和可逆的行动，从而加强遗传研究，并提供了一个独特的机会，揭示信号通路在幼虫和成虫生理学的作用。目前，在成年斑马鱼中研究基因功能的方法包括产生转基因热休克驱动系、使用二元基因激活如Gal 4-UAS系统或使用遗传重组如Cre重组酶来激活基因表达。复杂的组织特异性基因敲除目前在斑马鱼中是不可行的，因此限制了对信号通路的研究， 早期发育，此时基因产物可以用反义寡核苷酸敲低。本提案的目的是确定新的FGF和TGF β通路的小分子调节剂作为工具来剖析这些信号通路在斑马鱼幼体和成体修复和再生中的作用。FGF和TGF β信号通路在再生、修复和伤口愈合中至关重要，但它们作为潜在药理学靶点的开发尚待阐明它们在这些事件中的精确分子机制。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，并代表了开发新的再生疗法的起点。最终，我们将为斑马鱼社区提供一套独特的工具来研究发育后期和成年斑马鱼疾病模型。这些研究将提供经验证的探针，用于在组织修复和再生模型中以确定的特异性和体内活性增强FGF和TGFb信号传导。拟议的工作分为三个具体目标，这利用了研究人员对这一多PI提案的补充专业知识。目的1：我们将确定激活FGF信号通路的化合物。目的2：我们将鉴定激活TGF β信号通路的化合物。目标3：我们将在我们实验室常用的再生模型中测试新化合物的功效。

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.