Functional Analysis of Signaling Pathways Controlling Limb Regeneration

尾纲两栖动物（蝾螈和蝾螈）在动物王国中是独一无二的，因为它们有能力完美地再生整个器官，如四肢、大脑和心脏。例如，如果肢体在其长度上的任何地方被截肢，一个完美的替代物将在大约1-2个月内再生，这个再生的替代物将包含原来在那里的骨头、软骨、关节、肌肉、神经、血管和皮肤，所有这些都以完全相同的关系相互联系，因此肢体将再次工作。该项目的目标是确定生长因子和信号分子如何驱动这一过程。人们早就知道，神经提供了一种刺激肢体再生细胞增殖的因素，最近已经确定了几种候选因素。该项目在一种基于伤口表皮基因表达的新实验中对这些候选物质进行功能测试。发现刺激细胞增殖的因素将对哺乳动物系统产生巨大影响，因为缺乏细胞增殖是组织再生的主要障碍。除了神经因子外，还有两条额外的信号通路被用来指导再生过程。这些也将使用最新的基因敲除技术来检查，以确定这些途径如何与其他已知信号相互作用。这个项目将影响到几个科学界和广大公众，因为再生具有巨大的公众吸引力。这种兴趣的例子包括，这些最近的蝾螈再生研究在电视节目和其他公共媒体上得到了强调，并得到了公众的好评。此外，该项目还包括专门设计的小型研究项目，每年有4-6名本科生参与，并促进代表性不足的少数民族和妇女的积极参与。本项目的第一部分主要研究截肢后形成的顶端表皮作为再生的关键调节因子的功能。转录组学分析已经确定了在顶端表皮中表达的肢体特异性基因，包括Wnt、BMP和FGF，以及两个新的途径：IGF和RA。这些新途径将通过反义morpholinos抑制、药物抑制和Crispr/Cas9靶向诱变来检测，并通过qPCR和原位杂交鉴定与其他途径的相互作用。RA信号下游的转录网络也将通过RNA-Seq进行检测，以确定该信号分子的靶点。第二个目标是发现神经营养控制肢体再生的分子基础。由于顶端表皮是神经的主要靶点，我们将采用一种新的方法来筛选12种潜在的神经营养因子，以了解它们对体外根尖表皮基因表达的影响。在这个屏幕上包括7种新的神经营养因子，这些因子是通过微阵列从感觉神经分泌因子中鉴定出来的。成功的候选基因将进一步进行体内分析，包括肢体再生过程中的表达模式和转基因动物的过表达。

Urodele amphibians (newts and salamanders) are unique throughout the animal kingdom in their ability to perfectly regenerate whole organs such as limbs, brains and the heart. For example, if the limb is amputated anywhere along its length a perfect replacement will regenerate in about 1-2 months and this regenerate will contain the bones, cartilage, joints, muscles, nerves, blood vessels and skin that were originally there and all in the exactly the same relationship to each other so that the limb will work again. The goal of this project is to determine how growth factors and signaling molecules drive this process. It has long been known that the nerves supply a factor that stimulates the proliferation of the regenerating cells in the limb, and several candidates have been identified recently. This project functionally tests each of these candidates in a novel assay based on gene expression in the wound epidermis. The identification of a factor that stimulates proliferation would have a huge impact on mammalian systems where the lack of proliferation is the major impediment to tissue regeneration. In addition to the nerve factor, two additional signaling pathways are utilized to guide the regenerative process. These will also be examined using the latest gene knockout techniques to determine how these pathways interact with other known signals. This project will impact several scientific communities, and the public at large because regeneration has huge public appeal. Examples of this interest include that fact that these recent axolotl regeneration studies were highlighted in TV programs and other public media, and received public acclaim. Additionally, this project includes specifically-designed mini-research projects that involve 4-6 undergraduates per year, and promotes active involvement of under-represented minorities and women.The first part of this project concentrates on the function of the apical epidermis that forms after limb amputation as a crucial regulator of regeneration. Transcriptomic analysis has identified the limb-specific genes expressed in this apical epidermis, including Wnt, BMP and FGF - as well as two novel pathways: IGF and RA. These new pathways will be examined by inhibition using antisense morpholinos, pharmacological inhibition and Crispr/Cas9 targeted mutagenesis to identify interactions with other pathways by qPCR and in situ hybridization. The transcriptional network downstream of RA signaling will also be examined by RNA-Seq to identify targets of this signaling molecule. The second objective is to discover the molecular basis of the neurotrophic control of limb regeneration. A novel approach will be used to screen 12 potential neurotrophic factors for their effect on gene expression in the apical epidermis in vitro since the apical epidermis is the primary target of the nerve. Included in this screen are 7 novel neurotrophic factors that were identified in a microarray for secreted factors from sensory nerves. Successful candidates will be taken further using in vivo analyses including expression patterns during limb regeneration and over-expression in transgenic animals.