Transcriptional regulatory mechanisms of vertebrate regeneration

为什么人类不能再生受损的中枢神经系统组织，而其他脊椎动物可以 容易？在这个建议中，我们通过定义细胞内在机制来瞄准这个基本问题， 使热带爪蟾的脊髓得以再生。这个物种的蝌蚪能够再生 脊髓组织和运动功能损伤后，而成年动物不能。我们会利用这个时间 再生能力，以了解再生通常如何进行，以及为什么它可能 失败这种独特的生物学加上功能和基因组分析的深层可用工具集 使X。tropicalis是一个独特的强大的再生分析系统。脊髓的中心目标 再生研究的目的是确定使神经发生和轴突再生的细胞内在因子。 我们对该系统的初步分析揭示了对这些因素和基因的新见解。 可能形成再生能力基础的调节机制。首先，我们发现， 数以千计的基因组区域迅速转变为可接近的染色质构象，然后出乎意料地 一种难以接近的构造，在再生的最初几个小时内。这些重组发生在 高度富集FoxO1和Ascl1结合位点的区域，具有先锋活性的因子， 在神经祖细胞功能中的重要作用。第二，表达分化神经元特异性基因 并且令人惊讶地独立于神经诱导和神经发生基因 activation.基于这些观察结果，我们假设再生能力依赖于三个因素， 特征：1）强大的神经祖细胞群体，2）神经祖细胞中染色质重塑的快速爆发 由Ascl1、FoxO1和其他先驱因子进行的细胞，和3）激活神经元特异性基因， 允许轴突发生和现有分化神经元中的神经元生长。在本提案中，我们将测试这些 通过鉴定在分离的神经细胞中介导染色质重塑的转录因子来预测， 祖先我们将使用功能缺失突变体功能性地测试Ascl1和FoxO1在再生中的作用 对于这些因素。然后我们将确定是否上调轴突发生基因在再生蝌蚪 代表神经元修复或神经发生，并询问这些基因是否被上调， 胚胎基因调控元件或再生特异性调控元件。最后，我们将确定 成年青蛙的再生失败是否是由于缺乏神经祖细胞，未能启动染色质 重塑或不能上调神经元形态发生基因。通过系统地描述 事件，定义再生能力的爪蟾，我们希望确定分子机制， 成为人类脊髓损伤患者更有效治疗的目标。

Why do humans fail to regenerate injured central nervous system tissues, when other vertebrates do so readily? In this proposal we take aim at this fundamental question by defining the cell-intrinsic mechanisms that enable spinal cord regeneration in the frog Xenopus tropicalis. Tadpoles of this species are able to regenerate spinal cord tissues and motor function following injury, while adult animals cannot. We will exploit this temporal competence to regenerate in order to understand how regeneration normally proceeds as well as why it might fail. This distinctive biology coupled with the deep set of available tools for functional and genomic analysis makes X. tropicalis a uniquely powerful system for analysis of regeneration. A central goal of spinal cord regeneration research is to identify the cell-intrinsic factors that enable neurogenesis and axon regeneration. Our preliminary analyses in this system have uncovered new insights into these factors and the gene regulatory mechanisms that may form the basis for regenerative competence. First, we have found that tens of thousands of genomic regions shift rapidly to an accessible chromatin conformation, and then unexpectedly to an inaccessible conformation, within the first few hours of regeneration. These rearrangements take place in regions that are heavily enriched for binding sites of FoxO1 and Ascl1, factors that have pioneer activity and critical roles in neural progenitor function. Second, genes specific to differentiated neurons are expressed within hours of amputation, and are surprisingly independent of neural induction and neurogenesis gene activation. Based on these observations, we hypothesize that regenerative competence relies on three features: 1) a robust neural progenitor population, 2) a rapid burst of chromatin remodeling in neural progenitor cells carried out by Ascl1, FoxO1, and other pioneer factors, and 3) activation of neuronal specific genes that allow axonogenesis and neuronal growth in existing differentiated neurons. In this proposal, we will test these predictions by identifying the transcription factors that mediate chromatin remodeling in isolated neural progenitors. We will functionally test the role of Ascl1 and FoxO1 in regeneration using loss-of-function mutants for these factors. We will then identify whether upregulation of axonogenesis genes in regenerating tadpoles represents neuronal repair or neurogenesis, and interrogate whether these genes are upregulated using embryonic gene regulatory elements or regeneration-specific regulatory elements. Finally, we will identify whether regeneration in adult frogs fails due to lack of neural progenitors, failure to initiate chromatin remodeling, or failure to upregulate neuronal morphogenesis genes. By systematically characterizing the events that define regeneration competence in Xenopus, we expect to identify molecular mechanisms that can be targeted for more effective therapeutics in human spinal cord injury patients.