Transcriptional control of cell plasticity and inflammation in regeneration.

摘要 脊髓损伤(SCI)导致哺乳动物不可逆的感觉和运动功能丧失。相比之下，斑马鱼 自然再生完全横断的脊髓(SC)。因为这种自然现象背后的机制 虽然对斑马鱼再生的研究还很少，但我们实验室已经对再生斑马鱼SC的转录组进行了测序。 在初步数据中，我完成了基于CRISPR/Cas9的反向遗传筛查，以识别 斑马鱼干细胞再生所必需的。总体而言，这些数据集中在两个关键过程中 斑马鱼的先天SC再生：神经胶质桥接和促再生炎症。这项提议将 描述参与这两个过程的三个基因。 首先，在再生的斑马鱼SC中，专门的胶质细胞桥接SC损伤并为轴突提供支架 重生。在初步数据中，我发现桥接神经胶质细胞及其假定的前体强烈表达上皮细胞 至间充质(EMT)成分。EMT是再生和伤口修复的基本过程，而EMT 在这些过程中增强多种细胞类型的增殖和可塑性。在我的CRISPR/Cas9屏幕中， 我在已发表的文献中发现了两个与EMT相关的基因：早期生长反应1(Egr1)和 JunB原癌基因b(Junb)。在本提案的目标1中，我将探索EMT相关基因的表达 在Egr1和Junbb下游。我假设Egr1和Junbb正在建立EMT监管网络 对脊髓损伤后的神经胶质细胞桥接是必需的。第二，斑马鱼免疫系统支持成功 脊髓损伤后的再生，尽管促再生的确切炎症途径不是很好 明白了。在我的CRISPR/Cas9屏幕上，我发现转录和免疫反应调节剂(TCIM)是一种 SC再生因子。脊髓损伤后，TCIM在SC中强烈上调，并由小胶质细胞和 巨噬细胞，成年斑马鱼对脊髓损伤作出反应的主要免疫细胞。TCIM突变体未完全康复 损伤后的功能或解剖学上的反应，并在脊髓损伤后有高度炎症反应。在这个目标2中 提议，我将首先确定炎症何时是促进再生的，并且是野生型胶质细胞桥接所必需的 斑马鱼。其次，我将确定TCIM下游的促再生炎症途径如下 SCI。我推测，TCIM的作用是抑制抗再生炎症通路，同时促进促再生炎症反应。 斑马鱼SC损伤后的再生性炎症。总之，这些研究将开始揭示 斑马鱼SC再生的先天能力的途径和机制。 这项建议旨在为莫卡莱河独特的成年斑马鱼SC再生系统提供培训 WUSM的实验室。我在WUSM拥有先进的设施，我周围有一群强大的 能够提供最高级别的指导和培训的科学家。这些经历将会训练我 先进和独立的研究方法、科学写作和交流以及教学/指导 成功成长为独立学术研究岗位所需的技术。

ABSTRACT Spinal cord injury (SCI) causes irreversible sensory and motor function loss in mammals. In contrast, zebrafish naturally regenerate a fully transected spinal cord (SC). Because the mechanisms underlying this natural regeneration remain understudied, our lab has sequenced the transcriptome of the regenerating zebrafish SC. In preliminary data, I completed a CRISPR/Cas9-based reverse genetic screen to identify genes that are necessary for SC regeneration in zebrafish. Collectively, these data converged on two key processes during innate SC regeneration in zebrafish: glial bridging and pro-regenerative inflammation. This proposal will characterize three genes that are involved in these two processes. First, in the regenerating zebrafish SC, specialized glia bridge the SC lesion and provide a scaffold for axon regrowth. In preliminary data, I found bridging glia and their presumptive precursors strongly express epithelial to mesenchymal (EMT) components. EMT is a process fundamental to regeneration and wound repair, and EMT enhances the proliferation and plasticity of many cell types during these processes. In my CRISPR/Cas9 screen, I identified two genes that have been linked to EMT in published literature: early growth response 1 (egr1) and junb proto-oncogene b (junbb). In Aim 1 of this proposal, I will explore the EMT-related gene expression downstream of egr1 and junbb. I hypothesize that egr1 and junbb are establishing the EMT regulatory network necessary for glial bridging following SCI. Second, the zebrafish immune system supports successful regeneration after SCI, although the precise inflammatory pathways that are pro-regenerative are not well understood. In my CRISPR/Cas9 screen, I identified transcription and immune response modulator (tcim) as a SC regeneration factor. Following SCI, tcim is strongly upregulated in the SC and expressed by microglia and macrophages, the primary immune cells responding to SCI in adult zebrafish. tcim mutants do not fully recover functionally or anatomically post-injury and have a hyper-inflammatory response following SCI. In Aim 2 of this proposal, I will first determine when inflammation is pro-regenerative and necessary for glial bridging in wild-type zebrafish. Second, I will determine the pro-regenerative inflammatory pathways downstream of tcim following SCI. I hypothesize that tcim acts to dampen anti-regenerative inflammatory pathways, while enhancing pro- regenerative inflammation in the zebrafish SC post-injury. Together, these studies will begin to uncover the pathways and mechanisms that underlie the innate ability for the zebrafish SC to regenerate. This proposal is designed to provide training in a unique adult zebrafish SC regeneration system in the Mokalled lab at WUSM. The facilities available to me at WUSM are advanced, and I am surrounded by a strong group of scientists who can provide mentorship and training at the highest level. These experiences will train me in advanced and independent research methodology, scientific writing and communication, and teaching/mentoring techniques necessary for successful growth into an independent academic research position.