Transcriptional control of cell plasticity and inflammation in regeneration.

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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.