Elucidating the gene regulatory networks that drive neural regeneration

项目摘要/摘要 目前还没有有效的治疗方法来替代受损的视网膜神经元，这反映了一种根本的 人类在中枢神经系统内建立强大的再生反应。为了解决这个问题，它将是 对于理解可能阻碍或推动神经元再生的基因调控机制的多样性至关重要 在脊椎动物的背景下。胚胎羊膜具有从细胞再生视网膜神经元的暂时性能力 在视网膜损伤时，如果外源性FGF2供应，视网膜色素上皮(RPE)将受到损伤。这 再生机制在鸡胚胎发育的第4天(E4)很容易被诱导，但RPE 神经能力在胚胎第5天(E5)时丧失。拟议研究的首要目标是 RPE细胞分化时丧失其神经能力的基因调控的轮廓变化。 特定目标1将询问跨E4/E5的RPE细胞内的转录因子调节活性 通过整合基因表达分析、染色质可及性分析和 转录因子结合试验。初步的散装和单核rna-seq数据集显示急性 神经视网膜转录因子在E4和E5的激活，如PAX6、ASCL1和VSX2。在……里面 相比之下，与RPE成熟度相关的基因，如OTX2和色素沉着基因，在E5中升高 RPE不依赖于视网膜切除术和FGF2治疗。同样，染色质的可及性表明更广泛的 OTX2和相关的同源异型盒转录因子结合位点的失调。在为期1年的F99阶段， 在完整和经FGF2处理的RPE细胞中，OTX2结合活性将在E4和E5阶段被描绘出来。另外， 单核RNA测序将捕捉RPE细胞在不同转录状态下的表达 E4和E5的分化和FGF2治疗反应。这些结果将被集成到一个模型中 描述了RPE基因调控格局的变化如何最终导致神经能力的丧失。在……里面 特定目的2，中枢神经系统成体脊椎动物模型中存在的基因调控网络 将对再生进行询问，以启发诱导哺乳动物再生的新途径。这个K00 阶段将侧重于关键研究技能的发展，包括多种组学数据分析方法， 空间转录和单细胞表观基因组学以及跨物种基因组学技术 转录学。K00阶段将在直接支持这些的环境中花费长达4年的时间 申请。将同时追求特定的职业发展目标，如教育学 发展、多样性外展倡议和赠款撰写。总而言之，这些目标围绕着一项职业生涯 将导致形成独立研究计划并产生影响的研究的发展计划 专注于扩大人类中枢神经系统的再生能力。

Project Summary / Abstract There are no effective treatments to replace damaged retinal neurons, reflecting a fundamental inability for humans to mount robust regenerative responses within the central nervous system. To address this, it will be critical to understand the diversity of gene regulatory mechanisms that can impede or drive neuron regeneration across vertebrate contexts. Embryonic amniotes have a transitory ability to regenerate retinal neurons from cells of the retinal pigment epithelium (RPE) if supplied with exogenous FGF2 at the time of retinal injury. This mechanism of regeneration can be readily induced at embryonic day 4 (E4) of chicken development, but RPE neural competence is lost by embryonic day 5 (E5). The overarching objective of the proposed research is to profile changes in gene regulation that dispossess RPE cells of their neural competency as they differentiate. Specific aim 1 will interrogate transcription factor regulatory activity within RPE cells across the E4 / E5 developmental window by integrating gene expression analysis, chromatin accessibility profiling, and transcription factor binding assays. Preliminary bulk and single nuclei RNA-seq datasets revealed the acute activation of neural retina transcription factor profiles at both E4 and E5, such as PAX6, ASCL1, and VSX2. In contrast, genes associated with RPE maturity, such as OTX2 and pigmentation genes, were elevated in the E5 RPE independently of retinectomy and FGF2 treatment. Similarly, chromatin accessibility suggested wider dysregulation of OTX2 and related homeobox transcription factor binding sites. During the 1-year F99 phase, OTX2 binding activity will be profiled in intact and FGF2-treated RPE cells at E4 and E5 stages. Additionally, single nuclei RNA-sequencing will capture the heterogeneous transcriptional states of RPE cells during differentiation and FGF2 treatment response at E4 and E5. These results will be integrated to into a model that describes how changes in the RPE gene regulatory landscape culminates in a loss of neural competency. In specific aim 2, the gene regulatory networks present in adult vertebrate models of central nervous system regeneration will be interrogated to inspire novel routes for the induction of mammalian regeneration. This K00 phase will focus on the development of key research skills, including multi-omics data analysis approaches, techniques for spatial transcriptomics and single cell epigenomics, and cross-species genomics / transcriptomics. Up to 4 years will be spent on the K00 phase in an environment directly supportive of these applications. Specific professional development objectives will be concurrently pursued, such as pedagogical development, diversity outreach initiatives, and grant writing. Together, these aims encompass a career development plan that will lead to formation of an independent research program and result in impactful research focused on expanding human central nervous system regenerative capacity.