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Identification of gene regulatory networks that control proliferative and neurogenic competence in mammalian Müller glia

鉴定控制哺乳动物穆勒神经胶质细胞增殖和神经发生能力的基因调控网络

基本信息

批准号：
10636825
负责人：
Seth Blackshaw
金额：
$ 51.19万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-08-01 至 2024-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10636825
关键词：
Adult CRISPR/Cas technology Catalogs Cell Cycle Cells Chick Chromatin Clustered Regularly Interspaced Short Palindromic Repeats Competence Dedications Degenerative Disorder Disease Family Gene Expression Gene Expression Profile Generations Genes Gliosis Goals Growth Factor Hour Individual Injury Mammals Mediating Molecular Morphology Muller&apos s cell Mus NFIA gene Natural regeneration Neuroglia Photoreceptors Predictive Factor Process Proliferating Radial Repression Rest Retina Retinal Diseases Retinal Dystrophy Retinal Photoreceptors Treatment Factor Work Zebrafish cell type cold blooded vertebrate combinatorial design efficacy validation gene function gene regulatory network hatching insight loss of function morphogens mutant neurogenesis novel strategies overexpression regenerative therapy regenerative treatment response to injury retinal damage retinal neuron retinal progenitor cell retinal rods therapy development transcription factor

项目摘要

Project Summary Müller glia of cold-blooded vertebrates can re-enter the cell cycle and rise to photoreceptors following retinal injury, while mammals have lost this ability. As part of the NEI Audacious Goals Initiative, we have conducted a ü comprehensive analysis of injury-induced changes in gene expression and chromatin accessibility in zebrafish, chick and mouse M ller glia, allowing us to identify both evolutionarily consüerved and species-specific gene regulatory networks that regulate glial reprogramming. This has identified a set of dedicated gene regulatory networks in mice tühat restrict proliferative and neurogenic competence in M ller glia. We aim to use these findings to gain a more complete insight into the molecular mechanisms that regulate neurogenic competence in mammalian M ller glia, and to develop treatments that can maximize generation of glial-derived photoreceptors while simultaneously not depleting the number of existing glia. To do this, we propose to generate individual loss of function mutants of the top candidate negative regulators of proliferative and ü neurogenic competence using AAV-mediated CRISPR/Cas9 gene disruption. We will first validate efficacy of sgRNAs targeting individual TFs, comprehensively profile chanüges in geüne expression in reactive M ller glia following loss of function of these genes, and characterize the fate of M ller glia-derived cells. We will then conduct combinatorial loss of function of negative regulators of M ller glia reprogramming to enhance generation ü of glial-derived retinal progenitor cells in wildtype and Nfia/b/x-deficient mice. Finally, we will combine CRISPR- mediated loss of function analysis with overexpression of Ascl1, Crx and Nrl to enhance generation of M ller glia-derived rod photoreceptors in both wildtype and dystrophic retina. We predict that these studies may substantially advance cell-based regenerative treatments aimed at restoring retinal photoreceptors lost due to blinding diseases.

项目概要冷血脊椎动物的穆勒胶质细胞可以重新进入细胞周期并继视网膜之后上升为光感受器受伤，而哺乳动物已经失去了这种能力。作为 NEI 大胆目标计划的一部分，我们开展了一项 ü 综合分析损伤引起的斑马鱼基因表达和染色质可及性变化，鸡和小鼠 M ller 神经胶质细胞，使我们能够识别进化保守基因和物种特异性基因调节神经胶质细胞重编程的调节网络。这已经确定了一套专门的基因调控小鼠中的网络限制了米勒神经胶质细胞的增殖和神经源性能力。我们的目标是利用这些研究结果可以更全面地了解调节神经源能力的分子机制哺乳动物米勒神经胶质细胞，并开发可以最大限度地产生神经胶质细胞的治疗方法光感受器，同时不耗尽现有神经胶质细胞的数量。为此，我们建议产生增殖和增殖的顶级候选负调节因子的个体功能丧失突变体 ü 使用 AAV 介导的 CRISPR/Cas9 基因破坏的神经发生能力。我们将首先验证功效靶向单个 TF 的 sgRNA，全面分析反应性 M ller 胶质细胞中基因表达的变化这些基因功能丧失后，并表征 M ller 胶质细胞衍生细胞的命运。我们随后将进行 M ller 胶质细胞负调节因子功能的组合丧失重新编程以增强生成 ü 野生型和 Nfia/b/x 缺陷小鼠中神经胶质源性视网膜祖细胞的变化。最后，我们将结合CRISPR- 通过过表达 Ascl1、Crx 和 Nrl 介导的功能丧失分析来增强 M ller 的生成野生型和营养不良型视网膜中的神经胶质细胞衍生的杆状光感受器。我们预测这些研究可能大幅推进基于细胞的再生治疗，旨在恢复由于视网膜光感受器而丧失的视网膜光感受器致盲疾病。

项目成果

期刊论文数量（16）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Ectopic insert-dependent neuronal expression of GFAP promoter-driven AAV constructs in adult mouse retina.

DOI：
10.3389/fcell.2022.914386
发表时间：
2022
期刊：
Frontiers in cell and developmental biology
影响因子：
5.5
作者：
通讯作者：

A potential role for somatostatin signaling in regulating retinal neurogenesis.

DOI：
10.1038/s41598-021-90554-3
发表时间：
2021-05-26
期刊：
Scientific reports
影响因子：
4.6
作者：
Weir K;Kim DW;Blackshaw S
通讯作者：
Blackshaw S

IReNA: Integrated regulatory network analysis of single-cell transcriptomes and chromatin accessibility profiles.

DOI：
10.1016/j.isci.2022.105359
发表时间：
2022-11-18
期刊：
ISCIENCE
影响因子：
5.8
作者：
Jiang, Junyao;Lyu, Pin;Li, Jinlian;Huang, Sunan;Tao, Jiawang;Blackshaw, Seth;Qian, Jiang;Wang, Jie
通讯作者：
Wang, Jie

Updates and challenges of axon regeneration in the mammalian central nervous system.

DOI：
10.1093/jmcb/mjaa026
发表时间：
2020-10-01
期刊：
Journal of molecular cell biology
影响因子：
5.5
作者：
Qian C;Zhou FQ
通讯作者：
Zhou FQ

Control of neurogenic competence in mammalian hypothalamic tanycytes.

DOI：
10.1126/sciadv.abg3777
发表时间：
2021-05
期刊：
Science advances
影响因子：
13.6
作者：
Yoo S;Kim J;Lyu P;Hoang TV;Ma A;Trinh V;Dai W;Jiang L;Leavey P;Duncan L;Won JK;Park SH;Qian J;Brown SP;Blackshaw S
通讯作者：
Blackshaw S