Mechanisms regulating the plasticity of postmitotic cells in mammalian retina

项目摘要/摘要 治疗视网膜疾病的一个有希望的策略是产生所需的神经元类型，这些类型是 在疾病期间受损或丢失，并开发基于细胞替代的治疗方法。一个全面的 了解不同的视网膜类型是如何在发育过程中形成的，这在很大程度上可以帮助这些治疗 战略。虽然有丝分裂的视网膜前体细胞(RPC)被认为在本质上是不同的，但 视网膜细胞不能在RPC中确定。许多新生的有丝分裂后细胞仍然是可塑性的。它目前不是 明确新生的有丝分裂后细胞如何达到最终的命运状态。我们建议揭开基因和 调控新生有丝分裂后细胞在发育过程中命运决定的途径 它们是否也能促进成年视网膜的细胞重编程。 我们收集了新生的视网膜有丝分裂后细胞中丰富的基因，基于发表的 单细胞rna-seq数据，并开发了新的方法来研究它们在新生儿中的功能。 利用基于逆转录病毒的遗传方法和光片显微镜观察体内的有丝分裂后细胞。一根锌手指 转录因子Myt1(髓鞘转录因子1)被发现在新生有丝分裂后细胞中富含， 但不是RPC，在发育过程中；它可以促进神经发生，特别是双极细胞的形成，而 在新生儿期抑制新生有丝分裂后细胞的神经胶质命运。我们假设Myt1确保 神经元在新生的有丝分裂后细胞中的命运，并有助于成熟视网膜中的神经元重新编程。 在拟议的研究中，我们将通过两个目标来检验这一假说。在Aim1中，我们将阐明 Myt1在命运决定中的作用，并测试Myt1通过滴定促进双极细胞命运的假设，但 未完全关闭Notch信号通路并主动抑制新生大鼠神经胶质细胞基因 有丝分裂后细胞。在AIM2中，我们将确定Myt1与Ascl1和Brn2是否可以直接促进 成熟小鼠视网膜Müler神经胶质细胞重编程为神经元。 综上所述，这一建议旨在阐明哺乳动物视网膜中有丝分裂后细胞的可塑性。我们 将重点了解锌指转录因子Myt1如何促进新生的特定神经元命运 出生的有丝分裂后细胞在发育过程中的作用及Myt1能否增强神经元 成熟视网膜中神经胶质细胞的重编程。这项工作将提高我们对基础生物学和 为视网膜神经元再生提供新的候选基因和可能性。值得注意的是，基因突变 人类Myt1基因与发育中的眼-耳-脊椎谱性疾病相关 有眼部缺陷的疾病，如小眼球。Myt1在视网膜发育中的作用 也可以揭示疾病的机制。

PROJECT SUMMARY/ABSTRACT One of the promising strategies to treat retinal diseases is to generate desired neuronal types, which are damaged or lost during disease, and develop cell replacement-based therapies. A comprehensive understanding of how distinct retinal types are formed during development can greatly inform these therapeutic strategies. While mitotic retinal progenitor cells (RPCs) are thought to be intrinsically different, the fates of retinal cells are not determined in RPCs. Many newly born postmitotic cells are still plastic. It is currently not clear how newly born postmitotic cells attain their final fate states. We propose to uncover the genes and pathways that regulate fate decisions in newly born postmitotic cells during development and determine whether they can also promote cell reprogramming in adult retina. We collected genes that are enriched in newly born postmitotic cells in the retina based on published single cell RNA-seq data, and developed novel methods to study their function specifically in newly born postmitotic cells in vivo by utilizing retroviral-based genetic approach and light sheet microscopy. A zinc finger transcription factor Myt1 (Myelin transcription factor 1) was found to be enriched in newly born postmitotic cells, but not RPCs, during development; it can promote neurogenesis, especially the formation of bipolar cells, while repressing glial fate in newly born postmitotic cells at neonatal stages. We hypothesize that Myt1 ensures the neuronal fates in newly born postmitotic cells and can contribute to neuronal reprogramming in mature retina. In the proposed studies, we will test this hypothesis through two aims. In Aim1, we will elucidate the function of Myt1 in fate determination, and test the hypothesis that Myt1 promotes bipolar cell fates by titrating down, but not completely shutting down Notch signaling pathway, and by actively repressing glial genes in newly born postmitotic cells. In Aim2, we will determine whether Myt1 together with Ascl1 and Brn2 can promote direct reprogramming of Müller glial cells into neurons in mature mouse retina. Taken together, this proposal aims to elucidate the plasticity of postmitotic cells in mammalian retina. We will focus on understanding how zinc finger transcription factor Myt1 promotes specific neuronal fates in newly born postmitotic cells during development and determining whether Myt1 can enhance neuronal reprogramming from glial cells in mature retina. This work will improve our understanding of basic biology and provide new candidate genes and possibilities for the regeneration of retinal neurons. Notably, mutations in human Myt1 gene are associated with Oculo-Auriculo-Vertebral Spectrum diseases, which are developmental disorders with ocular defects such as microphthalmia. Elucidating the function of Myt1 in retinal development can also shed light on the disease mechanisms.