Math5 Regulatory Pathway in Retinal Development

描述（由申请人提供）：由于目前没有有效的细胞替代疗法，由视网膜神经元永久性丧失引起的退行性视网膜疾病构成了巨大的公共卫生风险。从干细胞或视网膜前体细胞的从头视网膜再生可能导致视网膜变性疾病的潜在补救措施。确定视网膜神经元形成所必需的基因和遗传过程的研究将为视网膜再生提供基础。本研究旨在揭示青光眼患者视网膜神经节细胞（retinal ganglion cells，RGCs）发生的分子机制。本提案的长期目标是了解Atonal类碱性螺旋-环-螺旋（bHLH）转录因子Math 5在视网膜神经发生过程中的作用及其与其他转录因子在视网膜发育过程中调节细胞命运选择的相互作用。敲除研究表明，Math 5的缺失导致RGC形成的缺失以及无长突和视锥光感受器细胞产生的同时增加，这意味着Math 5在促进RGC分化途径和抑制非RGC分化途径中的双重作用。此外，我们已经表明Math 5正调控RGC特异性转录因子如Brn 3b和Isll的表达，并且负调控非RGC因子如Bhlhb 5、NeuroD、Math 3和Ngn 2。该提议的目的1旨在表征Bhlhb 5在无长突细胞和锥双极细胞的选择性亚型的发育中的作用。使用Bhlhb 5-Cre（Cre重组酶）小鼠进行谱系分析以测试Bhlhb 5表达是否定义无长突和锥双极亚型。在目标2中，将在视网膜特异性Isl 1敲除小鼠中研究Isl 1在视网膜发生中的作用。此外，通过敲入Math 5基因座中的Isl 1来评估Math 5和Isl 1共表达在促进视网膜前体形成RGC中的协同效应。目的3是为了测试Math 5及其已知下游靶基因的调控关系。使用抗HA和遗传修饰的Math 5-HA小鼠的染色质免疫沉淀（ChIP）实验将用于鉴定Math 5的直接靶基因以及发现新的Math 5靶基因。或者，他莫昔芬诱导的Math 5（Math 5ER，Math 5和雌激素受体配体结合结构域的融合物）将用于鉴定在他莫昔芬和蛋白质合成抑制剂放线菌酮存在下由Math 5直接激活的基因。

DESCRIPTION (provided by applicant): Degenerative retinal diseases resulting from permanent loss of retinal neurons pose a great public health risk as no effective cell replacement therapies are currently available. De novo retinal regeneration from stem cells or retinal precursors could lead to a potential remedy for retinal degeneration diseases. Studies to identify genes and genetic processes essential for the formation of retinal neurons will provide the foundation for retinal regeneration. This proposal aims to reveal the molecular mechanisms governing the generation of retinal ganglion cells (RGCs), a group of neurons degenerated in glaucoma diseased eyes. The long-term objective of this proposal is to understand the role of an Atonal-class basic helix-loop-helix (bHLH) transcription factor, Math5, in the process of retinal neurogenesis and its interactions with other transcription factors in regulating the cell fate choices during retinal development. Knockout studies have shown that loss of Math5 leads to the absence of RGC formation and a concurrent increase in the generation of amacrine and cone photoreceptor cells, implying the dual roles of Math5 in promoting RGC differentiation pathway and inhibiting non-RGC differentiation pathways. Furthermore, we have shown that Math5 positively regulates the expression of RGC- specific transcription factors like Brn3b and Isl1, and negatively regulates non-RGC factors such as Bhlhb5, NeuroD, Math3, and Ngn2. The Aim 1 of this proposal is designed to characterize the role of Bhlhb5 in the development of selective subtypes of amacrine and cone bipolar cells. Lineage analysis using Bhlhb5-Cre (Cre recombinase) mice will be carried out to test if Bhlhb5 expression defines amacrine and cone bipolar subtypes. In Aim 2, the role of Isl1 in retinal genesis will be investigated in retina-specific Isl1 knockout mice. In addition, the synergistic effects of Math5 and Isl1 co-expression in promoting RGC formation from retinal precursors will be assessed by knocking-in Isl1 in Math5 locus. The Aim 3 is proposed to test the regulatory relationship of Math5 and its known downstream target genes. Chromatin immunoprecipitation (ChIP) experiments using anti- HA and genetically modified Math5-HA mice will be used to identify the direct target genes of Math5 as well as to discover novel Math5 target genes. Alternatively, a Tamoxifen-inducible Math5 (Math5ER, a fusion of Math5 and estrogen receptor ligand-binding domain) will be used to identify genes directly activated by Math5 in the presence of Tamoxifen and protein synthesis inhibitor, cycloheximide.