The Roles of LIM-Homeodomain Transcription Factors in Retinal Development

LIM-同源域转录因子在视网膜发育中的作用

• 批准号: 9229030
• 负责人: Lin Gan
• 金额: $ 38.46万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-03-01 至 2021-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9229030
• 关键词: Alpha Cell; Amacrine Cells; Axon; Blindness; Cell Differentiation process; Cell Lineage; Cells; Characteristics; Contrast Sensitivity; Coupled; Data; Dendrites; Detection; Development; Dyes; Electrophysiology (science); Enterobacteria phage P1 Cre recombinase; Event; Failure; Family; Ganglia; Ganglion Cell Layer; Gene Targeting; Genes; Genetic; Genetic Transcription; Individual; Injection of therapeutic agent; Inner Nuclear Layer; Inner Plexiform Layer; Knock-in; LHX2 gene; Link; Location; Massive Parallel Sequencing; Mediating; Molecular; Morphology; Mus; Neuraxis; Neurons; Pathway interactions; Pattern; Photoreceptors; Play; Process; Property; Proteins; Regulatory Pathway; Reporter; Retina; Retinal; Retinal Cone; Retinal Defect; Role; Shotgun Sequencing; Stratification; Structure; Subgroup; Synapses; Technology; Vision; Visual Perception; Visual impairment; base; cell type; chromatin immunoprecipitation; homeodomain; in vivo; interest; member; neural circuit; novel; null mutation; object motion; patch clamp; public health relevance; retinal neuron; retinal rods; retinogenesis; transcription factor; transcriptome; transcriptome sequencing; visual information; visual processing

 DESCRIPTION (provided by applicant): Our accurate vision depends on the flow of visual information through precisely wired synaptic connections among axons and dendrites of retinal neurons with unique morphological and functional properties. In the vertebrate retina, each of the six neuronal cell types: ganglion, amacrine, bipolar, horizontal, and rod and cone photoreceptor cells are further divided into subtypes based on location, morphology and function. Of all retinal neurons, amacrine cells are the most diverse group with >30 subtypes being identified so far. They represent ~40% of neurons both in the inner nuclear layer (INL) and the ganglion cell layer (GCL), make up a majority of synapses in the inner plexiform layer (IPL), and contribute to a majority of visual processing in the retina. One of the key questions i how the many retinal neuronal subtypes are produced and wired during development. In this proposal, we focus on the amacrine cells associated with the sublaminar layer 3 (S3) of the IPL. The S3 sublamina separates the ON and OFF laminas in the IPL but its cellular makeup and function is poorly understood. Here, we have demonstrated LHX9, a LIM-homeodomain transcription factor, is expressed early in retinogenesis and its expression is tightly confined toa few amacrine cells in the INL and the GCL. In our preliminary study, we have shown that these LHX9+ cells are a subgroup of GABAergic amacrine cells and express GAD67 but not GAD65. LHX9-expressing cells are also LHX2-expressing subgroup of amacrine cells. Targeted deletion of Lhx9 in mice results in a nearly complete loss of these LHX2-expressing amacrine cells and strikingly, in the absence of the S3 sublamina, suggesting that LHX9 could be expressed in and be required for the development of a unique, S3-stratifying amacrine subtype cells. Interestingly, our preliminary data show that bNOS expression is significantly down-regulated in the Lhx9-null retina, suggesting a loss of bNOS-subtype of amacrine cells that are known to project in the S3 sublamina. Being a transcription factor with a known function in neuronal subtype development in the central nervous system, LHX9 likely plays a critic role in amacrine subtype specification and offers us a unique opportunity to ultimately elucidate the genetic pathway governing the formation of the S3 sublamina and its associated neural circuitry. In this proposal, we will fully characterize the subtype identity of these LHX9-expressing amacrine subtypes and will identify its circuitry within the retina. Second, we will analyze the retinal defects of Lhx9-null mutation, particularly the effect on amacrine subtype specification, differentiation of these Lhx9-expressing S3 stratifying amacrine cells, and the change in the functional properties and circuitry of the Lhx9-lineage cells. To elucidate the LHX9 regulatory pathway in the S3-stratifying amacrine cells, we will perform RNA-Seq of control and Lhx9-null retinas and use LHX9 ChIP-Seq to screen for downstream target genes of LHX9 and to identify the transcriptional network. Together, these studies will define the role of LHX9 in regulating the formation and neural circuitry of S3 sublamina and elucidate the transcriptional events that occur downstream of LHX9.

 描述（由申请人提供）：我们的准确视觉取决于视觉信息通过具有独特形态和功能特性的视网膜神经元的轴突和树突之间的精确有线突触连接的流动。在脊椎动物视网膜中，六种神经元细胞类型中的每一种：神经节细胞、无长突细胞、双极细胞、水平细胞以及视杆细胞和视锥细胞根据位置、形态和功能进一步分为亚型。在所有的视网膜神经元中，无长突细胞是最多样化的一组，迄今已鉴定出超过30种亚型。它们占内核层（INL）和神经节细胞层（GCL）中神经元的约40%，构成内网状层（IPL）中的大部分突触，并对视网膜中的大部分视觉处理做出贡献。 关键问题之一是在发育过程中如何产生和连接许多视网膜神经元亚型。在这个提议中，我们专注于与IPL的亚层3（S3）相关的无长突细胞。S3亚板层将IPL中的ON和OFF板层分开，但其细胞组成和功能知之甚少。在这里，我们已经证明LHX 9，一个LIM-homeodomain转录因子，在视网膜发生的早期表达，并且其表达被严格地限制在INL和GCL中的一些无长突细胞中。在我们的初步研究中，我们已经表明，这些LHX 9+细胞是GABA能无长突细胞的一个亚群，表达GAD 67，但不表达GAD 65。表达LHX 9的细胞也是表达LHX 2的无长突细胞亚群。在小鼠中靶向缺失Lhx 9导致这些表达LHX 2的无长突细胞几乎完全丧失，并且在缺乏S3亚层的情况下，这表明LHX 9可以在独特的S3分层无长突亚型细胞中表达并且是其发育所需的。有趣的是，我们的初步数据显示，bNOS的表达显着下调Lhx 9无效的视网膜，这表明损失的bNOS亚型的无长突细胞，已知的项目在S3亚层。作为一种在中枢神经系统神经元亚型发育中具有已知功能的转录因子，LHX 9可能在无长突亚型特化中起关键作用，并为我们提供了一个独特的机会，以最终阐明控制S3亚层形成及其相关神经回路的遗传途径。在这项提案中，我们将充分表征这些LHX 9表达无长突亚型的亚型身份，并将确定其在视网膜内的电路。其次，我们将分析Lhx 9-null突变的视网膜缺陷， 特别是对无长突细胞亚型特化、这些表达Lhx 9的S3分层无长突细胞的分化以及Lhx 9谱系细胞的功能特性和电路的变化的影响。为了阐明LHX 9在S3-分层中的调节途径， 在无长突细胞中，我们将对对照和Lhx 9缺失的视网膜进行RNA-Seq，并使用LHX 9 ChIP-Seq筛选LHX 9的下游靶基因并鉴定转录网络。总之，这些研究将确定LHX 9在调节S3亚层的形成和神经回路中的作用，并阐明LHX 9下游发生的转录事件。