The Roles of LIM-Homeodomain Transcription Factors in Retinal Development

 描述(申请人提供)：我们准确的视觉依赖于视觉信息的流动，通过具有独特形态和功能特性的视网膜神经元轴突和树突之间精确的有线突触连接。在脊椎动物视网膜中，六种神经细胞类型：神经节细胞、无长突细胞、双极细胞、水平细胞、视杆细胞和视锥细胞根据位置、形态和功能被进一步划分为亚型。在所有视网膜神经元中，无长突细胞是最多样化的一组，迄今已发现30种亚型。它们占内核层(INL)和节细胞层(GCL)神经元的40%左右，构成了内丛状层(IPL)的大部分突触，并参与了视网膜的大部分视觉处理。关键问题之一是如何在发育过程中产生和连接许多视网膜神经元亚型。在这项建议中，我们集中在与IPL的板层下第3层(S3)相关的无长突细胞。S3亚板将IPL的开、关板分开，但对其细胞组成和功能知之甚少。在这里，我们已经证明了LHX9，一个LIM同源结构域转录因子，在视网膜发生的早期表达，它的表达紧密地局限于INL和GCL中的少数无长突细胞。在我们的初步研究中，我们发现这些LHX9+细胞是GABA能无长突细胞的一个亚群，表达GAD67而不表达GAD65。表达LHX9的细胞也是表达LHX2的无长突细胞亚群。在小鼠中靶向缺失LHX9会导致这些表达LHX2的无长突细胞几乎完全丧失，并且令人惊讶的是，在没有S3亚层的情况下，LHX9可能在一个独特的、S3分层的无长突细胞中表达，并且是发育所必需的。有趣的是，我们的初步数据显示，在Lhx9缺失的视网膜中，bnos的表达显著下调，这表明已知的在S3亚板投射的bnos亚型的无长突细胞丢失。作为一个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，LHX9可能在无长突细胞亚型的指定中起着关键作用，并为我们最终阐明控制S3亚层及其相关神经回路形成的遗传途径提供了一个独特的机会。在这项建议中，我们将充分表征这些表达LHX9的无长突细胞亚型的亚型同一性，并确定其在视网膜内的回路。第二，我们将分析Lhx9零突变的视网膜缺陷， 特别是对无长突细胞亚型规范的影响，这些表达Lhx9的S3复层无长突细胞的分化，以及Lhx9系细胞功能特性和回路的变化。阐明LHX9在S3-层积中的调控途径 在无长突细胞中，我们将进行对照和Lhx9缺失视网膜的RNA-Seq，并使用LHX9芯片-序列来筛选LHX9的下游靶基因，并鉴定转录网络。总之，这些研究将确定LHX9在调节S3亚板的形成和神经回路中的作用，并阐明LHX9下游发生的转录事件。

 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.