LIM-HD/bHLH Combinatorial Code in Motoneurons

描述（由申请人提供）：我们的长期目标是了解发育中的中枢神经系统（CNS）中的组合转录调控网络，其在指定大量不同的神经元和神经胶质细胞类型中发挥重要作用。LIM同源结构域（LIM-HD）和碱性螺旋-环-螺旋（bHLH）蛋白是胚胎中枢神经系统中调节细胞类型特化的相对较好表征的转录因子。然而，这些因子调节基因表达的分子机制及其靶基因的身份仍然知之甚少。我们希望探讨这些重要的问题，特别是集中在他们的作用，产生脊髓运动神经元（MN）。我们的研究结果表明，LIM-HD因子Lhx 3和Isl 1指定两个不同的神经元的胚胎脊髓的组合方式。Lhx 3和LIM辅因子NLI（核LIM相互作用物）形成V2-中间神经元（IN）特异性四聚体。与此相反，Lhx 3和NLI形成MN指定的六聚体时，共表达的Isl 1。在MN规范，bHLH蛋白Ngn 2/NeuroM功能同步的六聚体，虽然这种新的串扰的分子机制尚未完全理解。我们最近的筛选工作导致发现的基因组片段与NLI：lsl 1：Lhx 3六聚体结合位点，其中许多是连接到候选MN基因。这些结果导致了我们的中心假设，这一建议：六聚体直接调节多个MN基因与bHLH因子的影响MN规范。我们将剖析这一假设，揭示的分子基础，指导MN规格的组合表达的转录因子。具体而言，研究提出了探索不同的功能方面的六聚体/bHLHs与他们的候选靶基因。总的来说，拟议的研究应该为神经系统的正确形成的基本原则和治疗各种神经元损伤和疾病的实用信息提供重要的见解。值得注意的是，LIM-HD/bHLH因子还参与指定发育中CNS的其他区域中的细胞命运（例如，视网膜和前脑）以及非中枢神经系统器官，如心脏、胰腺和垂体，这突出了拟议研究的普遍重要性。

DESCRIPTION (provided by applicant): Our long-term goal is to understand the combinatorial transcriptional regulatory networks in the developing central nervous system (CNS), which play essential roles to specify a large number of distinct neuronal and glial cell types. LIM homeodomain (LIM-HD) and basic helix-loop-helix (bHLH) proteins are relatively well characterized transcription factors regulating cell type specification in the embryonic CNS. However, both the molecular mechanism by which these factors regulate gene expression and the identity of their target genes remain poorly understood. We wish to explore these important issues by specifically focusing on their role in generating spinal motoneurons (MNs). Our results demonstrate that LIM-HD factors Lhx3 and Isl1 specify two distinct neurons of the embryonic spinal cord in a combinatorial manner. Lhx3 and the LIM cofactor NLI (for nuclear LIM-interactor) form a V2-interneuron (IN)-specifying tetramer. In contrast, Lhx3 and NLI form a MN-specifying hexamer when co expressed with Isl1. During MN specification, bHLH proteins Ngn2/NeuroM are functionally synchronized with the hexamer, although the molecular mechanism underlying this novel crosstalk is not fully understood. Our recent screening effort led to the finding of genomic fragments with NLI:lsl1:Lhx3 hexamer binding sites, many of which are linked to candidate MN genes. These results led to our central hypothesis of this proposal: the hexamer directly regulates multiple MN genes in conjunction with bHLH factors to effect MN specification. We will dissect this hypothesis to uncover the molecular basis that directs MN specification in response to a combinatorial expression of transcription factors. Specifically, studies are proposed to explore the diverse functional aspects of the hexamer/bHLHs with their candidate target genes. Overall, the proposed studies should provide critical insights into the basic principles for the proper formation of the nervous system and practical information to treat a variety of neuronal injuries and diseases. Of note, LIM-HD/bHLH factors are also involved with specifying cell fates in other regions of the developing CNS (e.g., retina and forebrain) as well as non-CNS organs such as heart, pancreas and pituitary, which highlights the general importance of the proposed studies.