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）的组合转录调控网络，它在确定大量不同的神经元和胶质细胞类型中起着重要作用。LIM同源结构域（LIM- hd）和碱性螺旋-环-螺旋（bHLH）蛋白是调控胚胎中枢神经系统细胞类型的转录因子。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。我们希望通过特别关注它们在产生脊髓运动神经元（MNs）中的作用来探索这些重要问题。我们的研究结果表明，LIM-HD因子Lhx3和Isl1以组合方式指定胚胎脊髓的两种不同的神经元。Lhx3和LIM辅助因子NLI（核LIM-相互作用因子）形成一个v2 -中间神经元（IN）指定四聚体。相反，Lhx3和NLI与Isl1共表达时形成mn指定六聚体。在MN规范过程中，bHLH蛋白Ngn2/NeuroM在功能上与六聚体同步，尽管这种新型串扰的分子机制尚不完全清楚。我们最近的筛选工作导致发现具有NLI:lsl1:Lhx3六聚体结合位点的基因组片段，其中许多与候选MN基因相关。这些结果导致了我们这一建议的中心假设：六聚体直接调节多个MN基因与bHLH因子一起影响MN规格。我们将剖析这一假设，以揭示在转录因子组合表达的反应中指导MN规范的分子基础。具体来说，我们建议研究六聚体/bHLHs及其候选靶基因的不同功能方面。总的来说，拟议的研究应该为神经系统正确形成的基本原理和治疗各种神经元损伤和疾病的实用信息提供关键的见解。值得注意的是，LIM-HD/bHLH因子也参与了发育中的中枢神经系统其他区域（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）的细胞命运，这凸显了拟议研究的总体重要性。

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.