LIM-HD/bHLH Combinatorial Code in Motoneurons

描述(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，这些网络在指定大量不同的神经元和神经胶质细胞类型方面发挥着至关重要的作用。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控细胞类型规范的相对较好的转录因子。然而，这些因子调节基因表达的分子机制及其靶基因的特性仍然知之甚少。我们希望通过特别关注它们在脊髓运动神经元(MN)产生中的作用来探讨这些重要问题。我们的结果表明，LIM-HD因子Lhx3和Isl1以组合的方式指定了胚胎脊髓中两个不同的神经元。LHX3和LIM辅因子NLi(核LIM相互作用因子)形成V2-中间神经元(IN)特异性四聚体。相反，当Lhx3和Nli与Isl1共表达时，形成MN指定的六角体。在MN规范过程中，bHLH蛋白Ngn2/Neurm在功能上与六角体同步，尽管这种新的串扰背后的分子机制尚不完全清楚。我们最近的筛选工作导致发现了带有NLI：LSL1：Lhx3六聚体结合位点的基因组片段，其中许多与候选MN基因相关联。这些结果导致了我们对这一提议的中心假设：六角体直接调控多个MN基因，与bHLH因子一起影响MN规范。我们将剖析这一假设，以揭示指导MN规范的分子基础，以响应转录因子的组合表达。具体地说，研究是为了探索六聚体/bHLHs及其候选靶基因的不同功能方面。总体而言，拟议的研究应该为神经系统的正确形成提供关键的洞察力，并为治疗各种神经元损伤和疾病提供实用信息。值得注意的是，LIM-HD/bHLH因子也参与了指定发育中的中枢神经系统其他区域(例如视网膜和前脑)以及非中枢器官如心脏、胰腺和垂体的细胞命运，这突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.