Molecular development and diversity of callosal projection neurons

 描述(申请人提供)：这项拟议研究的长期目标既是阐明对新皮质穹隆投射神经元(CPN)发育和多样性的中央分子控制，也是为了确定与CPN有关的疾病的潜在原因和治疗方法。CPN是广泛分布的大脑半球间锥体神经元，其轴突通过穹隆体连接到两个大脑半球。CPN在高水平的联想、整合、认知、行为、感觉和运动功能中发挥关键作用，其基础是精确的、特定区域的CPN亚型连通性和多样性。CPN发育的中断与多种认知和行为障碍的缺陷有关，包括胼胝体发育不全、自闭症谱系障碍和精神分裂症。目前，CPN亚型和连接性的显著多样性是如何确定的，以及它们如何分化形成高度精确和特定的电路，目前尚不清楚。我们的实验室最近发现了一组组合表达的基因，这些基因既将CPN定义为一个广泛的种群，又在胚胎和出生后早期发育过程中识别了CPN的新亚群(Arlotta，Neuron，2005；Molyneaux，J Neurosci，2009)。在我们以前工作和大量初步数据的基础上，我们建议对Cited2进行深入而严格的功能研究，Cited2是一种新发现的转录协同调控因子，我们假设它在CPN的区域和功能特异性亚型的发展中具有重要功能，特别是体感CPN，其功能障碍可能导致CPN连通性和多样性的障碍。我们已经完成了对Cited2在新皮质发育中功能的相当实质性的、高度鼓舞人心的初步研究，导致了这样的假设：Cited2调控和精炼CPn精确发育和多样性的两个阶段，1)广泛地在基础前体细胞中调节浅层CPn的产生，2)以区域受限的方式精炼体感(S1)CPn的独特身份和发育。为了进一步研究这一假说，我们建议：目的1)研究Cited2缺失Cited2的CPN轴突靶向的发育和精确度；目的2)确定Cited2在祖细胞中与Cited2功能不同的有丝分裂后特异性功能(S)；目的3)通过用于双种群分析的新型嵌合、重组酶遗传操纵技术(“BEAM”)，研究Cited2功能在S1CPN发育和连通性中的独立性；以及目的4)通过全新的创新方法研究WT vs Cited2CKO S1CPN在轴突发育过程中的生长锥RNA和蛋白质组，以直接从机制上了解CPN电路的发育。总之，这些拟议的研究将为分子控制CPN亚型的发育和多样性提供实质性的见解，这些亚型具有不同的连接性、功能和皮质信息的整合，这些过程在多种人类疾病中集中中断。目前，对CPN多样性的这种控制基本上是未知的。这项研究将有助于了解皮质的组织、功能、进化，并有可能预防、诊断和未来人类发育障碍的治疗。

 DESCRIPTION (provided by applicant): The long-term goals of the proposed research are both to elucidate central molecular controls over development and diversity of neocortical callosal projection neurons (CPN), and to identify potential causes and therapeutic approaches to disease involving CPN. CPN are the broad population of interhemispheric pyramidal neurons whose axons connect the two cerebral hemispheres via the corpus callosum. CPN play key roles in high-level associative, integrative, cognitive, behavioral, sensory, and motor functions, based on precise, area-specific CPN subtype connectivity and diversity. Disruptions in CPN development are correlated with deficits in multiple cognitive and behavioral disorders, including agenesis of the corpus callosum, autism spectrum disorders, and schizophrenia. Currently, how the remarkable diversity of CPN subtypes and connectivity is specified, and how they differentiate to form highly precise and specific circuits, is unknown. Our lab recently identifieda combinatorially-expressed set of genes that both define CPN as a broad population, and identify novel subpopulations of CPN during embryonic and early postnatal development (Arlotta, Neuron, 2005; Molyneaux, J Neurosci, 2009). Building on our previous work and substantial preliminary data, we propose deep and rigorous functional investigation of Cited2, one newly identified transcriptional co-regulator that we hypothesize functions importantly in development of areally- and functionally-specific subtypes of CPN, notably somatosensory CPN, and its dysfunction might contribute to disorders of CPN connectivity and diversity. We have completed quite substantial, highly motivating preliminary studies of Cited2 function in neocortical development, leading to the hypothesis that Cited2 regulates and refines two stages of precise CPN development and diversity, functioning 1) broadly in basal progenitors to regulate generation of superficial layer CPN, and 2) in an areally-restricted manner to refine distinct identity and development of somatosensory (S1) CPN. To further investigate this hypothesis, we propose to: Aim 1) investigate development and precision of Cited2-null CPN axonal targeting; Aim 2) identify potential postmitotic-specific function(s) of Cited2 that are distinct from Cited2 function in progenitors; Aim 3) investigate CPN-autonomy of Cited2 function during S1 CPN development and connectivity, via novel mosaic, recombinase-based genetic manipulation technology ("BEAM") for dual population analysis; and Aim 4) investigate growth cone RNA and proteomes of WT vs Cited2 cKO S1 CPN during axonal development via an entirely new and innovative approach, to gain direct mechanistic understanding of CPN circuit development. Together, these proposed studies will provide substantial insight into molecular controls over development and diversity of CPN subtypes with distinct connectivity, function, and integration of cortical information, processes centrally disrupted in multiple human disorders. Such controls over CPN diversity are currently essentially unknown. This research will contribute to understanding of cortical organization, function, evolution, and potentially toward prevention, diagnosis, and future therapy of human developmental disorders.