Subplate-dependent mechanisms of cortical circuit assembly

项目总结/摘要 大脑皮层功能需要在发育过程中正确组装电路连接。辅助板 神经元位于灰质和白色物质交界处，在大脑皮质的组装中起着重要的作用 电路，特别是在指导丘脑皮层轴突和形成感觉地图。亚板神经元具有 也与胼胝体的形成有关三种非细胞自主机制， 已经提出了支持轴突寻路的神经元：1）开拓性的，其中亚板神经元延伸第一 轴突并为皮质轴突铺设路径; 2）共成束，其中亚板下行轴突紧密 与上行轴突相互作用（“握手”）以引导相互连接; 3）细胞外基质，其中 亚板神经元为轴突在新生白色物质中生长提供基质。底板消融研究 显示亚板神经元是不可或缺的。然而，亚板神经元介导的遗传基础 轴突引导在很大程度上是未知的，部分原因是缺乏遗传途径亚板神经元在胚胎 电路发展阶段。在最近的一项研究中（Doyle等人，PNAS，2021），我们报告了一种遗传学策略， 靶向亚板神经元。用这种方法来询问亚板神经元中基因的必要性和充分性， 介导的电路组装，我们发现染色质重塑Arid 1a是必不可少的布线 亚板神经元的功能。人类ARID 1A突变是Coffin-Siris综合征的原因， 以胼胝体发育不全为特征的疾病。我们发现皮质Arid 1a缺失导致胼胝体发育不全， 丘脑皮质轴突走错让人联想到基板消融。这些错误连接的表型与 底板神经元转录特性的破坏，以及底板神经元布线功能的缺陷， 包括亚板-丘脑皮质轴突“握手”和细胞外基质。因此，在Arid 1a中，我们确定了一个 亚板神经元依赖性轴突引导功能的多功能调节剂-一个关键发现， 为亚板神经元的分子和机制研究打开了大门。在初步研究中， 策略，我们进一步确定了一个额外的调节子介导的电路布线的转录因子 Sox 5。人类SOX 5突变导致Lamb-Shaffer综合征，一种神经发育障碍， 智力残疾。在这里，我们将利用Arid 1a和Sox 5提供的特殊机会， 研究亚板神经元。我们将利用我们在分子遗传学，电路神经生物学，基因组学， 染色质生物学，以鉴定Arid 1a和Sox 5的转录组和基因组靶点（目的1和2）， 获得候选基因在亚板神经元依赖性轴突指导（目标3）的功能理解。一个关键 我们工作的意义是亚板神经元的缺陷可能是神经元损伤的一个未被充分认识的因素， 神经发育障碍中的电路错误布线，包括与染色质失调相关的那些。 因此，成功完成对亚板神经元的拟议研究将阐明基本机制 以及发育性脑障碍中基板功能障碍的潜在后果。

PROJECT SUMMARY/ABSTRACT Cerebral cortex function requires correct assembly of circuit connectivities during development. Subplate neurons, strategically positioned at the gray and white matter interface, play essential roles in assembling cortical circuits, notably in guidance of thalamocortical axons and formation of sensory maps. Subplate neurons have also been implicated in corpus callosum formation. Three non-cell autonomous mechanisms by which subplate neurons support axon pathfinding have been proposed: 1) pioneering, wherein subplate neurons extend the first axons and lay a path for cortical axons to follow; 2) co-fasciculation, wherein subplate descending axons closely interact (“handshake”) with ascending axons to guide reciprocal connectivity; 3) extracellular matrix, wherein subplate neurons provide a substrate for axon growth in nascent white matter. Subplate ablation studies have shown subplate neurons to be indispensable. However, the genetic underpinnings of subplate neuron-mediated axon guidance are largely unknown, in part due to a lack of genetic access to subplate neurons at embryonic stages of circuit development. In a recent study (Doyle et al., PNAS, 2021), we reported a strategy to genetically target subplate neurons. Using this approach to interrogate gene necessity and sufficiency in subplate neuron- mediated circuit assembly, we discovered that the chromatin remodeler Arid1a is essential for the wiring functions of subplate neurons. Human ARID1A mutations are a cause of Coffin-Siris syndrome, a developmental disorder characterized by callosal agenesis. We found that cortical Arid1a deletion led to callosal agenesis and thalamocortical axon misrouting reminiscent of subplate ablation. These miswiring phenotypes coincided with disruptions in the transcriptional identity of subplate neurons, and deficits in subplate neuron wiring functions, including subplate-thalamocortical axon “handshake” and extracellular matrix. Thus, in Arid1a, we identified a multifunctional regulator of subplate neuron-dependent axon guidance functions – a key discovery that opens doors to molecular and mechanistic studies on subplate neurons. In preliminary studies using the same genetic strategy, we further identified an additional regulator of subplate-mediated circuit wiring in the transcription factor Sox5. Mutations in human SOX5 cause Lamb-Shaffer syndrome, a neurodevelopmental disorder characterized by intellectual disability. Here, we will leverage the exceptional opportunities that Arid1a and Sox5 provide to study subplate neurons. We will use our expertise in molecular genetics, circuit neurobiology, genomics, and chromatin biology, to identify the transcriptomic and genomic targets of Arid1a and Sox5 (Aims 1 and 2) and gain a functional understanding of candidate genes in subplate neuron-dependent axon guidance (Aim 3). A key implication of our work is that deficits in subplate neurons may be an underappreciated contributor to neural circuit miswiring in neurodevelopmental disorders, including those associated with chromatin dysregulation. Successful completion of the proposed study on subplate neurons will thus illuminate fundamental mechanisms of circuit development, and the potential consequences of subplate dysfunction in developmental brain disorders.