Layer 5 extratelencephalic (ET) neurons are present across neocortical areas and send axons to multiple subcortical targets^1,2,3,4,5,6. Two cardinal subtypes exist^7,8: (1) Slco2a1-expressing neurons (ET_dist), which predominate in the motor cortex and project distally to the pons, medulla and spinal cord; and (2) Nprs1- or Hpgd-expressing neurons (ET_prox), which predominate in the visual cortex and project more proximally to the pons and thalamus. An understanding of how area-specific ET_dist and ET_prox emerge during development is important because they are critical for fine motor skills and are susceptible to spinal cord injury and amyotrophic lateral sclerosis^9,10,11,12. Here, using cross-areal mapping of axonal projections in the mouse neocortex, we identify the subtype-specific developmental dynamics of ET neurons. Whereas subsets of ET_prox emerge by pruning of ET_dist axons, others emerge de novo. We outline corresponding subtype-specific developmental transcriptional programs using single-nucleus sequencing. Leveraging these findings, we use postnatal in vivo knockdown of subtype-specific transcription factors to reprogram ET neuron connectivity towards more proximal targets. Together, these results show the functional transcriptional programs driving ET neuron diversity and uncover cell subtype-specific gene regulatory networks that can be manipulated to direct target specificity in motor corticofugal pathways.

第 5 层端脑外 （ET） 神经元存在于新皮层区域，并将轴突发送到多个皮层下靶点^1,2,3,4,5,6。存在两种主要亚型^7,8：（1） 表达 Slco2a1 的神经元 （ET_dist），主要存在于运动皮层，并向远端投射到脑桥、延髓和脊髓;（2） 表达 Nprs1 或 Hpgd 的神经元 （ET_prox），它们在视觉皮层中占主导地位，并更靠近脑桥和丘脑。了解区域特异性 ET_dist 和 ET_prox 在发育过程中如何出现很重要，因为它们对精细运动技能至关重要，并且容易受到脊髓损伤和肌萎缩侧索硬化症的影响^9,10,11,12。在这里，使用小鼠新皮层中轴突投射的跨区域映射，我们确定了 ET 神经元的亚型特异性发育动力学。虽然 ET_prox 的子集是通过修剪 ET_dist 轴突出现的，但其他亚群是从头出现的。我们使用单核测序概述了相应的亚型特异性发育转录程序。利用这些发现，我们使用亚型特异性转录因子的出生后体内敲低来重编程 ET 神经元连接，朝向更近端的靶标。总之，这些结果显示了驱动 ET 神经元多样性的功能性转录程序，并揭示了细胞亚型特异性基因调控网络，这些网络可以纵以指导运动皮质通路中的靶标特异性。