Molecular programs guiding arealization of descending cortical pathways

Layer 5 extratelencephalic (ET) neurons are present across neocortical areas and send axons to multiple subcortical targets^1-6. 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^9-12. 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.