Developmental molecular controls over arealization of descending cortical motor pathways [AbeP_GEO]

Layer 5 extratelencephalic (ET) neurons are a main class of neocortical projection neurons that predominate in the motor cortex and send their axon to multiple proximal and distal targets including the thalamus, pons, medulla and spinal cord1-6. Precise connectivity of ET neurons is critical for fine motor control; they are central to loss of function upon spinal cord injury and specifically degenerate in amyotrophic lateral sclerosis7-10. ET neurons consist of several subtypes of cells with distinct laminar and areal locations, molecular identities, connectivities, and functions11,12. Two cardinal subtypes of ET neurons have been identified: neurons that express Nprs1 or Hpgd and project proximally to the pons and thalamus (ETprox), and neurons that express Slco2a1 and project more distally to the pons, medulla and spinal cord (ETdist)11. Despite their critical function, how these neuronal subtypes emerge during development and acquire their area-specific distributions remains unaddressed. Here, using combinations of anatomical labeling, MAPseq mapping13, and single- nucleus transcriptomics across developing cortical areas, we reveal that these two subtypes of ET neurons are present at birth along opposite antero-posterior cortical gradients. We first characterize area-specific developmental axonal dynamics of ETprox and ETdist neurons and find that the former can emerge by pruning of subsets of ETdist neurons. We next identify area- and ET neuron subtype-specific developmental transcriptional programs to identify key target genes in vivo and predict their gene regulatory networks. Finally, we reprogram ET neuron subtype- specific connectivity from motor to visual-like, by generating more proximal connections through postnatal in vivo knockdown of three subtype-specific transcription factors. Together, these findings delineate the functional transcriptional programs controlling ET neuron diversity across cortical areas and provide a molecular blueprint to investigate and direct the developmental emergence of corticospinal motor function.