Developmental molecular controls over arealization of descending cortical motor pathways

Abstract Layer 5 extratelencephalic (ET) neurons are a main class of neocortical projection neurons that predominate in the motor cortex and send their axon to the pons and spinal cord, and collaterals to the thalamus and multiple deep subcerebral structures. 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 select neurodegenerative disorders. ET neurons consist of several types of cells with distinct laminar and areal locations, molecular identities, connectivities, and functions. Within layer 5 of the cortex, two cardinal subtypes of ET neurons have been identified: “ET lower” neurons, which express Slco2a1 and project to distal targets including the spinal cord, and “ET upper“ neurons, which express Nprs1 or Hpgd and project more proximally to the pons and thalamus. 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 mapping, 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 ET lower and ET upper neurons and find that the latter can emerge by pruning of subsets of ET lower neurons. We next identify area- and ET neuron type-specific developmental transcriptional programs to identify key target genes in vivo. Finally, we reprogram ET neuron area-specific connectivity from motor to visual by postnatal in vivo combinatorial knockout of three key type-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 control.