Molecular specification of cortico-brainstem versus corticospinal projection neurons in development

Abstract Skilled motor control requires precise connections between subcerebral projection neurons (SCPN) in the cerebral cortex and their appropriate subcerebral targets in the brainstem or spinal cord. The brainstem is an important motor control center and cortical projections to the brainstem serve distinct motor control functions than corticospinal projections. However, mechanisms controlling cortico-brainstem versus corticospinal projections during development remain unknown. Here, we show that the transition between the brainstem and cervical cord distinguishes cortico-brainstem from corticospinal neurons from the earliest stages of SCPN axon extension in white matter. We used high throughput single-cell RNA sequencing of FACS-purified SCPN, retrogradely labeled from either the cerebral peduncle (labeling both cortico-brainstem and corticospinal neurons) or the cervical cord (labeling corticospinal neurons only) at critical times of axon extension. We identify that cortico-brainstem and corticospinal neurons are molecularly distinct: We establish Neuropeptide Y ( Npy ) as specifically enriched in cortico-brainstem neurons in lateral cortex, while CART prepropeptide ( Cartpt ) delineates cervical-projecting corticospinal neurons. Our results highlight molecular specification of cortico-brainstem vs. corticospinal projections well before these axons reach their appropriate segmental target and suggest a broad molecular program over SCPN axon targeting to distinct subcerebral targets early in development. These findings are likely to inform future investigations of motor circuit development, as well as approaches aimed at enhancing motor recovery after central nervous system damage. Highlights Cortico-brainstem neurons (CBN) limit their axon extension to supraspinal levels from the earliest time points of white matter axon extension in development. CBN can be molecularly delineated from corticospinal neurons (CSN) even at these initial time points. Molecular diversification of developing subcerebral projection neurons occurs across at least two axes: cortical location (medial vs. lateral) and projection targeting specificity (brainstem vs. spinal) Within lateral cortex, Neuropeptide Y ( Npy ) is expressed by CBN, while CART prepropeptide ( Cartpt ) expression delineates cervical-projecting CSN. Abstract Figure