✍🏼 Julia Kaiser, Payal Patel, Friederike Dündar, Jimena Perez-Tetuan, Nirupama Angira, Eytan Sieger, Vibhu Sahni

🏠 Burke Neurological Institute, White Plains, NY

📑 bioRxiv (2022)

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.

How is the WOLF used in this study
In this study, the WOLF Cell sorter was used to purify retrogradely labeled layer V projection neurons for high-resolution single-cell transcriptomic analysis. Following retrograde labeling of subcerebral projection neurons (SCPN) or corticospinal neurons (CSN) with CTB 555, cortical tissue was dissected, enzymatically and mechanically dissociated into single-cell suspensions, and filtered to remove debris. CTB-positive neurons were then enriched using the WOLF sorter with optimized size thresholds and a two-step sorting strategy—first at high cell concentration to rapidly enrich labeled cells, followed by a second sort at lower concentration to improve purity and reduce contamination. Approximately 15,000 labeled neurons were collected per sort and immediately processed for downstream 10x Genomics single-cell RNA sequencing. The gentle microfluidic sorting approach enabled efficient isolation of viable, projection-defined neuronal populations, allowing the authors to define transcriptional differences between brainstem- and spinal cord–projecting layer V neurons during early postnatal development.