Gene expression programs underlying spinal commissural neuron differentiation and axon growth across the midline

Commissural neurons in the spinal cord project axons across the midline to fulfill various critical functions in somatosensory information processing and left-right coordination of motor output. These neurons have long served as a powerful model system for axon pathfinding, driving the identification of molecules and mechanistic principles for neuronal wiring that apply widely across neural circuits and model organisms. Aside from their shared initial projection path, commissural neurons are highly heterogenous, which has hampered lineage-based methods for accessing them, and most prior studies have been limited to small subpopulations of dorsal commissural neurons. The shared transcriptional programs for axonal midline crossing and the divergent gene expression signatures that define commissural neuron subtypes have therefore remained largely elusive. Here, we combine a genetic labeling strategy with single-cell RNA sequencing, birthdating, and complementary methods to study the production and transcriptomic trajectories of developing commissural neurons in the mouse embryonic spinal cord. We identify molecularly distinct commissural neuron subtypes that reveal stratification of this neuronal population beyond progenitor identities and temporal cohorts of neurogenesis, and we provide evidence that axon laterality is not linked to neuronal birthdate. We also uncover a large-scale gene expression switch that coincides with commissural axon growth across the midline and underlies differential axonal protein expression for guidance toward and away from the midline. Our findings elucidate both shared and subtype-specific gene expression programs that drive commissural neuron differentiation and axon midline crossing, and they provide a valuable resource for future studies of commissural neuron development and function.