Abstract
Motor neurons (MNs) connect the central nervous system to skeletal muscles, enabling movement. In zebrafish, the three MN subtypes, RoP, MiP, and CaP, are defined anatomically, but their molecular identities remain underexplored. Using single-cell transcriptomics of developing Tg(mnx1:gfp) zebrafish embryos, where MNs are specifically labeled, we identified 14 clusters, including differentiating primary MNs. Subclustering and pseudotime analysis resolved seven primary MN subtypes, that were assigned to early, intermediate, and terminal differentiation states, each with distinct gene expression profiles, involving transcription factors and membrane receptors associated with axon pathfinding and cell fate. Spatial validation using smiFISH confirmed subtype-specific marker expression within the spinal cord. Protein-protein interaction analysis revealed potential interactions between subtype-specific receptors and extracellular signaling molecules present in the motor axon environment. These findings define molecular signatures of primary MN subtypes, providing new insights into their identity and roles in motor axon decision-making during zebrafish development.