Distinct differentiation trajectories leave lasting impacts on gene regulation and function of V2a neurons.

V2a interneurons are excitatory neurons found throughout the hindbrain and spinal cord, two regions that arise from distinct progenitors during embryonic development. Whether this lineage difference shapes mature gene regulation and function is unknown. V2a neurons show plasticity after spinal cord injury and are candidates for cell therapy. We differentiated human stem cells into V2a neurons through hindbrain- and spinal cord-like progenitor routes and profiled them by single nucleus multiomic sequencing. The two lineages showed distinct transcription factor motif enrichment and differentially expressed genes governing axon growth and calcium handling. Inducing V2a transcription factors directly, bypassing developmental patterning, produced a population unlike either lineage, confirming progenitor history is not interchangeable. Using CellOracle and lentiviral knockdown, we identified CREB5 and TCF7L2 as regulators specific to the spinal-like lineage. These results show that progenitor origin shapes V2a identity and reveal new regulators of neural diversity along the anterior-posterior axis.