Distinct differentiation trajectories leave lasting impacts on gene regulation and function of V2a neurons [multiome_scATAC-seq]

In development, early regionalization steps segregate populations of cells and direct them to specific cell fates. In some instances, this process separates populations of progenitors which eventually become analogous cell types. V2a neurons are a population of excitatory interneurons which are found throughout the hindbrain and spinal cord, two regions of the CNS that arise from different progenitors in development. Plasticity in the V2a population after spinal cord injury has increased interest in this population as a therapeutic target. We therefore set out to examine how progenitor lineage influences V2a gene regulation and ultimately function. To do so, we differentiated V2a neurons from human stem cells via two distinct progenitor populations. Single nucleus multiomic analysis showed that different V2a lineages were enriched for different transcription factor motifs and differentially expressed hundreds of genes relevant to neuron function, such as axon growth and calcium handling. Attempting to ‘skip’ developmental patterning by induced transcription factor expression yielded a population unlike either developmentally relevant population, highlighting the importance of following developmental steps in establishing cell identities in vitro. Lastly, we employed the CellOracle tool and lentiviral knockdown to perturb lineage-specific gene regulatory networks and identify CREB5 and TCF7L2 as regulators unique to a spinal-like lineage. Overall, this work highlights how small differences in differentiation protocols can influence mature cell fate and uncovers new regulators of neural diversity along the anterior-posterior axis of the central nervous system.