UNLABELLED: Dorsal interneurons (dIs) in the spinal cord encode the perception of touch, pain, heat, itch, and proprioception. While previous studies using genetic strategies in animal models have revealed important insights into dI development, the molecular details by which dIs arise as distinct populations of neurons remain incomplete. We have developed a resource to investigate dI fate specification, by combining a single-cell RNA-Seq atlas of mouse ESC-derived dIs with pseudotime analyses. To validate this in silico resource as a useful tool, we have used it to first identify novel genes that are candidates for directing the transition states that lead to distinct dI lineage trajectories, and then validated them using in situ hybridization analyses in the developing mouse spinal cord in vivo . We have also identified a novel endpoint of the dI5 lineage trajectory, and found that dIs become more transcriptionally homogenous during terminal differentiation. Together, this study introduces a valuable tool for further discovery about the timing of gene expression during dI differentiation and uses it to clarify dI lineage relationships. SUMMARY STATEMENT: Pseudotime analyses of embryonic stem cell-derived dorsal spinal interneurons reveals both novel regulators and lineage relationships between different interneuron populations.
