Transcriptomic analysis of spinal V1 interneurons informs their multifunctional role in motor output.

Neural circuits in the spinal cord are composed of diverse populations of interneurons that play crucial roles in shaping motor output. However, the extent of interneuron heterogeneity and how this diversity relates to functional aspects of movement remain unclear. Here, through a focus on mouse spinal V1 interneurons, we show that loss of the V1 transcription factor En1 selectively disrupts the frequency of rhythmic locomotor output but does not disrupt flexion/extension limb movement, thereby decoupling two key functional roles ascribed to this neuronal population. To investigate the cellular basis of these deficits, we generated a single-nucleus transcriptomic atlas of V1 interneurons across postnatal development. Our analysis reveals age-dependent transcriptional changes while also demonstrating that their core molecular taxonomy perdures into adulthood. Notably, En1 deficiency selectively perturbed a single subset of V1Pou6f2 interneurons, thereby identifying a possible cellular substrate for influencing locomotor speed. Beyond serving as a molecular resource, our study highlights how deep neuronal profiling provides an entry point for understanding the multifunctional nature of heterogeneous interneuron populations.