Project Summary: Neural circuits in the spinal cord serve as the conduit through which the nervous system controls muscle contraction to implement behavior. Defining spinal circuit organization is therefore central to understanding the neural control of movement. One major challenge in resolving how spinal circuits direct motor output is the highly heterogeneous nature of spinal interneurons, which shape fundamental elements of limb movement underlying locomotion and skilled forelimb behaviors. Because our ability to resolve distinct interneuron cell types remains limited, little is known about the synaptic and circuit organization of spinal interneurons or their functional contributions to motor output. We recently discovered that spinal V1 interneurons, the largest inhibitory interneuron population in the spinal motor system, constitute a molecularly heterogeneous group that can be segregated into at least four mutually exclusive subsets (clades) defined by expression of the transcription factors Foxp2, MafA, Pou6f2, and Sp8. V1 clades exhibit restricted and highly stereotyped positions in the spinal cord, and several show distinct electrophysiological signatures. As such, V1 interneurons represent an ideal system in which to explore general principles of interneuron identity and circuitry governing motor output, of relevance to other classes of spinal interneurons. Motivated by our discovery of V1 interneuron diversity, this proposal aims to (1) define the molecular and cellular identity of these clades and the mechanisms through which this diversity arises, (2) test the hypothesis that descending motor pathways from the brain differentially innervate V1 clades, and (3) investigate how V1 interneurons influence one key aspect of motor control – the speed of rhythmic locomotor output. Together, the proposed experiments address a fundamental gap in knowledge about the identity, circuit organization, and function of interneurons in the spinal motor system, and serve as a foundation for future efforts aimed at dissecting the contributions of specific interneuron cell types to motor behavior, of relevance for developmental motor disorders and spinal cord injury.