Retrograde tracing reveals segmental organization of sensory and sympathetic neurons innervating mouse limb bones with exploratory transcriptomic associations

Sensory and sympathetic nerves contribute to skeletal homeostasis, bone repair, and pain transmission, but the segmental origins of neurons innervating individual limb bones remain unclear. Here, we combined multiplex retrograde fluorescent tracing with three-dimensional imaging of solvent-cleared organs (3DISCO) to map sensory and sympathetic neurons projecting to mouse limb bones. Tracer-positive dorsal root ganglia (DRG) and sympathetic ganglia (SG) neurons were quantified across spinal segments, and normalized distributions were analyzed using dominant segments, dominance index, Shannon entropy, and hierarchical clustering. Upper-limb bones mainly received lower cervical and upper thoracic inputs, whereas lower-limb bones were associated with lumbar inputs. Sensory neurons innervating the humerus, radius, femur, and tibia were distributed across C4-T1, C5-C8, L1-L6, and L1-L5 DRG, respectively. Sympathetic neurons showed more restricted distributions, with humerus, radius, femur, and tibia inputs concentrated in T1-T2, T1, L4-L5, and L4 SG, respectively. Exploratory integration with public bone transcriptomic datasets suggested candidate molecular signatures related to developmental patterning, metabolic/cytoskeletal annotations, and repair- or immune-associated processes. This study provides a segmental anatomical reference for neuro skeletal communication.