Mapping Somatosensory Afferent Circuitry to Bone Identifies Neurotrophic Signals Required for Fracture Healing

A central feature of bone injury is the sensation of pain, transmitted by nociceptive sensory nerves that innervate the skeleton. Indeed skeletal-innervating peripheral neurons are known to regulate skeletal repair and pathophysiology. Here, using retrograde peripheral nerve labeling and single cell RNA sequencing (scRNA-seq), we precisely identify the unique molecular signature of skeletal-innervating sensory neurons and the shifting neuronal transcriptomic landscape after bone fracture. Two methods to surgically or genetically denervate fractured bones were used in combination with scRNA-seq to implicate defective mesenchymal cell proliferation and osteodifferentiation as underlying the poor bone repair capacity in the presence of attenuated skeletal innervation. Multi-tissue scRNA-seq and interactome analyses implicated neuron-derived FGF9 as a potent regulator of fracture repair, a finding confirmed by in vitro assessments of neuron-to-skeletal mesenchyme interactions. Identification of FGF9 as a novel, neuron-derived skeletal growth factor uncovers a potential target for improving tissue repair. Overall design: To identify skeleton-innervating neurons, an engineered virus with enhanced tropism for peripheral neurons was injected into the midshaft of the ulnar periosteum of 14-week-old mice (3.5µl AAV-PHP.S-tdTomato). 4 weeks after injection, retrograde labeling of dorsal root ganglion (DRG) neurons with AAV-PHP.S-tdTomato was evaluated using whole-mount immunohistochemistry, scRNA-Seq and RNAscope (DRGs at C7, C8, T1 levels were used corresponding to the innervation pattern of the forelimb). To characterize temporal transcriptomic responses to ulnar stress fracture retrogradely labeled whole DRGs were harvested and dissociated at 1-, 14- and 56- days post-stress fracture and subjected to scRNA-Seq. To uncover ulna callus resident cells that may interact with ulna-innervating sensory neurons, callus single-cell transcriptomics atlas from control and denervated callus were generated 14 days post fracture. Denervation of callus was achieved by two methods: surgical neurectomy of ulna neurectomy and chemical inhibition by administrating small molecule 1NMPP1 to TrkAF592A mice. Interaction modalities were performed to reveal potential skeleton-innervating neuron-derived ligands that are involved in fracture repair.