Mapping somatosensory afferent circuitry to bone identifies neurotrophic signals required for fracture healing.

The pain associated with bone fracture is mediated by somatosensory neurons, which also appear to be required to initiate bone regeneration. To characterize neuroanatomical circuitry mediating skeletal nociception and regeneration, we profiled dorsal root ganglia (DRG) neurons innervating murine bones using single-cell transcriptomics before and after fracture. CGRP+ and Aβ-Field LTMR neurons were the most represented classes of bone-innervating neurons. Dynamic changes in sensory neuron response to injury reflected the phasic nature of bone repair, including expression of morphogens such as Tgfb1, Fgf9, and Shh. Innervation loss resulted in poor bone repair and was associated with defective mesenchymal cell proliferation and osteodifferentiation. Finally, we identified fibroblast growth factor 9 (FGF9) as a major regulator of fracture repair that could be leveraged to promote bone repair. Editor's summary: Sensory neurons innervating bones can modulate the healing process (bone regeneration) after a fracture. Xu et al. used single-cell transcriptomics to characterize the bone-innervating dorsal root ganglia (DRG) neurons before and after an ulnar stress fracture in mice (see the Perspective by Rosen and Gori). The experiments revealed temporally dynamic responses of the DRG neurons after the fracture. Moreover, the authors identified fibroblast growth factor 9, released by sensory neurons, as a major neural regulator in bone regeneration after injury. These findings show that pathways activated by bone-innervating neurons after an injury might be exploited for accelerating bone fracture repair. —Mattia Maroso INTRODUCTION: The profound pain accompanying bone fracture is mediated by somatosensory neurons, which also appear to be required to initiate bone regeneration. Primary somatosensory neurons comprise a diverse subset of neurons, which communicate information about the external environment and internal state to the central nervous system, enabling perception and reaction to a wide range of stimuli including pain. RATIONALE: Most work in skeletal neurobiology has focused on understanding bone nociceptive pathways, but recent studies provide evidence that sensory nerves also function to initiate bone formation during skeletal morphogenesis. To what extent such bone morphogenic and nociceptive actions are mediated by distinct neuronal pathways has been difficult to study in part due to lack of tractable model systems for studying nerve-bone interactions and the extensive heterogeneity of peripheral sensory neurons. RESULTS: To characterize neuroanatomical circuitry mediating skeletal nociception and regeneration, dorsal root ganglia (DRG) neurons innervating murine long bones were profiled by retrograde nerve labeling and single-cell transcriptomics before and after experimental fracture. Highest labeling across CGRP+ and Aβ-Field LTMR neurons was identified, which have nociceptive or mechanoreceptive functions. Dynamic changes associated with sensory neuron response to injury reflected the phasic nature of bone repair. At early time points, DRG neurons showed signatures of pain perception and inflammatory responses. At later time points, DRG neurons demonstrated transcriptomic changes more characteristic of a regenerative response, including mitogenic, angiogenic and osteogenic signals. This includes expression of morphogens in the fracture reparative phase such as Tgfb1, Fgf9, and Shh. Two methods to surgically or genetically denervate fractured bones were used to implicate defective mesenchymal cell proliferation and osteodifferentiation as underlying poor bone repair with loss of innervation. Finally, multitissue single-cell RNA-sequencing and interactome analyses implicated neuron-derived fibroblast growth factor 9 (FGF9) as a potent regulator of fracture repair, confirmed by in vivo sensory nerve–specific ablation studies. CONCLUSION: In sum, somatosensory neurons innervating bone are a group of neurons with nociceptive and mechanoreceptive functions, which are transcriptionally responsive to bone injury in a temporal dynamic fashion and positively regulate fracture healing through FGF9-FGFR signaling. Optically cleared whole-mount image of peripheral nerves interacting with a fractured ulna.: Red, Beta III Tubulin (TUBB3)–stained nerves. Green, autofluorescence in bone fracture site. [ABSTRACT FROM AUTHOR]