Spatial transcriptomics reveals a role for sensory nerves in preserving cranial suture patency through modulation of BMP/TGF-β signaling – pnas.org

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Edited by Clifford J. Tabin, Harvard Medical School, Boston, MA, and approved September 2, 2021 (received for review February 15, 2021)
Sensory nerves innervate bone and are required for normal skeletal development, but the molecular mechanisms through which nerves exert these essential functions have remained obscure. Here, we validate a spatial transcriptomics platform, which allowed us to map changes in the molecular landscape across the developing cranium of mice in which sensory nerve innervation is genetically blocked. We show that sensory, nerve-derived signals, including follistatin-like 1, coordinate cranial bone patterning by regulating the proliferation and differentiation of bone precursor cells within the suture mesenchyme.
The patterning and ossification of the mammalian skeleton requires the coordinated actions of both intrinsic bone morphogens and extrinsic neurovascular signals, which function in a temporal and spatial fashion to control mesenchymal progenitor cell (MPC) fate. Here, we show the genetic inhibition of tropomyosin receptor kinase A (TrkA) sensory nerve innervation of the developing cranium results in premature calvarial suture closure, associated with a decrease in suture MPC proliferation and increased mineralization. In vitro, axons from peripheral afferent neurons derived from dorsal root ganglions (DRGs) of wild-type mice induce MPC proliferation in a spatially restricted manner via a soluble factor when cocultured in microfluidic chambers. Comparative spatial transcriptomic analysis of the cranial sutures in vivo confirmed a positive association between sensory axons and proliferative MPCs. SpatialTime analysis across the developing suture revealed regional-specific alterations in bone morphogenetic protein (BMP) and TGF-β signaling pathway transcripts in response to TrkA inhibition. RNA sequencing of DRG cell bodies, following direct, axonal coculture with MPCs, confirmed the alterations in BMP/TGF-β signaling pathway transcripts. Among these, the BMP inhibitor follistatin-like 1 (FSTL1) replicated key features of the neural-to-bone influence, including mitogenic and anti-osteogenic effects via the inhibition of BMP/TGF-β signaling. Taken together, our results demonstrate that sensory nerve-derived signals, including FSTL1, function to coordinate cranial bone patterning by regulating MPC proliferation and differentiation in the suture mesenchyme.
Author contributions: R.J.T., Z.L., A.W.J., and T.L.C. designed research; R.J.T., Z.L., X.-W.W., L.R., Q.Z., and S.N. performed research; R.J.T., Y.-H.C., C.R.U., and P.C. contributed new reagents/analytic tools; R.J.T., Z.L., A.V., F.-Q.Z., P.C., A.W.J., and T.L.C. analyzed data; and R.J.T., A.W.J., and T.L.C. wrote the paper.
Competing interest statement: 10X Genomics provided supplies and expert consultation for the study. A.W.J. is a paid consultant for Novadip and Lifesprout LLC. This arrangement has been reviewed and approved by Johns Hopkins University in accordance with its conflict-of-interest policies.
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This article contains supporting information online at https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.2103087118/-/DCSupplemental.
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