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. 2021 Oct 19;118(42):e2103087118.
doi: 10.1073/pnas.2103087118.

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

Robert J Tower  1 , Zhu Li  1 , Yu-Hao Cheng  2 , Xue-Wei Wang  1 , Labchan Rajbhandari  3 , Qian Zhang  1   4 , Stefano Negri  5 , Cedric R Uytingco  6 , Arun Venkatesan  3 , Feng-Quan Zhou  1 , Patrick Cahan  2 , Aaron W James  7 , Thomas L Clemens  8   4
Affiliations

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

Robert J Tower et al. Proc Natl Acad Sci U S A. .

Abstract

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.

Keywords: TrkA; calvarial bone; cranial suture; skeletal innervation; spatial transcriptomics.

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Conflict of interest statement

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.

Figures

Fig. 1.
Fig. 1.
TrkA signaling is essential for maintaining cranial suture patency. (A) Whole-mount calvaria images of the sagittal suture within NGF-eGFP reporter animals subjected to TUBB3 immunohistochemical staining. (Scale bar, 500 µm.) (B) Whole-mount calvaria images of the coronal and lambdoid sutures within NGF-eGFP reporter animals subjected to TUBB3 immunohistochemical staining. (Scale bar, 100 µm.) (C) Coronal section of P0 sagittal suture stained for TUBB3. (Scale bar, 100 µm.) (D) Quantification of TUBB3+ nerve area within the OF or midline (Mid) suture mesenchyme. n = 4 to 5 mice/genotype. (E) Calvarial, whole-mount skeletal staining (top-down view) conducted at P0 following treatment with 1NMPP1, initiated at the time of conception. Oc, occipital bone and Pa, parietal bone. (F) Average suture width among posterior frontal and sagittal sutures within control (Cont) and TrkAF592A mice. n = 5 to 9 mice/genotype. (G) Micro-CT reconstructions of P21 calvaria (top-down view) and sagittal suture (coronal cross-section), following treatment with 1NMPP1, initiated at P6. Numbers below indicate the average sagittal suture width. (H) Percent of sagittal suture closure within Cont and TrkAF592A mice. (I) 2D average suture width between flanking parietal bones relative to the anterior–posterior position. n = 7 mice/genotype. Graphs represent average values ± SD, *P < 0.05, and **P < 0.01.
Fig. 2.
Fig. 2.
TrkA+ sensory nerves promote the proliferative expansion of the cranial sutures via a spatially restricted, secreted ligand. (A) Proliferation and innervation of the P0 sagittal suture among control (Cont) and TrkAF592A mutant animals, visualized via EdU-labeling and TUBB3 immunohistochemical staining. (Scale bar, 200 µm.) (B) Quantification of EdU incorporation within the OF or midline (Mid) suture mesenchyme among Cont and TrkAF592A mutant animals. (Scale bar, 50 µm.) n = 4. (C) Proliferation of primary MPCs following direct coculture with DRG axons using a microfluidic device, in which chambers are connected via microchannels. (Scale bar, 10 µm.) TUBB3 immunohistochemical staining is used to visualize axons. (D) Spatial distribution of EdU+ MPCs in relation to the nearest DRG axon. n = 4. (E) Quantification of EdU labeling in MPCs following treatment with DRG-derived, neural conditioned media (CM). n = 4. (Scale bar, 25 µm.) (F) MAR of developing parietal bones in Cont and TrkAF592A mutant calvaria. n = 8. Graphs represent average values ± SD, *P < 0.05, **P < 0.01, and ***P < 0.001.
Fig. 3.
Fig. 3.
In vivo spatial transcriptomics confirms the attenuation of proliferative markers in the sagittal suture of TrkAF592A mice. (A) Spatial feature plot and suture complex quantification of Tubb3 expression. (B) Cell-cycle gene Ccna2 and predictive cell-cycle phase scoring of spots within the sagittal suture of control (Cont) and TrkAF592A mice. (C) Representative calvarial suture with spots analyzed using SpatialTime. (D) Validation of SpatialTime analysis using known suture/progenitor (Top) and osteogenic (Bottom) markers. n = 5 to 7 mice/genotype. (E) Changes in cell-cycle scoring across SpatialTime. Relative SpatialTime regions aligning to the midline (Mid) suture, OF, and flanking parietal bone (Bone) are denoted above graph.
Fig. 4.
Fig. 4.
Spatial transcriptomics demonstrate a disruption in BMP/TGF-β signaling in TrkAF592A calvaria. (A) Violin plot and spatial expression data of BMP-signaling module scoring among control (Cont) and TrkAF592A calvaria, including BMP activators, BMP inhibitors, and overall BMP signaling. (B) Heatmap of BMP activator and inhibitor genes expressed across SpatialTime among Cont and TrkAF592A calvaria. (C) Overall BMP activation, based on module scoring across SpatialTime from midline (Mid) suture to flanking parietal bone (Bone) among Cont and TrkAF592A calvaria. Relative SpatialTime regions aligning to the Mid suture, OF, and flanking Bone are denoted above graph. n = 5 to 7 mice/genotype. (D) Canonical BMP signaling, as detected via immunofluorescent staining for p-SMAD1/5/9 using coronal cross-sections of the sagittal suture and Bones. (Scale bar, 200 µm.) (E) Violin plot and spatial expression of TGF-β signaling module scoring among Cont and TrkAF592A calvaria, including TGF-β activators, TGF-β inhibitors, and overall TGF-β signaling. (F) Heatmap of TGF-β activator and inhibitor genes expressed across SpatialTime among Cont and TrkAF592A calvaria. (G) Overall TGF-β activation based on module scoring across SpatialTime, from Mid suture to flanking Bone among Cont and TrkAF592A calvaria. Relative SpatialTime regions aligning to the Mid suture, OF, and flanking Bone are denoted above graph. n = 5 to 7 mice/genotype. (H) Canonical TGF-β signaling, as assessed by immunofluorescent staining for p-SMAD2 using coronal cross-sections of the sagittal suture and bones. (Scale bar, 200 µm.)
Fig. 5.
Fig. 5.
FSTL1 is secreted by TrkA+ sensory nerves and regulates MPC proliferation and differentiation. (A) UMAP projection of merged scRNAseq datasets of E18.5 calvarial bone resident cells (21) and lumbar DRG neurons (19). (B) Violin plot of cluster marker genes. (C) Candidate secreted factors showing the enriched expression in DRG neurons relative to other cell populations. (D) Subclustering of DRG cells into distinct neural subtypes. PEP, peptidergic nociceptors; TH, tyrosine hydroxylase containing; NP, nonpeptidergic nociceptors; and NF, neurofilament. (E) Violin plot of DRG subpopulation marker genes. (F) Feature and violin plot showing the expression of TrkA (encoded by Ntrk1) by PEP and NP neural subtypes. (G) Violin plot showing the expression of candidate genes in different neural subtypes. (H) Expression of candidate genes showing the enriched expression in TrkA+ neural subtypes in control DRG cell bodies or following direct, axonal coculture with MPCs. (I) Proliferation of MPCs following treatment with rmFSTL1 (250 ng/mL) for 48 h. (J) Gene expression following early osteogenic induction with or without rmFSTL1. The dotted line denotes values obtained at day 0 of osteogenic induction. (K) Alizarin red (AR) staining and quantification of MPCs following 21 d of osteogenic differentiation with or without rmFSTL1 (250 ng/mL). n = 4. (L) Proliferation of MPCs following treatment with fresh neural media or media condition by nontargeting (NT CM) or Fstl1-targeted (KD CM) siRNA. n = 3. Graphs represent average values ± SD, *P < 0.05, **P < 0.01, and ***P < 0.001. a.u., arbitrary units; EC, endothelial cells.
Fig. 6.
Fig. 6.
Overview of sensory, nerve-mediated regulation of suture plasticity. The suture is maintained by a balance of expansive progenitor cell proliferation and inward closing mineralization along the bony plates. TrkA+ sensory nerves, guided to the cranial suture by NGF, secrete factors such as FSTL1, regulating progenitor activation and expansion and inhibiting BMP-mediated terminal differentiation. TrkA inhibition impairs sensory innervation and reduces suture mesenchyme expansion, resulting in premature suture closure.
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