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.