Axon guidance cue SLIT2 regulates the murine skeletal stem cell niche through sympathetic innervation

Abstract

Sympathetic tone is a central signaling axis inhibiting osteogenesis; however, the combination of durable local and systemic sympathetic effects on bone argues that multiple mechanisms, including yet-undiscovered pathways, are involved. Here, we found that sympathetic nerves constituted a component of the skeletal stem cell (SSC) niche: mice with conditional deletion of the classical axonal repellent Slit2 in sympathetic nerves (Slit2th mice), but not in bone stem/progenitor cells or sensory nerves, showed osteopenia due to an increase in sympathetic innervation and an associated decrease in SSCs. Mice with increased skeletal sympathetic innervation displayed impaired SSC niche function in an SSC orthotopic transplantation and engraftment system. Follistatin-like 1 (FSTL1) is a SLIT2-regulated soluble factor suppressing SSC self-renewal and osteogenic capacity. Accordingly, ablation of Fstl1 in sympathetic neurons enhanced SSC-driven osteogenesis and attenuated the bone loss seen in Slit2th mice. Together, the findings indicate that SLIT2 is a regulator of a sympathetic nerve-mediated SSC niche.

Keywords: Bone biology; Bone development; Neuroscience.

Figure 1. Bone loss, impaired bone formation, and sympathetic hyperinnervation in Slit2^syn1 mice.

(A and B) Representative μCT images of trabecular bone in the distal femur (A) and BV/TV with relative quantitative analysis of bone parameters (B) in Slit2^fl/fl and Slit2^syn1 male mice at 8 weeks of age. Slit2^fl/fl, n = 11; Slit2^syn1, n = 10. Scale bars: 500 μm. (C) Representative images of Von Kossa/Van Gieson staining and BV/TV of the L3 vertebral bone in 8-week-old Slit2^fl/fl and Slit2^syn1 male mice. n = 7 per group. Scale bars: 500 μm. (D and E) Representative images of calcein double labeling (D) and quantification of histomorphometric parameters of the L3 vertebrae (E) in Slit2^fl/fl and Slit2^syn1 male mice at 8 weeks of age. MAR, trabecular mineral apposition rate (μm/day); BFR/BS, bone formation rate/bone surface (μm³/μm/yr); Ob.S/BS, osteoblast surface/bone surface (%); (No.Oc./B.Pm, osteoclast number/bone perimeter. MAR and BFR/BS: n = 6 per group; Ob.S/BS: n = 7 per group; No.Oc./B.Pm: n = 6 per group. Scale bars: 100 μm. (F) Representative confocal images of immunofluorescence staining with TH (red) and DAPI (blue) and quantitative analysis of relative quantity of TH⁺ sympathetic nerves in femur sections from 3-week-old Slit2^fl/fl and Slit2^syn1 male mice. Arrowheads indicate TH⁺ sympathetic nerve fibers. n = 10 per group. Scale bars: 400 μm. (G) Images of tibiae prior to and after tissue clearing. Representative images of whole-tissue TH immunofluorescence labeling of femurs from 3-week-old Slit2^fl/fl and Slit2^syn1 male mice. Scale bars: 400 μm. Error bars indicate mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, by unpaired, 2-tailed Student’s t test for 2-group comparisons.

Figure 2. Slit2^th but not Slit2^adv mice recapitulate the skeletal and nerve phenotypes of Slit2^syn1 mice.

(A and B) Representative μCT images of trabecular bone in the distal femur (A) and relative quantitative analysis of BV/TV and relative quantitative analysis of bone parameters (B) in male control (Th-cre and Slit2^fl/fl) and Slit2^th mice at 8 weeks of age. Th-cre, n = 6; Slit2^fl/fl; n = 14; Slit2^th; n = 15. Scale bars: 500 μm. (C and D) Representative μCT images of trabecular bone in the distal femur (C) and relative quantitative analysis of BV/TV and of bone parameters (D) in male control (Adv-cre and Slit2^fl/fl) and Slit2^adv mice at 8 weeks of age. Adv-cre, n = 5; Slit2^fl/fl, n = 13; Slit2^adv. n = 12. Scale bars: 500 μm. (E) Representative confocal images of immunofluorescence staining with TH (red) and DAPI (blue) and quantitative analysis of relative TH⁺ sympathetic nerves in femur sections from 3-week-old Slit2^fl/fl and Slit2^th male mice. Slit2^fl/fl, n = 15; Slit2^th. n = 17. Scale bars: 500 μm. (F) Representative images of whole-femur immunofluorescence labeling of TH⁺ sympathetic nerves in 3-week-old Slit2^fl/fl and Slit2^th male mice. Scale bars: 500 μm. (G) Representative images of Von Kossa/Van Gieson staining and BV/TV of the L3 vertebral bone in 8-week-old Slit2^fl/fl and Slit2^th male mice. n = 7 per group. Scale bars: 500 μm. (H and I) Representative images of calcein double labeling (H) and quantification of histomorphometric parameters of L3 vertebrae in Slit2^fl/fl and Slit2^th male mice at 8 weeks of age. MAR trabecular mineral apposition rate (μm day⁻¹); BFR/BS, bone formation rate/bone surface (μm³μm⁻²yr⁻¹); Ob.S/BS, osteoblast surface/bone surface (%); No.Oc./B.Pm, osteoclast number/bone perimeter (I). MAR and BFR/BS: n = 6 per group; Ob.S/BS and No.Oc./B. Pm: n = 6 per group; Scale bars: 100 μm. Error bars indicate mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, by ordinary 1-way ANOVA for multiple-group comparisons (B and D). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, by unpaired, 2-tailed Student’s t test for 2-group comparisons (E,G, and I).

Figure 3. Sympathetic innervation negatively controls SSC abundance.

(A) Representative flow cytometry plots and relative frequency of SSCs from femurs of 3-week-old Slit2^fl/fl and Slit2^th male mice. n = 5 per group. (B) Representative flow cytometry and quantitative analysis of the relative frequency of Ki-67⁺ fractions of SSCs (expressed as a proportion of the total Lin-CD31-CD51+ progenitor population) isolated from the femurs of 3-week-old Slit2^fl/fl and Slit2^th male mice. n = 4 per group. (C) Timeline of the 6-OHDA chemical sympathectomy mouse models. (D) Representative flow cytometry plots and relative frequency of SSCs in the femurs of 3-week-old WT male mice treated with 6-OHDA or vehicle as neonates. n = 7 per group. (E) Statistical analysis of body weight of 3-week-old WT male mice treated with 6-OHDA or vehicle as neonates. n = 7 per group. (F) Representative confocal images of immunofluorescence staining with TH (red) and DAPI (blue) and quantitative analysis of TH⁺ sympathetic nerves in femur sections from 3-week-old WT male mice treated with 6-OHDA or vehicle at the neonatal stage. n = 4 per group. Scale bars: 500 μm. (G) Representative confocal images of immunofluorescence staining with CGRP (green) and DAPI (blue) and quantitative analysis of CGRP⁺ sensory nerves in femur sections from 3-week-old WT male mice treated with 6-OHDA or vehicle as neonates. n = 6 per group. Scale bars: 500 μm. (H) Representative flow cytometry showing the relative frequency of femur SSCs in 3-week-old WT male mice after sympathectomy surgery. n = 7 per group. Sham, sham surgery group; Den, denervation group. (I) Representative confocal images of immunofluorescence staining with TH and DAPI and quantitative analysis of TH⁺ sympathetic nerves in femur sections from 3-week-old WT male mice after sympathectomy surgery. n = 6 per group. Scale bars: 500 μm. Error bars indicate mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, by unpaired, 2-tailed Student’s t test for 2-group comparisons.

Figure 4. Sympathetic nerves are a component of the SSC niche.

(A) Experimental model of mice bone marrow cavity orthotopic transplantation for SSCs. Approximately 2 × 10⁴ SSCs and 1 × 10⁷ bone marrow cells (after RBC lysis) were transplanted into each recipient mouse. (AUTHOR: You may wish to clarify the label “Mice harvest” in A and E. Do you mean “Organoid harvest”?) (B) Representative flow cytometry plots and relative frequency of tdTomato⁺ SSCs from the femurs of Slit2^fl/fl-MIP-GFP and Slit2^th-MIP-GFP male mice 1 month after surgery. n = 4 per group. (C) Representative images and quantitative analysis of OSX⁺ osteoblast immunofluorescence staining in the femurs of Slit2^fl/fl-MIP-GFP and Slit2^th-MIP-GFP male mice 4 weeks after tdTomato⁺ SSC bone marrow cavity transplantation. td⁺ OSX⁺/BS, tdTomato⁺ OSX⁺/bone area. n = 4 per group. Scale bars: 100 μm. (D) Representative images and quantitative analysis of FABP4 immunofluorescence to visualize adipocytes in femurs of Slit2^fl/fl-MIP-GFP and Slit2^th-MIP-GFP male mice 4 weeks after orthotopic bone marrow cavity transplantation of tdTomato⁺ SSCs. N.td⁺ FABP4⁺/BC, number of tdTomato⁺FABP4⁺/bone cavity. n = 6 per group. Scale bars: 100 μm. (E) Experimental model of mouse renal subcapsular transplantation of SSCs. Approximately 1 × 10⁴ SSCs were transplanted beneath the renal capsule on one side of each recipient mouse. (F) Representative images of μCT and quantitative analysis of bone parameters 4 weeks after subcapsular SSCs transplantation in mouse kidney. n = 4 per group. Scale bars: 500 μm. (G) Immunofluorescence staining for osteopontin and TH in bone organoids derived from SSCs transplanted into the renal capsule of secondary recipient mice. Organoids were harvested 4 weeks after transplantation. Scale bars: 200 μm. Error bars indicate mean ± SEM. *P < 0.05, **P < 0.01, by unpaired, 2-tailed Student’s t test for 2-group comparisons.

Figure 5. FSTL1 secreted from sympathetic nerves inhibits SSC self-renewal and osteogenesis.

(A) GO enrichment analysis of genes differentially expressed in Slit2^th sympathetic neurons relative to Slit2^fl/fl sympathetic neurons. The significance values are based on a hypergeometric test. (B) Sympathetic neuron culture, TH⁺ (red) and βIII-tubulin⁺ (green) axon staining, and quantitative analysis in 3-week-old mice. Slit2^fl/fl, n = 18; Slit2^th. n = 18. Scale bars: 25 μm. (C) Crystal violet staining and quantitative analysis of CFU formation in SSCs stimulated with sympathetic neuronal conditioned medium. n = 3 per group. CM-Slit2^fl/fl, conditioned culture medium of sympathetic neurons in Slit2^fl/fl mice; CM-Slit2^th, conditioned culture medium of sympathetic neurons in Slit2^th mice. Scale bars: 5 mm. (D) Alizarin red staining and quantitative analysis of SSC mineralization activity after stimulation with conditioned medium from sympathetic neurons. n = 3 per group. Scale bars: 1 mm. (E) Expression of the soluble factors elevated in primary Slit2^th sympathetic neurons relative to Slit2^fl/fl sympathetic neurons. (F) mRNA levels of Fstl1 in primary Slit2^fl/fl and Slit2^th sympathetic neurons were analyzed by real-time PCR. n = 4 per group. (G) Protein levels of FSLT1 in primary Slit2^fl/fl and Slit2^th sympathetic neurons were analyzed by immunoblotting. n = 3 per group. (H) Results from ELISA for FSTL1 secretion in primary Slit2^fl/fl and Slit2^th sympathetic neurons. n = 3 per group. (I) Pellet formation assay and quantitative analysis of SSCs cultured for 8 days after treatment with recombinant FSTL1 or vehicle. n = 4 per group. Scale bars: 20 μm. (J) Representative μCT images and quantitative analysis of the bone volume of bone organoids 4 weeks after renal capsule transplantation of SSCs encapsulated in Matrigel and stimulated by treatment with recombinant protein FSTL1 or vehicle. n = 4 per group. Scale bars: 500 μm. (K) Representative flow cytometry plots and relative frequency of SSCs from femurs of 3-week-old Fstl1^fl/fl and Fstl1^th male mice. n = 5 per group. (L and M) Representative μCT images of trabecular bone in the distal femur (L) and relative quantitative analysis of bone volume/total volume (BV/TV) and relative quantitative analysis of bone parameters (M) in Fstl1^fl/fl, Fstl1^th, Slit2^th, and Slit2^thFstl1^th male mice at 8 weeks of age. Fstl1^fl/fl, n = 10; Fstl1^th, n = 11; Slit2^th, n = 15; Slit2^thFstl1^th, n = 10. Scale bars: 500 μm. Error bars indicate mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, by unpaired, 2-tailed Student’s t test for 2-group comparisons (B–D, F, and H–K). *P < 0.05, **P < 0.01, ***P < 0.001, by 1-way ANOVA followed by Tukey’s post hoc test for multiple comparisons (M).

Figure 6. Sympathetic hyperinnervation disrupts bone regeneration by impairing SSC expansion.

(A and B) Representative μCT images of bone regeneration 7 days after femoral bone marrow ablation and quantitative analysis of bone parameter in regeneration area in 6-week-old Slit2^fl/fl and Slit2^th male mice. n = 5. Scale bars: 1 mm. (C) Representative flow cytometry plots 7 days after femoral bone marrow ablation and relative frequency of SSCs from femurs of 6-week-old Slit2^fl/fl and Slit2^th male mice. n = 5. (D) Representative confocal images 7 days after femoral bone marrow ablation. Shown are TH (red) and DAPI (blue) signal and quantitative analysis of TH⁺ sympathetic nerves in femur sections. Marrow ablation was conducted in 6-week-old Slit2^fl/fl and Slit2^th male mice. n = 6; Scale bars, 500 μm. (E) Representative μCT images 21 days after femur fracture and quantitative analysis of callus bone volume. Fractures were performed in 6-week-old Slit2^fl/fl and Slit2^th male mice. n = 6. Scale bars: 2 mm. (F) H&E staining visualizing the callus formed 21 days after femur fracture in 6-week-old Slit2^fl/fl and Slit2^th male mice. Scale bars: 200 μm. (G) Representative flow cytometry plots 14 days after femur fracture and the relative frequency of SSCs from the femurs of 6-week-old Slit2^fl/fl and Slit2^th male mice. n = 5 per group. (H) Representative μCT images 21 days after femur fracture and quantitative analysis of bone callus volume. Fractures were performed in 6-week-old Fstl1^fl/fl and Fstl1^Dbh-cre-ert2 male mice. n = 5. Scale bars: 2 mm. (I) Representative flow cytometry plots 14 days after femur fracture and relative frequency of SSCs from femurs of 6-week-old Fstl1^fl/fl and Fstl1^Dbh-cre-ert2 male mice. Fstl1^fl/fl, n = 6; Fstl1^Dbh-cre-ert2, n = 5. Error bars indicate mean ± SEM. *P < 0.05, **P < 0.01, by unpaired, 2-tailed Student’s t test for 2-group comparisons.