Fig 1: MSR1-activated macrophage PI3K/AKT/GSK3β/β-catenin signaling promotes osteogenic differentiation of BMSCs. (A) Heat map of several genes encoding molecules involved in BMSC osteogenic differentiation was performed based on the results of RNA sequencing (MSR1 KO vs. WT). Blue and yellow colors represent low and high expression values, respectively.(B) The amount of secreted BMP4 in 24-h in the serum-free medium by MSR1 WT and MSR1 KO macrophages after co-culture, or MSR1 WT macrophages treated with LY294002 or ARQ 092 before co-culture was assessed by ELISA. Values are expressed as mean ± SD, ***p < 0.001. (C) The amount of secreted BMP4 in 24-h serum-free MSR1 BL, Vec, and OE RAW264.7 cells after co-culture, or MSR1 OE RAW264.7 cells treated with LY294002, ARQ 092 before co-culture was determined by ELISA. Values are expressed as mean ± SD, ***p < 0.001. (D-F) In the co-culture system, knockout of MSR1 or inhibition of PI3K/AKT/GSK3β/β-catenin signaling in macrophages impaired pro-osteogenic differentiation of BMSCs as observed by AR staining (D). Quantitative evaluation of AR staining results (E) and ALP activities (F) on day 7 and 14 was performed. BMSC without co-culture was used as the Con group. Values are expressed as mean ± SD, *p < 0.05, **p < 0.01. (G) mRNA expression levels of osteogenic biomarkers (Col1, ALP, Ocn and Runx2) in osteogenic differentiated BMSCs on day 14 were detected by qPCR in different groups. β-actin was used as an internal control. Values are expressed as mean ± SD, **p < 0.01, ***p < 0.001. (H-J) Inhibition of PI3K/AKT/GSK3β/β-catenin signaling in MSR1 OE RAW264.7 cells in the co-culture system decreased osteogenic differentiation of BMSCs as observed by AR staining (H). Quantitative evaluation of AR staining results (I) and ALP activities (J) on day 7 and 14 was performed. Values are expressed as mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001, ns indicates no significance. (K) mRNA expression levels of Col1, ALP, Ocn and Runx2 in osteogenic differentiated BMSCs on day 14 detected by qPCR in the indicated groups. β-actin was used as an internal control. Values are expressed as mean ± SD, **p < 0. 01, ***p < 0.001, ns indicates no significance.
Fig 2: Detection of key soluble morphogens in the system of sweat gland morphogenesis and the impact of BMP receptor inhibitor and EGF receptor inhibitor on sweat gland development in vitro. BMP4 and EGF demonstrated sharped difference in the medium of the sweat gland organogenesis system. BMP receptor inhibitor could block the formation of sweat gland in this system, while EGF receptor inhibitor significantly reduced the expression of K18. (a) The variation tendency of BMP4 and EGF in the medium of system. The error bar meant the standard error of BMP4 and EGF concentrations in different systems at different time points. (b) The impact of BMP receptor inhibitor and EFG receptor inhibitor on sweat gland morphogenesis. In comparison with the control group, EGF receptor inhibitor significantly reduced the expression of K18, but glandular structure was still observed. BMP receptor inhibitor completely blocks the expression of K18, and no glandular structure was observed. All the nuclei were counterstained with DAPI (DAPI: blue; K18: red; bars = 200 μm and 50 μm; K18: cytokeratin 18; IM: light microscope; H&E: hematoxylin-eosin staining; IF: immunofluorescence staining).
Fig 3: Blocking BMP4 signaling rescued enlarged tracheal lumen in Fstl1-deficient embryos(A and B) Whole-mount staining of E-cadherin (A) and quantification of the tracheal lumen diameters (B) in Fstl1+/+ and Fstl1−/− mouse tracheas after different doses of DMSO or dorsomorphin treatment in vivo. DMSO: Fstl1+/+, n = 7, Fstl1−/−, n = 4; dorsomorphin: E10.5–E13.5, Fstl1+/+, n = 5, Fstl1−/−, n = 4; E8.5–E13.5, Fstl1+/+, n = 4, Fstl1−/−, n = 4; E5.5–E13.5, Fstl1+/+, n = 4, Fstl1−/−, n = 3. Scale bars: 500 μm ∗, p < 0.05; ∗∗∗, p < 0.001. Differences between the groups were assessed using Student’s t tests.(C–E) Representative images of H&E staining (C), and the quantification of tracheal lumen circumference (D) and area (E) at E13.5 in Fstl1+/+ and Fstl1−/− mouse embryos with daily DMSO or dorsomorphin treatment in vivo from E5.5 to E13.5. n = 6. Scale bars, 100 μm ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001; ∗∗∗∗, p < 0.0001. Differences between the groups were assessed using one-way ANOVA.(F and G) Violin plots (F) and Rose plots (G) of mitotic spindle angles of tracheal epithelial cells at E13.5 in Fstl1+/+ and Fstl1−/− mouse embryos with daily DMSO or dorsomorphin treatment in vivo from E5.5 to E13.5. ∗∗, p < 0.01.Differences between the groups were assessed using one-way ANOVA. Data are expressed as the mean ± SEM.
Fig 4: Ectopic activation of BMP4 signaling in tracheal epithelial cells of the Fstl1-deficient embryos(A and B) Immunostaining for p-Smad1/5/8 was performed on E12.5 (A) and E13.5 (B) in Fstl1+/+ and Fstl1−/− mouse tracheal sections. n = 3. Arrows indicate the proliferating epithelial cells. Scale bars: 10 μm.(C and D) Western blot for p-Smad1/5/8 and p-ERK1/2 in E16.5 (C) and E18.5 (D) Fstl1+/+ and Fstl1−/− mouse tracheal tissues. Data are representative of two independent experiments.(E) Bar plots showing the expression of canonical BMP4 target genes Id1, Id2, and Id3 in Epcam+Sox2+ proximal airway epithelial cells from scRNA-seq in E18.5 Fstl1+/+ and Fstl1−/− lungs (GSE225463). Data are expressed as the mean ± SEM. Differences between the groups were assessed using Student’s t tests.
Fig 5: BMP4 signaling is downregulated because of the expression of Fstl1 in the tracheal mesenchyme(A and B) Violin plots showing the expression of Fstl1 (A) and Bmp4 (B) in published scRNA-seq data on mouse E8.5/9.0/9.5 foregut (GSE136689), E9.5/10.5/11.5 (GSE87038), and E12.5 (GSE119228) lungs.(C) RNAscope in situ hybridization of Fstl1 and Bmp4 on tracheal transverse sections in Fstl1+/+ and Fstl1−/− mouse embryos at E13.5, E15.5, and E18.5. Dashed boxes mark regions displayed at a higher magnification, and dashed lines delineate the boundary between the epithelium and mesenchyme. Scale bars: 50 μm.
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