Fig 1: Single-cell RNA sequencing reveals heterogeneity and distinct subsets of fibroblasts.a Diagram depicting the generation of tdTomatorPßC mouse and PDGFRß+ cell-specific expression of tdTomato from 8-week old and their analyses at 10-week old. b Illustration of droplet-based single-cell RNA sequencing (scRNA-seq) of PDGFRß+tdTomato+ IntSCs sorted from the small intestine of tdTomatorPßC mice. c Visualization of unsupervised clustering of 7 distinct PDGFRß+ IntSC clusters by Uniform manifold approximation and projection (UMAP) in the small intestine of tdTomatorPßC mice. vFB, intestinal villi fibroblast; FB, fibroblast; SMC, smooth muscle cell; MC, mural cell. d Heatmap displaying the scaled expression patterns of top ten differentially expressed genes for random sampled cells (maximum thousand cells) for each indicated clusters. e List of representative marker genes of each of the seven PDGFRß+ IntSC clusters (left) and violin plots (right) showing the expression of top-ranking marker genes for each cluster. Log normalized read counts as y-axis (normalized expression). f UMAP visualization of unsupervised clustering of seven distinct PDGFRß+ IntSC clusters in the small intestine of Lats1/2i?-tdTomatorPßC mice. g Gene expression levels of Vegfc and Vegfa in tdTomatorPßC and Lats1/2i?-tdTomatorPßC mice projected on UMAP plot. Note that specific subsets of PDGFRß+ IntSCs (vFB1-3) in the small intestine of Lats1/2i?-tdTomatorPßC mice show higher expression of Vegfc compared with those of tdTomatorPßC mice. h, i Representative images of PDGFRß+ IntSCs in WT mouse reveal expressions of each fibroblast-specific markers: PAI-1+ vFB1, Serpina3n+ vFB2, P2X1+ vFB3, Ackr4+ or Grem1+ FB4, and PDGFRa+ Sox6+ FB5. Each white box in the lower left corner is a magnified view. White asterisks indicate lacteals and white arrowheads indicate each fibroblast cluster-specific cell type stained with the indicated marker. Similar findings were observed in n = 4 mice from two independent experiments. Scale bars, 20 µm. j Schematic images depicting the anatomic distribution of indicated markers for vFB1-3, FB4, FB5, SMC, MC, capillary plexus, and lacteal in intestinal villi of adult WT mouse. vFB1-3 are uniformly distributed around the lacteal, whereas FB4 is mainly located in the submucosal area and FB5 is mostly placed under the intestinal epithelium. Black dashed boxes are magnified in the right panels.
Fig 2: vFB1-3 are distinct cell types secreting VEGF-C.a Representative images of vFB3 (P2X1+Serpina3n+PDGFRß+) and vFB1-2 (P2X1-Serpina3n+PDGFRß+) in WT mouse. Similar findings were observed in n = 4 mice from two independent experiments. White and yellow dotted boxes are magnified in the upper right and lower right panels, respectively. Red and yellow arrowheads indicate vFB3 and vFB1-2, respectively. Scale bar, 20 µm. b Representative images of vFB1 (Serpina3n+Fosb+Shisa3+PDGFRß+) and vFB2 (Serpina3n+Fosb-Shisa3+PDGFRß+) in WT mouse. Similar findings were observed in n = 4 mice from two independent experiments. White and yellow dotted boxes are magnified in the upper right and lower right panels, respectively, for each image. White and yellow arrowheads indicate vFB1 and vFB2, respectively. Scale bars, 20 µm. c Representative images of Vegfc single-molecule fluorescence in situ hybridization (smFISH) in intestinal villi of tdTomatorPßC and Lats1/2i?- tdTomatorPßC mice. Similar findings were observed in n = 4 mice/group from two independent experiments. White dotted boxes are magnified in the right panel. Scale bars, 20 µm. d Representative images of Vegfc smFISH in vFB1-3 of WT and Lats1/2i?- tdTomatorPßC mice. Similar findings were observed in n = 4 mice/group from two independent experiments. White and yellow dotted boxes are magnified in the upper right and lower right panels, respectively, for each image. Scale bars, 20 µm.
Fig 3: mF4-31C1 inhibits osteoclast formation and bone loss in Osx-tTA;TetO-Vegfc mice.(A) Schematic showing when mice received normal water and doxycycline water. Osx-tTA;TetO-Vegfc mice were treated (3x/week) with vehicle or mF4-31C1 (VEGFR3 function-blocking antibody) from P21 to P35. (B,C) Representative images of TRAP stained femurs from vehicle-treated and mF4-31C1-treated mice. (D) Graph showing the number of osteoclasts per mm of bone for vehicle-treated (7.37 ± 2.088, n = 5) and mF4-31C1-treated (2.552 ± 0.6893, n = 5) mice. (E,F) Representative images of H and E stained femurs from vehicle-treated and mF4-31C1-treated mice. (G) Graph showing cortical bone porosity of femurs for vehicle-treated (0.07244 ± 0.02468, n = 5) and mF4-31C1-treated (0.006375 ± 0.007087, n = 4) mice. (**p<0.01, unpaired student’s T-test).
Fig 4: Mechanical stretching induces YAP/TAZ activation and VEGF-C secretion in IntSCs.a Diagram for primary culture of IntSCs derived from WT or Yap/Tazi?PßC mice for 2 days and their analyses at 2 days after 100% EtOH or 5 µM of hydroxy-tamoxifen (4-OHT) treatment. b Phase-contrast image of freshly isolated primary mouse IntSCs from WT mice without passage (P1). Similar results were observed in four independent experiments. Scale bar, 200 µm. c Representative images of YAP and TAZ with phalloidin+ actin filaments in unstretched and stretched IntSCs. Similar findings were observed in four independent experiments. Scale bars, 50 µm. d Comparison of VEGF-C protein concentration in the culture medium of unstretched and stretched IntSCs derived from WT or Yap/Tazi?PßC mice. Dots indicate value from four independent experiments and horizontal bars indicate mean ± SD. P value versus unstretched by two-tailed Mann–Whitney U test. e Schematic images depicting upregulation and secretion of VEGF-C and other factors upon nuclear translocation and activation of YAP/TAZ and subsequent binding to TEAD after mechanical stimuli in PDGFRß+ IntSCs.
Fig 5: Bone lymphatics in Osx-tTA;TetO-Vegfc mice disappear following the withdrawal of VEGF-C.(A) Schematic showing when mice received normal water and doxycycline water. One cohort of Osx-tTA;TetO-Vegfc mice received normal water from E18.5 to P35 (On 35d). A second cohort of Osx-tTA;TetO-Vegfc mice received normal water from E18.5 to P35 and then doxycycline water from P35 to P38 (On 35d/Off 3d). A third cohort of Osx-tTA;TetO-Vegfc mice received normal water from E18.5 to P35 and then doxycycline water from P35 to P42 (On 35d/Off 7d). (B) Graph showing the relative VEGF-C mRNA levels in tibias from mice. (C) Schematic showing when mice received normal water and doxycycline water. Osx-tTA;TetO-Vegfc mice received normal water from E18.5 to P35 (On 35d) or normal water from E18.5 to P35 and then doxycycline water for 3 (On 35d/Off 3d), 7 (On 35d/Off 7d), 28 (On 35d/Off 28d), or 56 days (On 35d/Off 56d). (D–I) Representative images of ribs stained with an anti-podoplanin antibody. The dashed lines separate the bone from the periosseous muscle. (J) Graph showing lymphatic vessel index values for ribs in Osx-tTA mice (0 ± 0.0; n = 5), Osx-tTA;TetO-Vegfc mice that received normal water for 35 days (152.5 ± 29.56; n = 5), and Osx-tTA;TetO-Vegfc mice that received normal water for 35 days and then doxycycline water for 3 (62.25 ± 51.7; n = 4), 7 (24.08 ± 21.26; n = 4), 28 (0 ± 0.0; n = 5) or 56 (0 ± 0.0; n = 3) days. (K) Graph showing lymphatic vessel index values for periosseous muscle in Osx-tTA mice (3.61 ± 1.974; n = 5), Osx-tTA;TetO-Vegfc mice that received normal water for 35 days (92.45 ± 34.63; n = 5), and Osx-tTA;TetO-Vegfc mice that received normal water for 35 days and then doxycycline water for 3 (99.29 ± 23.37; n = 4), 7 (74.84 ± 18.98; n = 4), 28 (92.67 ± 24.2; n = 5) or 56 (72.17 ± 14.05; n = 3) days. (**p<0.01, ***p<0.001, ****p<0.0001, ANOVA followed by Dunnett’s multiple comparisons test. Values were tested against values for Osx-tTA mice). ND = Not Detected.
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