Tgfβ signaling stimulates glycolysis to promote the genesis of synovial joint interzone in developing mouse embryonic limbs

Figures

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  Fig. 1.. scRNA-seq identifies interzone cells in mouse embryonic knee joint primordia. (A) scRNA-seq uncovers diverse cell types in knee joint primordia of E13.5 versus E14.5 mouse embryos. IZ cells, interzone cells. (B) Violin plots of select marker genes for each cluster. (C) Reclustering of chondrocytes, fibroblasts, and interzone cells as identified in (A). The clusters are further analyzed in (D) to (H). (D) Violin plots of select marker genes for each cluster. (E) Monocle trajectory analysis with chondrocyte cluster 0 as origin. (F) Heatmap of top 10 DEGs enriched in each cluster. Red bracket denotes top genes in interzone cell cluster 1. (G) GSEA plots showing enrichment of glycolysis and hypoxia pathways but decrease in OXPHOS in interzone cells over all other clusters.

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  Fig. 2.. Interzone cells are enriched with Glut1 and Glut3 proteins and are highly glycolytic. (A and B) Immunofluorescence staining for Glut1 and Glut3 on frozen sections of hindlimbs. Dotted circles denote presumptive joint region. Boxed regions in lower magnification images were amplified at a higher magnification. (C) Glucose consumption and lactate production by knee joint primordial cells versus adjacent chondrocytes (Chon) from E13.5 hindlimbs. F, femur; T, tibia. **P < 0.01, n = 3, Student’s t test. Error bars: SD.

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  Fig. 3.. Deletion of Glut1 and Glut3 causes severe joint malformation. (A) Whole-mount skeletal staining at P0. Red arrows denote defects in knee and elbow joints. (B) Safranin O staining of sections through various planes of knee joints at P0. Black dashed line: meniscus; red dashed line: ligament. *: dead tissue. (C) Von Kossa staining of central sections through the knee joint. Red arrow: ectopic mineralization. (D) RNAscope of Prg4 on lateral sections of the knee. (E) EdU and TUNEL staining on knee sections at E13.5. White arrow denotes dead cells. G1, Glut1; G3, Glut3; DCKO, Prx1Cre;Glut1f/f;Glut3f/f. F, femur; T, tibia.

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  Fig. 4.. Glut1/3 deletion disrupts interzone formation. (A to D) RNAscope for Gdf5 and Col2a1 on knee [(A) to (C)] or elbow (D) sections of Prx1Cre-mediated Glut1/3 deletion mutants versus controls at E12.5 (A), E13.5 [(B) and (D)] and E14.5 (C). *: cell death area. (E and F) RNAscope for Wnt4 and Wnt9a on knee sections at E12.5 (E) or E13.5 (F). (G) Confocal images of hindlimb sections showing red fluorescence in skeletal elements and knee joint primordia of Col2-Cre;Ai9 but not Ai9 embryos. (H) RNAscope of Gdf5 on knee sections of Col2Cre-mediated Glut1/3 deletion mutants versus controls at E13.5. Green line denotes normal height of developing interzone in the control embryo. G1, Glut1; G3, Glut3; DCKO, Prx1Cre;Glut1f/f;Glut3f/f. F, femur; T, tibia; H, humerus; U, ulna; Fi, fibula.

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  Fig. 5.. Deletion of Glut1/3 with Gdf5Cre does not impede joint development but causes postnatal loss of articular cartilage. (A) Confocal fluorescent images of knee joint sections from Gdf5Cre;Ai9 embryos. (B and C) Safranin O staining of knee sections from newborn (B) or 2-month-old mice (C) with genotypes as indicated. Boxed areas shown at higher magnification below. Double-headed arrows in (C) denote thinning of articular cartilage in tibial lateral condyle of mutant mice, with quantification shown to the right. (D) Confocal fluorescent images of knee joint sections from 2-month-old mice with genotypes as indicated. Red: Gdf5-lineage cells including articular chondrocytes; Green: TUNEL staining. Note increased dead articular chondrocytes (yellow nuclei) in the lateral tibia plateau of mutant mice, with quantification shown to the right. 1: No cre, 2: Gdf5Cre; Ai9;G1f/f;G3f/+, and 3: Gdf5Cre;Ai9;G1f/f;G3f/f. **P < 0.01, Student’s t test, n = 3 mice, one section from each mouse. Error bars: SD. F, femur; T, tibia.

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  Fig. 6.. Glut1/3 deletion suppresses Tgfβ signaling in Gdf5+ cells. (A) scRNA-seq of cells from joint regions of E14.5 embryos. DCKO, Prx1Cre;Glut1f/f;Glut3f/f. Red dashed lines denote Gdf5+ cells. (B) Violin plots of select marker genes for each cluster. (C) Relative abundance of interzone cells according to scRNA-seq. (D) GSEA reveals down-regulation of Tgfβ signaling pathway in Gdf5+ cells of DCKO versus Ctrl embryos. (E) RNAscope for Tgfβ2 (upper, dotted circles denote expression domain in joint region) and immunofluorescence for P-Smad2 (lower, dotted circles denote joint region) on knee sections at E13.5. Note the cell death in presumptive knee joint in DCKO samples. F, femur; T, tibia.

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  Fig. 7.. Deletion of Tgfβr2 disrupts interzone formation and knee joint development. (A) RNAscope on knee sections. Tgfβr2CKO: Prx1Cre;Tgfβr2f/f at E13.5. Green arrow denotes enriched Tgfβr2 expression in wild-type (WT) embryos but absent in Tgfβr2CKO. (B) Absence of meniscus (black dashed lines) and ligaments (red dashed lines) in the knee of Tgfβr2CKO at E18.5. (C) Loss of Prg4 mRNA in the knee joint of Tgfβr2CKO at E18.5. (D) Immunofluorescence staining detects enhanced Glut1 and Glut3 expression in normal knee joint region (dotted circle) but not in equivalent areas of Tgfβr2CKO at E13.5. F, femur; T, tibia; Fi, fibula.

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  Fig. 8.. Tgfβ2 stimulates glycolysis in primary rib and limb chondrocytes. (A and B) Representative Western blot images (A) and quantification (B) from primary rib chondrocytes after 12 hours of Tgfβ2 or vehicle (ctrl) treatment. N = 3. (C and D) Glucose consumption (C) and lactate production (D) by primary rib chondrocytes following Tgfβ2 treatment for various durations as indicated. N = 4. (E to J) Seahorse assays in primary rib chondrocytes following Tgfβ2 treatment for various durations as indicated. N = 4 or 5. (K and L) Glucose consumption (K) and lactate production (L) by primary limb chondrocytes cultured in hypoxia (5 or 1% O2) and treated with Tgfβ2 or vehicle (Ctrl) for 12 hours. N = 3. *P < 0.05 and **P < 0.01, Student’s t test [(B) to (D)] or one-way ANOVA [(F) and (H) to (L)]. Error bars: SD.

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  Fig. 9.. Tgfβ2 stimulates glycolysis through mTOR in primary rib chondrocytes. (A) Smad2 knockdown efficiency at mRNA (left) or protein level (right) by different shRNAs. Numbers in representative Western blot images indicate relative Smad2 levels normalized to β-actin. N = 3. (B and C) Quantification of glucose consumption and lactate production after 12 hour of Tgfβ2 or vehicle (Ctrl) treatment. N = 4. (D) Western blots showing effects of Smad2 knockdown on Glut1 after 12 hours of Tgfβ2 or vehicle (Ctrl) treatment. (E) Representative Western blot images and quantification of signal intensity showing temporal activation of Smad versus mTORC1 signaling by Tgfβ2. N = 3. (F) Representative Western blots images showing no effect by Smad2 knockdown on Tgfβ2-induced mTORC1 activation after 1 hour of Tgfβ2 or vehicle (ctrl) treatment. N = 3. (G and H) Effects of Torin1 on glucose consumption (G) and lactate production after 12 hours of Tgfβ2 or vehicle (Ctrl) treatment (H). N = 4. The inhibitor or its vehicle solution dimethyl sulfoxide (DMSO) was added to the cells for 12 hours (pretreatment) before either Tgfβ2 or its vehicle [0.1% bovine serum albumin (BSA)] was added. NS, not significant.…

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  Fig. 10.. Tgfβ stimulates glycolysis via mTOR-Hif1a activation to facilitate transition of chondrocytes to interzone cells. (A) Representative images and quantification of Western blots showing effects of Torin1 on Hif1a and Glut proteins after 12 hours of Tgfβ2 or vehicle (Ctrl) treatment. N = 3. (B and C) Effects of Hif1α inhibitors echinomycin (Echi) or KC7F2 on glucose consumption (B) and lactate production (C) after 12 hours of Tgfβ2 or vehicle (Ctrl) treatment. N = 4. (D) Representative Western blot images showing effects of Hif1a inhibitors on Glut proteins after 12 hours of Tgfβ2 or vehicle (Ctrl) treatment. The inhibitors or their vehicle solution DMSO were added to the cells for 12 hours (pretreatment) before either Tgfβ2 or its vehicle (0.1% BSA) was added. NS, not significant. *P < 0.05 and **P < 0.01, two-way ANOVA with Tukey’s multiple comparisons test. Error bars: SD. (E) A model for Tgfβ signaling to facilitate interzone formation from chondrocytes via stimulation of glycolysis.