Fig. 7

Loss of cardiac fibroblasts following scleraxis deletion in vivo. a–b Representative flow cytometry histograms of WT and Scx KO heart single-cell suspensions show cardiac cells stained for αSMA (a) or DDR2 (b) in WT (dotted line) and KO (solid line) mice. Unstained cell sample was used as control (shaded histogram). c Total αMHC⁺, αSMA⁺, CD31⁺ or DDR2⁺ stained events per 5 × 10⁴ events in WT or KO mice normalized to WT counts demonstrating normal cardiomyocyte counts but reduced numbers of fibroblasts; n = 3 independent samples per genotype. d Representative cardiac sections from WT and KO mice immunolabeled for DDR2 confirm the loss of DDR2⁺ cells; 63× objective, scale bar = 20 μm. e qPCR assay of Tcf21 mRNA expression in WT and Scx KO mice; n = 3 independent samples per genotype. f Cardiac sections as in (d) were immunolabeled for Twist1 expression (red; DAPI nuclear staining in blue); 20× objective, scale bar = 64 μm. g Cardiac mRNA from WT and Scx KO mice was assayed for expression of mesenchymal and epithelial marker genes by qPCR, indicating reduced epithelial-to-mesenchymal transition in KO hearts; n = 5 independent samples per genotype. h–i Cardiac proto-myofibroblasts were subjected to Scx knockdown (h) or over-expression (i) and EMT markers assessed by qPCR, indicating that Scx regulates mesenchymal marker gene expression; n = 3. j Scx dose-dependently transactivates the Snai1 and Twist1 proximal gene promoters as determined by luciferase assay or GFP western blot, respectively; n = 3. k Representative fibroblast/myofibroblast, mesenchymal, epithelial and tendon marker gene mRNA expression was assayed in A549 epithelial cells following Scx over-expression (AdScx) vs controls (AdGFP), assayed by qPCR; n = 3. l Scx mRNA expression was assayed by qPCR in A549 cells following treatment with 2.5 ng/mL TGFβ or vehicle; n = 4. m Mesenchymal marker gene mRNA expression was assayed in A549 cells following Scx dominant negative mutant over-expression (AdScxΔBD) vs controls (AdGFP), with or without treatment with 2.5 ng/mL TGFβ or vehicle; n = 3. *P < 0.05 vs WT (c, e, g), vs AdshLacZ (h), vs AdGFP (i, k, m), vs control transfected vector (j) or vs vehicle (l); #P <0.05 vs AdGFP + TGFβ (m). n Putative model of action of Scx. Top panel, Scx is required for cardiac fibroblast to myofibroblast phenotype conversion (solid arrow), and possibly for transition of epithelial precursors to the mesenchymal/fibroblast phenotype (dashed arrow). Bottom panel, Scx is sufficient to directly transactivate numerous genes that characterize the myofibroblast phenotype, and is required for TGFβ/Smad3-mediated gene expression by facilitating Smad3 and RNA polymerase II interaction at target gene promoters such as Col1α2