Fig. 5

The regeneration biosensor careg is dependent on TGFβ/Activin-β signalling.

(a–c) Immunofluorescence staining of careg:EGFP ventricle at 7 d.p.ci. with antibodies against GFP (green), pSmad3 (red) and Tropomyosin (blue) revealed the presence of TGFβ/Activin-β-activated cells in the careg:EGFP-expressing tissue. N=6. (d–g) careg:EGFP heart sections at 7 d.p.ci. treated with 0.1% DMSO or 20 μM SB431542, an inhibitor of TGFβ type I receptors, and immunostained with antibodies against GFP (green) and embCMHC (red). The intact myocardium was detected with F-actin staining (blue). In the magnified images, the upper dotted line demarcates a 100 μm-thick margin of the remaining myocardium from the injury border (lower dotted line). (h) Percentage of careg:EGFP⁺ and embCMHC⁺ area within a distance of 100 μm from the post-infarcted tissue in hearts at 7 d.p.ci. treated with DMSO or SB431542. The inhibitor treatment resulted in a significant reduction of careg:EGFP and embCMHC expression in the peri-injured zone, compared to control hearts treated with DMSO. N=8. ***P<0.001; unpaired t-test. Error bars correspond to s.e. of the mean (s.e.m.). (i–n) Live-imaging of careg:EGFP fins at 3 d.p.a. in a wild-type (WT) background treated with DMSO or SB431542, and in the fgf20a (dob) mutant background. careg:EGFP expression is suppressed in the stump by TGFβ inhibition, but it remains normal in fgf20a mutant fins. (o–t) Longitudinal sections of the fins shown in the left panels display a reduction of pSmad3-positive nuclei (red) and careg:EGFP in the stump of SB431542-treated fins, whereas both markers are unaltered in fgf20a mutants. BV, blood vessel. N=4.