Fig. 5

Fig. 5

Inhibition of the Mapk/Erk signalling pathway blocks regeneration and impairs autophagy induction in the blastema. (a and b) Inhibition of Mapk/Erk signalling using U0126 impaired caudal fin regeneration (panels a2 and b2 are magnifications from the central fin areas of the samples on panels a and b, respectively). (c and c2) Inhibition of Ras-MAPK signalling using a transgenic hs:DNRas reporter line also impaired regeneration and blastema formation. (d and e) Regeneration of caudal fin can be inhibited with SU5402, which also results in a decrease in GFP-Lc3 activity. (f–i) mkp3 expression does not depend on autophagic activity. Atg5MO-injected dorsal fin blastemas express mkp3 at levels comparable to that in uninjected ventral fins (f and g). Similarly, mkp3 staining displays a similar extent of labelling in DMSO-treated control (h) and autophagy-defective, bafilomycin A₁ (BafA₁)-treated backgrounds (i). (j–l) Serum-starved, DMSO-treated GFP-mRFP-LC3 HeLa cells show significant levels of autophagic activity, as denoted by the yellow and red puncta (autophagosomes and autolysosomes, respectively) (j and l). When 10μM or 100μM U0126 was added to the medium, we observed a drop in the autophagic activity (k and l). At concentrations comparable to our treatments, the effect of U0126 was highly significant (l). n=43 cells measured in both control and treated groups, ***P<0.001, unpaired t-test, error bars refer to S.E.M. (m) Schematic diagram showing the proposed interaction between the Fgf-Mapk/Erk signalling axis and autophagy. Injury activates Fgf signalling in the fin, which leads to the phosphorylation of Mapk/Erk. Activated Mapk/Erk regulates the sequestration step of autophagy, an essential step in the degradation of the cytosolic component. The small molecule reagents and the MO used in this study inhibit the process at the indicated steps. The three arrows from ‘ERK’ to ‘Autophagy’ indicate an indirect link between Mapk/Erk signalling and autophagy