Fig. 2.

Transient inhibition of ERK activation blocks regeneration initiation in planarians. a Phosphorylated ERK levels dramatically increased within 15 min post-amputation. b Amputation-induced ERK activation occurred even after depletion of stem cells by γ-irradiation (1 or 2 days post-irradiation (d.p.irr)) and after inhibition of protein synthesis with cycloheximide (CHX). c MEK activates ERK by phosphorylation (pERK); MEK inhibitors PD0325901 (PD) and U0126 (U0) prevent ERK activation by inhibiting MEK. d Treatment with PD and U0 effectively reduced pERK levels. e, f Inhibition of ERK activity severely (PD heads 44/100, trunks 33/100, tails 3/120; U0 heads 43/70, trunks 47/70, tails 11/70) or completely (PD heads 56/100, trunks 67/100, tails 117/120; U0 heads 27/70, trunks 23/70, tails 59/70) inhibited formation of blastemas (e) and anterior (notum expression in lower panel) and posterior (wnt1 expression in upper panel) regeneration poles (f). Black arrows: regeneration of new tissues in the blastema; red arrows: remodeling of existing tissues. g, h Other key regenerative responses, such as expression of early wound-induced genes (g), as well as stem cell proliferation (pH3, phospho-Histone H3 as a marker for mitotic cells) and accumulation of stem cells and progeny (SMEDWI-1) (h) were also severely affected by ERK inhibition. Sample numbers indicated in each image. h.p.a., hours post-amputation; d.p.a., days post-amputation; DMSO was used as the solvent control; scale bars: 200 μm