Fig. 2. Inactivation of MAPK blocks metamorphosis and associated apoptosis-dependent tail regression. (A) Double detection of apoptosis and nuclei in the tail of Ciona intestinalis during metamorphosis (at 28 hpf). Digitized images were merged to superimpose nuclei (blue) over the respective TUNEL-labelled field (TUNEL-positive nuclei appear in green). Notice that the nuclei of numerous cells of the tail extremity are TUNEL positive in the control panel (DMSO) and in p38 inhibitor (SB203580)-treated larvae. By contrast, TUNEL-positive nuclei were detected very rarely in the presence of the ERK inhibitor (U0126) or the JNK inhibitor (SP600125) in treated larvae. The white square corresponds to the region of higher magnification displayed in the lower panel. (B) Double detection of ERK phosphorylation (green) or JNK phosphorylation (red) and nuclei (blue) in larvae at 22 hpf. ERK phosphorylation was detected in papillae and JNK phosphorylation was detected in the CNS of larvae treated with DMSO at 22 hpf. Larvae treated with U0126 MEK inhibitor were negative for ERK activation in papillae, and the larvae treated with SP600125 JNK inhibitor were negative for JNK activation in the CNS (red). (C) Extracts from untreated larvae at 22 hpf and larvae treated with U0126 or SP600125 at this time were run on SDS-PAGE and western blotted with the anti-phosphorylated ERK and anti-phosphorylated JNK monoclonal antibodies. (D) U0126 MEK inhibitor and SP600125 JNK inhibitor blocked metamorphosis of C. intestinalis. From hatching, larvae were treated with 6 µm of U0126 or 10 µm of SP600125. In one condition (U0126^*) the treatment was repeated every 6 hours due to the loss of activity of the MEK inhibitor U0126. Data represent the mean of three independent experiments (400 animals per experiment) expressed as a percentage of the total number of larvae. Scale bars: 225 µm in A, upper panel; 50 µm in A, lower panel; 45 µm in B.