Fig. 5. Sushi antisense morpholino blocks apoptosis-dependent tail regression during metamorphosis. (A) Whole-mount in situ hybridization of Ci-sushi and Ci-Sccpb displaying, respectively, specific expression in tail and at the tip of the tail of Ciona intestinalis larvae. The white square corresponds to the region of higher magnification displayed in the right panel. (B) JNK activation controls Ci-sushi expression. Ci-sushi mRNA expression from hatching to metamorphosis. mRNA was extracted from embryos at various time points, reverse transcribed and semi-quantitative specific PCR was performed (see Materials and methods). Ci-sushi mRNA expression at 25 hpf was extracted from untreated embryos or from those treated with 10 µM SP600125, reverse transcribed and semi-quantitative specific PCR was performed (see Materials and methods). (C) Detection of apoptosis in the tail of C. intestinalis tadpoles at the onset of metamorphosis (28 hpf). Apoptotic cells were TUNEL labelled (TUNEL-positive nuclei appear green). Notice that numerous nuclei of cells of the tail extremity were TUNEL-positive in the control panel. By contrast, TUNEL-positive nuclei were detected very rarely in Ci-sushi-morpholino antisense-injected larva. At this time, tunic cells are TUNEL-positive in both cases (arrows), as described in our previous work (Chambon et al., 2002). (D) Model of the role played by the CNS in the regulation of apoptosis during metamorphosis. Ci-JNK activation in the CNS leads to Ci-sushi and Ci-Sccpb gene expression in epithelia. These genes are essential for initiating apoptosis at the onset of metamorphosis. ECM modification is also a result of Ci-JNK activation in the CNS, which could promote the induction of apoptosis through Ci-ERK activation in adjacent tissues. Scale bars: 200 µm.