Fig. 4

Morpholino-mediated depletion of map9 leads to increased apoptosis. (A-D) Zebrafish embryos were injected, or not (control), at the 1-cell stage with 1 pmol of MOex5 for observation at 8 hpf, or with 0.25 pmol MOex5 for observation at 24 hpf. TUNEL-positive cells (green) were counted in injected (B and D) and control embryos (A and C) (n = 10/assay). (E) Left panel, number of TUNEL-positive cells in the control and MOex5-injected embryos depicted in (A-D), n = 5 embryos per assay, total cell count ~1070 (*P < 0.005, **P < 0.02); right panel, tp53 inhibition does not rescue apoptosis induced by MOex5. Number of TUNEL-positive cells in MOex5- and MOex5 + MOtp53-injected embryos (n = 20 embryos per assay, total cell count ~564). (F) Map9 depletion does not induce tp53 expression. Embryos were injected at the 1-cell stage with 1 pmol of MOex5. RNA from 7 hpf map9 morphants and control embryos was used to quantify tp53 gene expression by RT-qPCR. Expression data were normalized to β-actin. Experiments were made in quadruplicate, n = 42 control embryos, and 50 MOex5 morphants at 7 hpf, and n = 39/44 embryos at 24 hpf. (G) Co-injection of MOex5 (1 pmol) and MOtp53 (0.5 pmol) does not rescue MOex5 effects. Embryo mortality was not rescued after tp53 depletion by morpholino. Embryo mortality after injection of a control MO (MOmis) was not significantly different compared with non-injected embryos. This experiment was performed in duplicate.