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Fig. 2 Effects of ectopic expression of ?Np53 on zebrafish embryos. (A) Steady-state ?Np53 message levels achieved by microinjection as compared to endogenous message levels in irradiated (20 Gy at 24 hpf) embryos; mRNA was isolated from 30 hpf embryos maintained in triplicate dishes of 60 embryos each and injected with 1-2000 pg of mRNA at the 2-4 cell stage. RT-PCR was performed as described in Material and Methods. (B) Embryo survival upon ectopic expression of ?Np53 mRNA. Survival was defined as the presence of a heartbeat. (C) Representative examples of malformations caused by ectopic expression of p53 isoforms as evident at 48 hpf. Embryos were anesthetized with 0.003% tricaine, placed on 3% methylcellulose on a glass depression slide and examined using a fluorescence microscope (Leica MZ16FA) at 10× magnification. (D) Effects on embryo survival of 1 ng of either zebrafish or human ?Np53 message either alone (upper panel) or in combination with zebrafish FLp53 mRNAs (lower panel). For control purposes, mRNAs encoding FLp53(z) and the functionally inactive M214K FLp53(z) mutant were included (upper panel). To ectopically express p53 isoforms, capped mRNAs were generated by cloning zebrafish cDNAs into pCS2+ and synthesized in vitro using the mMessage-mMachine-SP6 kit (Ambion).