Too much interference: Injection of double-stranded RNA has nonspecific effects in the zebrafish embryo

We have investigated the ability of double-stranded RNA (dsRNA) to inhibit gene expression in a vertebrate, the zebrafish, Danio rerio. Injection of d sRNA corresponding to the T-box gene tbx/16/spadetail (spt) into early wild -type embryos caused a rapid and dramatic loss of tbx16/spt mRNA in the bla stula. mRNAs from the papc, tbx6, and gata1 genes, which depend on tbx16/sp t function for their expression, were reduced, apparently mimicking the spt mutant phenotype. However, mRNAs from a number of genes that are unaffecte d by the spt mutation, such as beta catenin, stat3, and no tail, were also lost, indicating that the "interference" effect of tbx16/spt dsRNA was not restricted to the endogenous tbx16/spt mRNA. We compared the effects of inj ecting dsRNA from the zebrafish tbx16/spadetail, nieuwkoid/bozozok, and Bra chyury/no tail genes with dsRNA from the bacterial lacZ gene. In each case the embryos displayed a variable syndrome of abnormalities at 12 and 24 h p ostfertilization. In blind studies, we could not distinguish between the ef fects of the various dsRNAs. Consistent with a common effect of dsRNA, rega rdless of sequence, injection of dsRNA from the lacZ gene was likewise effe ctive in strongly reducing tbx16/spt and beta catenin mRNA in the blastula. These findings indicate that, despite published reports, the current metho dology of double-stranded RNA interference is not a practical technique for investigating zygotic gene function during early zebrafish development. (C ) 2000 Academic Press.