Double-stranded RNA injection produces null phenotypes in zebrafish

Zebrafish is a simple vertebrate that has many attributes that make it idea l for the study of developmental genetics. One feature that has been lackin g in this model system is the ability to disable specifically targeted gene s. Recently, double-stranded RNA has been used to silence gene expression i n the nematode Caenorhabditis elegans. We have found that expression of the green fluorescent protein (GFP) from a microinjected plasmid vector can be suppressed in zebrafish embryos by the coinjection of a double-stranded RN A that is specifically targeted to GFP. To determine that double-stranded R NA can attenuate endogenous gene expression, single-cell zebrafish embryos were injected with double-stranded RNA specifically targeted to Zf-T and Pa x6.1. We found that microinjection of double-stranded Zf-T RNA resulted in a high incidence of a phenotype similar to that of ntl. furthermore, Zf-T g ene expression could not be detected by in situ hybridization and the messa ge was decreased by 75% by semiquantitative RT-PCR in 12-h embryos that had been injected with the double-stranded RNA. Expression of the zebrafish ge nes sonic hedgehog and floating head was altered in the embryos microinject ed with the Zf-T double-stranded RNA in a manner that is remarkably similar to the zebrafish no-tail mutant. Microinjection of double-stranded RNA tar geted to Pax6.1 was associated with depressed expression of Pax6.1 and resu lted in absent or greatly reduced eye and forebrain development, similar to the phenotype seen in mouse mutants. Simultaneous injection of Pax6.1 and Zf-T resulted in embryos lacking notochords, eyes, and brain structures. (C ) 2000 Academic Press.