RESUMEN
By using retroviral insertional mutagenesis in zebrafish, we have identified a recessive lethal mutation in the not really started (nrs) gene. The nrs mutation disrupts a gene located in linkage group 3 that is highly homologous to the spinster gene identified in Drosophila and to spinster orthologs identified in mammals. In flies, spinster encodes a membrane protein involved in lysosomal metabolism and programmed cell death in the central nervous system and in the ovary. In nrs mutant fish embryos, we detect an opaque substance in the posterior yolk cell extension at approximately 1 day after fertilization. This material progressively accumulates and by 48 hr after fertilization fills the entire yolk. By day 3 of embryogenesis, mutant embryos are severely reduced in size compared with their wild-type siblings and they die a few hours later. By in situ hybridization, we show that the nrs mRNA is expressed in the yolk cell at the time the mutant phenotype becomes apparent. In wild-type embryos, nrs message is present maternally and zygotically throughout embryogenesis and is also detected in adult animals. In nrs homozygous mutant embryos, nrs transcripts are undetectable at the time the phenotype becomes apparent, indicating that the retroviral insertion has most likely abolished expression of the nrs gene. Finally, the nrs phenotype can be partially rescued by microinjection of nrs encoding DNA. These results suggest that the nrs mutation affects an essential gene encoding a putative membrane-bound protein expressed specifically in the yolk cell during zebrafish embryogenesis.