Characterization of vasa and dnd homologs in summer flounder, Paralichthys dentatus: Expression analysis and colocalization of PGCs during embryogenesis

Specification of primordial germ cells (PGCs) is particularly important for germline formation. Many maternal-effect genes such as vasa, dnd, and nanos have been identified. However, the research on distribution patterns of PGCs in marine fish is limited. Vasa has been widely used as a germ cell marker to identify its origination in teleosts because vasa RNA is a component of germ plasm. Dnd is known to be an RNA binding protein that protects germline-specific RNAs from degradation. In this study, we isolated full-length vasa and dnd cDNA from summer flounder to track germ cell origination and their expression patterns by RT-PCR and ISH. The results demonstrated that deduced amino acid sequence of Pdvas and Pddnd shared typically conserved motifs of their homologues and demonstrated high identities with other teleosts. Both vasa and dnd transcripts were exclusively detected in germ cells of the gonads. During embryogenesis, vasa and dnd RNA were located at the cleavage furrows of early cleavage stages, and then through proliferation and migration they eventually moved to a location at the predetermined genital ridge. Phylogenetic analysis revealed that summer flounder belongs to the Euteleostei species, but vasa/dnd transcripts localized at the cleavage furrows was similar to that in zebrafish (Osteriophysans). This suggests that germ cells differentiating at early embryogenesis have no direct relation with phylogenesis. At the same time, we found the spatio-temporal expression pattern of dnd was highly consistent with vasa during this process, which indicated the important function of dnd in keeping the target RNA from being degraded to maintain germ cell fate. These results will provide further understanding of germ plasm localization and PGC differentiation in teleosts, and facilitate germ cell manipulation in marine fishes.