Labelling Primordial Germ Cells(PGCs) And Analysis Of Its Migration Mechanism By Nanos3 In Olive Flounder(paralichthys Olivaceus)

The olive flounder(Paralichthys olivaceus) is an important cultured marine fish. Recently, to avoid the trend of degradation of genetical characterization, many breeding methods are successfully applied. But all of these breeding methods are time-consuming, tedious process, it is important to set up new methods. Methods that using primordial germ cells(PGCs), such surrogate propagation, transgenic fish and frozen preservation, are the trend in recent years.The labeling of PGCs is the premise of its using, and its marker gene is the key. We cloned nanos3 of olive flounder,a PGCs specific gene. RT-PCR and Whole-mount in situ hybridization were used to analyze its expression patterns during flounder embryogenesis. The results indicated nanos3 was consistently expressed during embryogenesis and was PGCs specific. So, it was reliable that nanos3 was PGCs specific marker gene in olive flounder.We simulated PGCs migration model according nanos3 expression pattern during embryogenesis. The PGCs migration model of olive flounder is almost similar with others teleosts fish. But at 15 somite stage, the PGC, aligned into two tight clusters at both side of body axis, aggregated into a single loose cluster and migrated posteriorward.At heart-beating stage, PGCs re-aligned into two tight clusters on both side of body axis and migrated posteriorward until reached the genital ridge. This PGCs migration pattern is different from the teleost fishes reported and it is a new model that combines elements of PGCs of medaka, herring and goby.We injected chimeric RNA containing green fluorescent protein(GFP) and olive flounder nanos3 3’UTR into zebrafish fertilized eggs and PGCs can be successfully visualized in vivo as early as the 8-somites stage. These results indicate that there is conserved regulation region in nanos3 3’UTR of olive flounder. Furthermore, Dnd binding region(U-rich region) and three predicted noncanonical miR-430 binding sites(GCACs) were predicated in nanos3 3’UTR of olive flounder. By injecting different length and mutant of olive flounder nanos3 3’UTR to zebrafish embryos, we found a 67bp(68-135bp) length functional region in olive flounder nanos3 3’UTR. We identified the GCAC1 and GCAC2 in this region might be the miR430 binding site and the U-rich region might be the Dnd binding region.Furthermore, we cloned sdf1 and cxcr4 and analyze their expression by whole-mount in situ hybridization during embryogenesis. Comparison of their expression patterns and the PGCs migration model showed that the cxcr4/sdfs guided the PGCs migration during somitogenesis. In particular, they play an important role in directing PGCs aggregation into a single loose cluster from the two elongated lines at 15 somite stage..