Medaka vasa is required for migration but not survival of primordial germ cells

Abstract

Vasa is essential for germline development. However, the precise processes in which vasa involves vary considerably in diverse animal phyla. Here we show that vasa is required for primordial germ cell (PGC) migration in the medakafish. vasa knockdown by two morpholinos led to the PGC migration defect that was rescued by coinjection of vasa RNA. Interestingly, vasa knockdown did not alter the PGC number, identity, proliferation and motility even at ectopic locations. We established a cell culture system for tracing PGCs at the single cell level in vitro. In this culture system, control and morpholino-injected gastrulae produced the same PGC number and the same time course of PGC survival. Importantly, vasa-depleted PGCs in culture had similar motility and locomotion to normal PGCs. Expression patterns of wt1a, sdf1b and cxcr4b in migratory tissues remained unchanged by vasa knockdown. By chimera formation we show that PGCs from vasa-depleted blastulae failed to migrate properly in the normal environment, whereas control PGCs migrated normally in vasa-disrupted embryos. Furthermore, ectopic PGCs in vasa-depleted embryos also retained all the PGC properties examined. Taken together, medaka vasa is cell-autonomously required for PGC migration, but dispensable to PGC proliferation, motility, identity and survival.

Accessibility of host cell lineages to medaka stem cells depends on genetic background and irradiation of recipient embryos

“…At present, it is not clear whether certain MES1 derivatives residing within the gonad are PGCs, because the low frequency of gonad colonization and high embryonic/post-hatching lethality prevented a functional test by germline transmission. Although the timely appearance, larger size, and location of these gonadal MES1 derivatives are not different from those of GFP-labeled PGCs in living embryos [32,35,37], the accessibility of the medaka germline to contribution by ES cell cultures remains an open question, because medaka PGCs might be formed by maternal factors [30]. Future work will determine whether MES1 expresses germ plasm components essential for PGC specification.…”

“…Similarly, in Lr medaka, transgenic RFP expression occurs strongly and specifically in the developing and adult liver (Zeng and Hong, unpublished). Transgenic line Vg expresses specifically GFP from the medaka Vasa promoter and labels migratory PGCs [32]. Vg and Lr were crossed separately with HB32C for three generations to an essentially (87.5%) HB32C background.…”

“…Irradiated and non-irradiated embryos were dechorionated and transplanted at the midblastula stage with approximately 100 MES1 cells [10,21] or non-cultivated blastomeres [16,21,32]. In some experiments, embryos transplanted with blastula cells were reared in the presence of 1 mM of phenylthiourea to prevent pigmentation for easier fluorescence observations.…”

“…For this, the olvas-gfp transgene was introduced into the HB32C background by crossing, producing Vg (Vasa-gfp) that specifically expresses GFP in PGCs ( Fig. 3a, b; [32]). Upon transplantation, Vg-blastomeres contributed to the germline of host strain af, as evidenced by GFP expression in the gonad (Fig.…”

Efficient Knockout of Transplanted Green Fluorescent Protein Gene in Medaka Using TALENs

“…Medaka is also at the forefront of research into cell transplantation and nuclear transplantation (Hong et al 2010;Liu et al 2011;Yi et al 2009), and this species demonstrates common characteristics of marine fish embryos, such as a hard egg membrane and high pressure inside the egg membrane, which makes it suitable for embryo manipulation techniques and gene editing. However, researches on gene knockout and gene editing of medaka still rely on the use of antisense morpholino oligos (Li et al 2009;Nasevicius and Ekker 2000). The emergence and development of artificial ribozyme technology led to sporadic reports of ZFN being used to knock out a transgene green fluorescent protein (GFP) (Ansai et al 2012) and an endogenous gene (gsdf) on the chromosome (Zhang et al 2014).…”