DEADSouth protein localizes to germ plasm and is required for the development of primordial germ cells in Xenopus laevis

Abstract

SummaryDEADSouth mRNA is a component of germ plasm in Xenopus laevis and encodes a DDX25 DEAD-box RNA helicase. To determine the intracellular localization of DEADSouth protein, we injected mRNA encoding DEADSouth tagged with mCherry fluorescent protein into fertilized eggs from transgenic Xenopus expressing EGFP fused with a mitochondrial targeting signal. The DEADSouth-mCherry fusion protein was localized to the germ plasm, a mitochondria-rich region in primordial germ cells (PGCs). DEADSouth overexpression resulted in a reduction of PGC numbers after stage 20. Conversely, DEADSouth knockdown using an antisense locked nucleic acid gapmer inhibited movement of the germ plasm from the cortex to the perinuclear region, resulting in inhibition of PGC division at stage 12 and a decrease in PGC numbers at later stages. The knockdown phenotype was rescued by intact DEADSouth mRNA, but not mutant mRNA encoding inactive DEADSouth helicase. Surprisingly, it was also rescued by mouse vasa homolog and Xenopus vasa-like gene 1 mRNAs that encode DDX4 RNA helicases. The rescue was dependent on the 3′ untranslated region (3′UTR) of DEADSouth mRNA, which was used for PGC-specific expression. The 3′UTR contributed to localization of the injected mRNA to the germ plasm, resulting in effective localization of DEADSouth protein. These results demonstrate that localization of DEADSouth helicase to the germ plasm is required for proper PGC development in Xenopus laevis.

Germes is involved in translocation of germ plasm during development of Xenopus primordial germ cells

“…The diameters of these PGCs were significantly larger than those of control PGCs (48.4±8.0 mm). Moreover, these phenotypes were very similar to those of DEADSouth knockdown (Yamaguchi et al, 2013).…”

“…To understand the mechanisms underlying germ plasm organization, it is important to investigate the phenotypes of embryos overexpressing GermesWT or GermesΔLZs for comparison with those of DEADSouth-knockdown embryos, particularly at stages 7-12 that are much earlier than the tailbud stage. Although it has been very difficult to directly observe the germ plasm in isolated PGCs, the use of mito-EGFP Xenopus enables such observations (Taguchi et al, 2012;Yamaguchi et al, 2013). Here, we demonstrate that translocation of the germ plasm is inhibited by overexpression of GermesWT or GermesΔLZs, and by knockdown of DEADSouth, suggesting that both Germes and DEADSouth proteins are involved in the translocation of germ plasm during MBT.…”

“…Interestingly, we could not detect any alteration in the PGCs of injected embryos until stage 7. The mislocalization of germ plasm in these PGCs was very similar to that in DEADSouth-knockdown PGCs (Yamaguchi et al, 2013). To elucidate the relationship among Germes, germ plasm translocation, and mitosis, we individually cultured PGCs isolated from stage 8 embryos injected with GermesWT-DS, and followed PGC mitosis for 11 hours (Fig.…”

“…Recently, we generated transgenic Xenopus expressing EGFP fused with a mitochondrial targeting signal (mito-EGFP) for observation of the germ plasm in living embryos (Taguchi et al, 2012), because germ plasm is enriched with mitochondria (Venkatarama et al, 2010;Elinson et al, 2011). Using mito-EGFP embryos, we demonstrated that DEADSouth protein localizes to the germ plasm, where it is required for proper PGC development (Yamaguchi et al, 2013). Interestingly, knockdown of DEADSouth mRNA causes expansion of the germ plasm, inhibition of germ plasm translocation and division of PGCs after MBT.…”

“…To elucidate the relationship among Germes, germ plasm translocation, and mitosis, we individually cultured PGCs isolated from stage 8 embryos injected with GermesWT-DS, and followed PGC mitosis for 11 hours (Fig. 4A) Yamaguchi et al, 2013) …”