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Germ plasm is found in germ-line cells of Xenopus and thought to include the determinant of primordial germ cells (PGCs). As mitochondria is abundant in germ plasm, vital staining of mitochondria was used to analyze the movement and function of germ plasm; however, its application was limited in early cleavage embryos. We made transgenic Xenopus, harboring enhanced green fluorescent protein (EGFP) fused to the mitochondria transport signal (Dria-line). Germ plasm with EGFP-labeled mitochondria was clearly distinguishable from the other cytoplasm, and retained mostly during one generation of germ-line cells in Dria-line females. Using the Dria-line, we show that germ plasm is reorganized from near the cell membrane to the perinuclear space at St. 9, dependent on the microtubule system.

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 mRNA and protein are components of the germ plasm in Xenopus laevis. Previously, based on phenotypic observations of tailbud embryos expressing intact and mutant Germes, it was suggested that Germes is involved in the organization of germ plasm (Berekelya et al., 2007). Recently, to observe the germ plasm in a living embryo, we generated transgenic Xenopus expressing EGFP fused with a mitochondrial targeting signal, because germ plasm is enriched with mitochondria (Taguchi et al., 2012). Using this transgenic Xenopus, we demonstrate that Germes plays an essential role in the translocation of germ plasm from the cortex to the perinuclear region in primordial germ cells during early gastrulation.

We investigated the intracellular localization of Xenopus Dead end protein (Dnd1) in primordial germ cells during early development by expressing the tagged protein in transgenic Xenopus embryos, with the germ plasm visualized. Dnd1 initially localized to the germ plasm in the cortex, moved to the perinuclear region together with the germ plasm after the midblastula transition, and then entered the nucleus. Using Dnd1 deletion mutants, we identified two distinct but overlapping regions of Dnd1 that were responsible for localization to either the germ plasm or nucleus. These Dnd1 regions appeared to function in primordial germ cell-and stage-specific manners. KEY WORDS: Dnd1, germline, germ plasm, nuage, nuclear localization signal, RNA-binding proteinThe Xenopus germline is specified by inheriting a special cytoplasm, germ plasm (GP), associated with the vegetal cortex of the egg. We demonstrated that the GP was sufficient for germline specification (Tada et al., 2012). It is divided almost equally into daughter blastomeres until the 4-cell stage and is then distributed unequally to one daughter blastomere at each successive cleavage until stage 9 (Whitington and Dixon, 1975). Subsequently, it moves from the cortex to the perinuclear region and is distributed equally into two daughter primordial germ cells (PGCs). Midblastula transition (MBT), when zygotic genes begin to be expressed in most blastomeres, is observed around stage 9. However, gene expression is repressed in PGCs at MBT, so PGC specification likely occurs later (Venkatarama et al., 2010). Xpat protein, a GP component, moves from the cortex to the perinuclear region and then enters the nucleus (Machado et al., 2005). These observations suggest that some signal from GP to the nucleus is required for PGC specification.Dead end (dnd) encoding an RNA-binding protein with an RNA recognition motif (RRM) has been identified as a germlinespecific gene in vertebrates including zebrafish, Xenopus and mice (Weidinger et al., 2003). In Xenopus, the dnd1 transcript is a GP component and is required for PGC migration (Horvay et al., 2006). Furthermore, mouse dnd1 is responsible for the Ter mutant, which causes high frequency teratoma generation in a particular genetic background (Youngren et al., 2005). Several reports have described the intracellular localization of Dead end protein; in chicks Dnd protein is present in the nuclei of PGCs and mature male germ cells (Aramaki et al., 2009). In mice, two isoforms, DND1a and DND1b, were identified and localized to the nucleus and cytoplasm when Int. J. Dev. Biol. 58: 793-798 (2014) Abbreviations used in this paper: Dnd, dead end protein; GP, germ plasm; GPS, germ plasm localization signal; PGC, primordial germ cell.expressed in HeLa and COS-7 cells, respectively (Bhattacharya et al., 2007). In zebrafish, Dnd was localized to germ cell granules (GCGs, probably equivalent to Xenopus GP) in the perinuclear region of PGCs after MBT. Interestingly, this localization was dependent upon the RNA recognition motif...