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Mech Dev
2006 Oct 01;12310:746-60. doi: 10.1016/j.mod.2006.07.006.
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Visualization of the Xenopus primordial germ cells using a green fluorescent protein controlled by cis elements of the 3' untranslated region of the DEADSouth gene.
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We succeeded in visualization of the primordial germ cells (PGCs) in a living Xenopus embryo. The mRNA of the reporter Venus protein, fused to the 3' untranslated region (UTR) of DEADSouth, which is a component of the germ plasm in Xenopus eggs, was microinjected into the vegetal pole of fertilized eggs and then the cells with Venus fluorescence were monitored during development. The behavior of the cells was identical to that previously described for PGCs. Almost all Venus-expressing cells were Xdazl-positive in the stage 48 tadpoles, indicating that they were PGCs. In addition, we found three sub-regions (A, B and C) in the 3' UTR, which were involved in the PGC-specific expression of the reporter protein. Sub-region A, which was identified previously as a localization signal for the germ plasm during oogenesis, participated in anchoring of the mRNA at the germ plasm and the degradation of the mRNA in the somatic cells. Sub-regions B and C were also involved in anchoring of the mRNA at the germ plasm. Sub-region B participated in the enhancement of translation.
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16945508
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Fig. 1. Visualization of PGCs in a whole embryo/tadpole microinjected with venus mRNA fused to 1.6 kb of DEADSouth 3′ UTR (DS3′UTR). (A) Schematic diagram of the microinjection of the mRNA into the vegetal pole of the fertilized egg (1 cell-stage). (B, D, and F–J) Expression of Venus protein (green) during development. (C and E) Whole-mount in situ hybridization for DEADSouth gene. (B–D) Vegetal view. (B and D) are the same embryo. Arrowheads in (C and D) indicate the pigmented dorsal lip. (E–I) Lateral view. (J) Dorso-lateral view. Each developmental stage is indicated (B–J). Arrows in (F–J) indicate the Venus-expressing cells. Scale bars are 250 μm in (B–D), 1 mm in (E–J).
Fig. 2. Behavior of Venus-expressing cells inside the embryos. (A) Sagittal manual section of gastrula at stage 11. bl, blastocoel; dl, dorsal lip; vl, ventral lip. (B) Transverse manual section of the neurula at stage 17. ar, archenteron. (C) Endodermal cell mass of the embryo at stage 20 dissociated in 1.25-fold diluted PBS(−) (Kataoka et al., 2005). Arrowheads in (A–C) indicate the Venus-expressing cells. (D) High magnification images of the Venus-expressing cells at stage 43 (tadpole). (E–G) Manual transverse section of the tadpoles at stages 34, 38 and 41. Fluorescent images were superimposed on bright field images. Scale bars are 250 μm in (A and B), 100 μm in (C), 50 μm in (D) and 250 μm in (E–G).
Fig. 3. Location of Venus-expressing cells in the tadpoles at stages 48 (A–D) and 52 (E–G). Paraffin-embedded tadpoles injected with DS3′UTR were sectioned transversely at the indicated level (inset of A) and immunostained with anti-Xdazl (red) and anti-GFP (green). All images are merged with Nomarski images. (A) A low-magnification image merged with anti-Xdazl and anti-GFP. (B-D) High-magnification image of the genital ridges in the box in (A). (B and E) Anti-Xdazl staining (red). (C and F) Anti-GFP staining (green). (D and G) are merged images of (B and C), and (E and F), respectively. High-magnification image of the gonad at stage 52 is shown in the inset of (E–G), respectively. g, gonad; i, intestine; l, lung; m, muscle; nc, notochord; sc, spinal cord. Scale bars are 250 μm in (A), 20 μm in (B–D), 50 μm in (E–G) and 20 μm in insets of (E–G), respectively.
Fig. 4. Venus-expressing cells in the gonads at the tadpole stage (ventral view). The mesonephros of the tadpole at stage 56 was immunostained in whole-mount simultaneously with anti-Xdazl (red in B and F) and anti-GFP (green in C and G). (A–D) Low-magnification images. (E–H) High-magnification images of the box in (A). (A and E) are bright-field images. (D) is a merged image of (B and C). (H) is a merged image of (E, F, and G). Auto-fluorescence was also observed. The arrowheads in (H) indicate the double positives for anti-Xdazl and anti-GFP (yellow). Note that several Xdazl-positive cells are Venus-negative. Scale bars, 500 μm in (A) and 200 μm in (E), respectively.
Fig. 6. Typical expression pattern of venus fused to variously deleted 3′ UTR of DEADSouth in stage 41 tadpoles. Whole-mount lateral views (left) are shown with high-magnification views of PGCs (right), respectively. Solid and dashed brackets indicate PGCs with and without fluorescence, respectively. The injected transcript is indicated on the upper right of each image. Refer to Fig. 5 for the transcripts. Scale bars, 1 cm and 250 μm in (H) and (H′), respectively.
Fig. 8. Expression of Venus protein in the stage 41 tadpoles injected with the transcripts of venus-β-globin fused to various sub-regions. Whole-mount lateral views (left) are shown with high-magnification views of PGCs (right), respectively. Solid and dashed brackets indicate PGCs with and without fluorescence, respectively. The injected transcript is indicated on the upper right of each image. Refer to Fig. 7C for the transcripts. Scale bars, 1 cm and 250 μm in (H) and (H′), respectively.
Fig. 9. Stability of the injected mRNA of venus-β-globin sub-region A, B and C. (Left) Northern blot analysis with radioactive venus DNA probe for total RNA at stages 1 (just after microinjection), 11 (gastrula) and 30 (tailbud). 18S rRNA bands are shown as a loading control (lower band in each row, stained with methylene blue). Closed and open arrowheads indicate the band of injected transcripts and the upper shifted band, respectively. The ratio of band intensity at stage 30 to that at stage 11 is also shown. Each ratio was the mean of duplicate experiments. UD indicates undetectable level. (Right) Whole-mount in situ hybridization (WISH) with digoxigenin-labeled anti-sense venus RNA probe. The embryos injected with a given transcript were allowed to develop until stage 30. The hybridization signal was blue. Brackets indicate the venus-positive PGCs. The percentage of embryos with venus-positive PGCs shown on the right was calculated from a total of 3 independent injections. The lowest row shows the result from an experiment in which venus-β-globin transcript fused to sub-region ABC (βG/ABC) was injected at the animal pole of the fertilized eggs. Scale bar, 1 mm.
