Ikenishi K and Yamakita S (2003), A trial for induction of supernumerary primordi...

XB-ART-4166

Dev Growth Differ 2003 Jan 01;455-6:417-26. doi: 10.1111/j.1440-169x.2003.00708.x.

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A trial for induction of supernumerary primordial germ cells in Xenopus tadpoles by injecting RNA of Xenopus vasa homologue into germline cells of 32-cell embryos.

Ikenishi K , Yamakita S .

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Whether overexpression of Xenopus vasa homologue or Xenopus vasa-like gene 1 (XVLG1) in germline cells of Xenopus embryos can induce supernumerary primordial germ cells (PGC) at tadpole stage was investigated. XVLG1 RNA (0.1-2.0 ng) and beta-gal RNA (0.5 ng) were injected into one of, usually, four germ plasm-bearing cells (GPBC) of 32-cell embryos, with the beta-gal RNA (2.0 ng) serving as both lineage tracer and control for XVLG1 RNA. The total number of PGC, including X-gal-stained and unstained PGC of injected and uninjected GPBC origins respectively, was examined in the experimental tadpoles developed from the injected embryos. The injected RNA, XVLG1 and beta-gal RNA, were translated, resulting in a large amount of corresponding proteins in presumptive PGC (pPGC) as well as in somatic cells derived from the injected GPBC. Nevertheless, the average number of total PGC per tadpole found in the experimental tadpoles from the XVLG1 RNA-injected embryos was not significantly different from that of beta-gal RNA-injected ones, irrespective of the injected dose of XVLG1 RNA. This indicates that the extra XVLG1 protein in pPGC is not sufficient to increase the number of PGC in the tadpoles.

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Species referenced: Xenopus
Genes referenced: ddx4 gal.2 mhc2-dab pgc tbx2
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	Fig. 1. An outline of an injection experiment with Xenopus vasa-like gene 1 (XVLG1) and -gal RNA. The injected RNA and their products were examined in embryos at stages 7–35. The total number of X-Gal stained and unstained primordial germ cells (PGC) were counted in experimental tadpoles (stage 46) from the injected embryos. See text for detail.
	Fig. 2. Detection of XVLG1 RNA in RNA-injected embryos at stages 12 (A and C) and 23 (B) by whole-mount in situ hybridization with the anti-sense probe. ( A) A strong signal representing XVLG1 RNA is observed in a cluster of cells in the endodermal cell mass derived from XVLG1 RNA-injected, single germ plasm-bearing cells (GPBC) of 32-cell embryos. Two cells (white arrows) with a granular cytoplasm around the nucleus, probably pPGC, are seen in the cluster. bc, blastocoele; Bar, 50 μm. (B) A strong signal is detectable mainly in a number of endoderm cells from XVLG1 RNA-injected embryos. gc, gut cavity; n, neural tube; s, somite; Bar, 250 μm. (C) No signal is observed in either pPGC (arrows), having neither a granular cytoplasm nor somatic endoderm cells from -gal RNA-injected, single GPBC of an embryo. Bar, 50 μm.

	Fig. 4. A section parallel to an animal- vegetal axis of a XVLG1 RNA-injected embryo at stage 12. XVLG1 protein is detected not only in a presumptive PGC (pPGC) (arrow), having a granular cytoplasm around the nucleus but also in many somatic cells (asterisks) derived from XVLG1 RNA-injected, single GPBC. Bar, 50 μm. Xenopus vasa-like gene 1 protein in the RNA-injected embryos by whole-mount immunostaining with the 2L-13 antibody. Color reaction is done with diaminobenzidine (DAB) as a substrate. The top is dorsal and the bottom ventral. bc, blastocoele.
	Fig. 5. (A) A t A transverse section through the presumptive mesonephros region of a XVLG1 RNA-injected embryo at stage 35. XVLG1 protein, mostly from the injected XVLG1 RNA, can be clearly seen in a cell at the lateral part (arrow) of the right-hand side and in a number of cells (asterisks) at the lateral and ventral parts of the left hand side in the endoderm cell mass. Those cells are derived from the injected GPBC. Bar, 100 μm. Panels B–D) display a higher magnification of the cell (arrow in A) seen in three consecutive sections. A strong color reaction is detected in the cell, showing characteristics of pPGC as described earlier (Kamimura et al., 1976), (i.e., it is roundish in external shape and has a granular cytoplasm next to the nucleus and a large intercellular space around it). Bar, 50 μm. (E) An adjacent section of the XVLG1 RNA-injected embryo in panel A. A cell (arrow) of uninjected GPBC origin, which seems to be unstained or nearly unstained, but shows similar morphologic characteristics to pPGC is also seen in the most dorsal part of the endoderm cell mass. Bar, 25 μm. (F) A transverse section of a -gal RNA-injected embryo at stage 35 is displayed. An unstained or nearly unstained cell (arrow), which is like pPGC, is observed in the dorsal part of the endodermal cell mass of the embryo, though XVLG1 protein was detected in pPGC at that stage by immunofluorescent microscopy in the previous study (Ikenishi et al., 1996). Staining was not detected in any cell of the embryo by whole-mount immunostaining. Bar, 25 μm. Method the same as in Figure 4. da, dorsal aorta; mg, midgut; n, neural tube; nt, notochord; s, somite.
	Fig. 6. A section of a -gal RNA-injected embryo at stage 12. XVLG1 protein is not recognized in any cell of the endodermal cells, including a cell with a granular cytoplasm or a pPGC (arrow). This was also the case in uninjected embryos. Bar, 50 μm. Method the same as in Figure 4; abbreviations as in Figure 5.
	Fig. 7. A section parallel to an animal-vegetal axis of a -gal RNA-injected embryo at stage 7. -galactosidase translated from the injected -gal RNA is already recognized with X-Gal staining in a number of cells (asterisks) beneath the blastocoele. bc, blastocoele; Bar, 100 μm.
	Fig. 8. A transverse section of a feeding tadpole (stage 46) from XVLG1 RNA-injected embryos. An X-Gal-stained PGC (black arrow) and an unstained PGC (white arrow) of injected and uninjected GPBC origins, respectively, are seen in the genital ridges. A considerable number of cells in the intestine are also stained, indicating that they are also of injected GPBC origin. i, intestine; Wd, Wolffian duct; Bar, 50 μm.
	Fig. 9. The proportions of X-Gal, stained (closed bar) and unstained (open bar) PGC in each of the experimental tadpoles from RNAinjected embryos. There is a variety in the number of X-Gal stained PGC among the experimental tadpoles from XVLG1 or -gal RNAinjected embryos.