Ikenishi K and Tanaka TS (2000), Spatio-temporal expression of Xenopus vasa homo...

XB-ART-10931

Dev Growth Differ 2000 Apr 01;422:95-103. doi: 10.1046/j.1440-169x.2000.00493.x.

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Spatio-temporal expression of Xenopus vasa homolog, XVLG1, in oocytes and embryos: the presence of XVLG1 RNA in somatic cells as well as germline cells.

Ikenishi K , Tanaka TS .

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The expression of Xenopus vasa homolog or XVLG1 was examined in oocytes and embryos by whole-mount in situ hybridization and reverse transcription polymerase chain reaction (RT-PCR). To confirm the results in embryos, both methods were also applied to explants of germ plasm-bearing cells (GPBC) from 32-cell embryos and to those of partial embryos deprived of GPBC. By hybridization, XVLG1 ribonucleic acid (RNA) was shown to be present throughout the cytoplasm in oocytes at stages I-III, except for the mitochondrial cloud. It was barely recognizable in a portion of germline cells of embryos at specific stages, notwithstanding that XVLG1 protein was present in those cells almost throughout their life-span. A weak signal for the RNA was detectable in some of the presumptive primordial germ cells (pPGC, descendants of GPBC from the gastrula stage onward) from the late gastrula (stage 12) to the hatching tadpole stage (stage 33/34), and in some of the PGC at stages 49-50. The results for pPGC were confirmed by the hybridization of explants of GPBC at equivalent stages in control embryos. In contrast, XVLG1 RNA was detected in certain somatic cells of embryos until stage 46. These observations were supported in part by the results of RT-PCR for embryos and explants. The possible role of the product of XVLG1 was reconsidered given its presence in both germline and somatic cells.

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Species referenced: Xenopus
Genes referenced: actl6a ddx4 gs17 mixer pgc tbx2

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	Fig. 1. (A) Whole-mount in situ hybridization of oocytes at stages I–VI with antisense or sense ribonucleic acid (RNA) probe for XVLG1 RNA. A signal representing XVLG1 RNA is clearly seen in stage I–III oocytes but nearly undetectable in stage IV–VI oocytes with the antisense probe (upper tier). The signal is not detected in oocytes at any stage when the sense probe is employed (lower tier). From left to right, oocytes at stages from I to VI are lined up in order. (B) A section of the whole-mount specimen of stage I–III oocytes hybridized with the antisense probe as shown in Fig. 1(A). XVLG1 RNA is distributed throughout the cytoplasm of stage I–III oocytes, except for the mitochondrial cloud (arrow) of the stage I oocyte. It seems to disappear with oocyte growth, but can be still observed moderately in stage II oocytes and faintly in stage III oocytes. Bar, 1 mm (A), 100 μm (B).

	Fig. 3. Explants of germ plasmbearing cells (GPBC) isolated from 32-cell stage (stage 6) embryos by in situ hybridization with antisense or sense ribonucleic acid (RNA) for XVLG1 RNA. (A) A ‘stage 7’ explant labeled with the antisense probe. No signal is detected either in cells having a granular cytoplasm or the germ plasm (arrowhead), nor in somatic cells. (B) A ‘stage 23’ explant labeled with the antisense probe. A signal is observed in a cell with granular cytoplasm (arrowhead), probably a presumptive primordial germ cell (pPGC), which is found in the periphery of the explant. (C) ‘Stage 10’ explants hybridized with the antisense or sense probe. A signal is clearly seen in the explants of the upper tier with the antisense probe while it is not detectable in those of the lower tier with the sense probe. (D) Section of a ‘stage 33/34’ explant labeled with the antisense probe. A signal is recognized in a group of cells, probably mesodermal cells, which were somewhat different morphologically from the endodermal cells occupying most of the explant. Bar, 50 μm (A), 20 μm (B), 1 mm (C), 100 μm (D).
	Fig. 4. Reverse transcription–polymerase chain reaction (RTPCR) analysis of XVLG1 ribonucleic acid (RNA) in Xenopus oocytes and embryos, and in explants of germ plasm-bearing cells (GPBC) and the partial embryos deprived of GPBC. See text for details concerning the conditions for RT-PCR. The PCR product was analyzed in a 2% agarose gel containing ethidium bromide (EtBr). (A) XVLG1 RNA is detected in stages I–III but not in stage VI oocytes. It is clearly observed in almost all embryos from the fertilized to the feeding tadpole stage, except that it was rare in unfertilized eggs, and stage 7 and 10 embryos. Expression of EF1 a is shown as a control for loading. (B) XVLG1 RNA is present in all partial embryos and all explants of GPBC, except for the ‘stage 7’ explants.
	Fig. 5. Reverse transcription–polymerase chain reaction (RTPCR) analysis of GS17, actin and mixer in explants of germ plasm-bearing cells (GPBC) and the partial embryos deprived of GPBC, and in whole embryos. The temporal expressions of ribonucleic acid (RNA) of these three genes in explants and the partial embryos are nearly consistent with those in whole embryos. This strongly suggests that gene expression in the in vitro culture system reflects that in vivo. See text for details.