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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.
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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.
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 âstage 7â 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 âstage 23â
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) âStage 10â 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 âstage 33/34â 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 transcriptionâpolymerase 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.
