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cDNAs specific to vegetal poles of Xenopus gastrula embryos were used as a probe to screen a gastrula vegetal pole cDNA library. One of the novel clones isolated had an RNA expression pattern consistent with it being a component of germ plasm and it was thus named Xpat (Xenopus primordial germ cell associated transcript). The open reading frame encodes a 35 kDa protein with no clear homologies. The RNA is localised to the vegetal pole throughout oogenesis and early cleavage. During gastrulation cells containing this message move internally and at tailbud stages they migrate in an antero-dorsal direction. Xpat mRNA is not detectable once the dorsal mesentery forms. We show that the 3'-UTR is required and is sufficient for localisation of exogenous RNA to the vegetal pole of oocytes. We propose that Xpat UTR-containing transcripts can be localised by the Vg1 or late pathway of mRNA localisation during stage III of oogenesis, but endogenous Xpat appears to be localised earlier by a mitochondrial cloud mechanism similar to that proposed for Xcat-2.
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9622619
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Fig. 1. Analysis of Xpat RNA expression. (A) Northern blot. Xpat is present only in vegetal poles at stage 10.5. The first three lanes contain dissected regions approximately equalised for rRNA input (eight vegetal poles, four animal poles and two equatorial regions). Each of the stage series represents RNA from two embryos. Xpat probing gives a single 4 kb band in unfertilised eggs, but later the band is very faint and no second peak of expression is observed. The gels are probed for 18S rRNA as a loading control. (B) RT-PCR. ODC is ornithine decarboxylase, a ubiquitous transcript (Bassez et al., 1990).
Fig. 3. Localisation of Xpat mRNA assessed by in situ hybridisation. (A) Stage III oocytes (Dumont, 1972); Xpat transcripts are localised to the vegetal pole from stage I (A), remaining there during stages II (B,C) and III (D). (E,F) In cleavage stage embryos the signal takes on a granular appearance in a small disc at the vegetal pole. During gastrulation (stage 10.5, G) the cells that contain Xpat move inside, and remain there through neurulation (stage 14, H) until tailbud (stage 26, I). At stage 35 (J) they have begun to move anteriorly and dorsally in the endodermal mass. This continues through stage 37 (K), until they are most dorsal by stage 40 (L). (G) Embryos are cleared. Scale bar=100 μm.
Fig. 4. Sections of Xpat location. Shown are 28-μm wax sections of whole-mount in situ hybridisations (Fig. 3). (A) Animalegetal sections (animal up). (D) Transverse sections (dorsal up). (A) At blastula stage 9, expression can be seen in cells at the vegetal pole. (B) These cells internalise during gastrulation (stage 10.5) and remain in the endoderm through neurulation (stage 16, C). At stage 36 the Xpat-containing cells are lateral (D). They move dorsally at stage 38 (E). Scale bar=100 mm.
Fig. 5. The 3′-UTR localises transcripts to the vegetal pole. (A) Localisation of endogenous and exogenous Xpat in stage III oocytes. In each case several whole-mount in situ hybridisations and one sectioned oocyte is shown. In all cases antisense LacZ was used as a probe, except in (i) where Xpat was used. Tagged RNA was injected into stage III oocytes, which were cultured under growth conditions for 5 days. (i) Endogenous Xpat is localised at the vegetal pole in a tight cortical disc. (ii) Transcripts containing the Xpat ORF and UTR localise to the vegetal pole. (iii) This is also true of transcripts containing only the UTR. (iv) Transcripts containing only the ORF are not localised within the oocyte. Scale bar=100 mm. (B) Schematic representation of the mRNAs injected.
