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Abstract
In Xenopus, the inheritance of germ plasm by a small subset of blastomeres during early development is thought to direct these cells into the germ cell lineage. We show that Xcat2 RNA, related to Drosophila nanos, is a germ plasm component that is translationally repressed during oogenesis. Xcat2 protein was not detected in oocytes at times prior to, or after its RNA was localized in germ plasm, suggesting Xcat2 RNA is functionally sequestered soon after transcription. Indeed, Xcat2 RNA is found in a dense non-polysomal compartment in oocytes. Repression of translation was not relieved by substituting the Xcat2 3'UTR with that of beta-globin. Immunodetection of Xcat2 protein during blastula and gastrula stages coincides with the time of symmetric segregation of the germ plasm and a net increase in the number of primordial germ cells. Xcat2 is capable of binding RNA in vitro and we propose that it may function to translationally regulate other RNAs specific to primordial germ cells.
Fig. 1. Xcat2 RNA follows the same distribution pattern as germ plasm. (A) Whole-mount in situ of a four-cell embryo; vegetal pole view showing Xcat2 positive germ plasm islands. (B-F) Whole-mounts of dissociated embryonic cells embedded in plastic and sectioned at 1 mm. (B) Cells from a stage 7 embryo. Note the one Xcat2 staining cell. (C) Higher magnification of pPGC shown in (B). Note that Xcat2 staining is in a yolk-free area, the germ plasm, close to the plasma membrane. (D) Cells from a stage 10 embryo showing one Xcat2 positive pPGC. (E) Higher magnification of pPGC seen in (D) showing the perinuclear Xcat2 staining. (F) Another Xcat2 positive pPGC at stage 10 at high magnification. Scale bars, (A) 200 um; (B) 50 um; (E-F) 20 um.
Fig. 4. Xcat2 RNA is not translated during oogenesis. (A) In situ analysis of Pre I and stage I oocytes for Xcat2 RNA. (1) Mixed Pre-I to stage II oocytes; arrow indicates group of Pre I oocytes. (2) Pre I to stage II oocytes hybridized with sense RNA probe (control). Scale bars, 50 mm. (B) Western blot analysis of oocyte extracts with Xcat2 depleted antiserum. Lanes 1, 2, 3, 8, 9 and 10 are lanes loaded with decreasing levels of rHTXcat2 protein in ng as a control for antiserum depletion. Lanes 1-7 were incubated with control depleted antiserum (depleted of anti-HisTag antibodies); lanes 84 were incubated with antiserum depleted of anti-Xcat2 antibodies. Each depleted antiserum was incubated 4 with the depleting antigen source. Oocyte equivalents per lane: Pre I, 500; stage I, 2000-4000 (25 ml); vegetal tips, 43; animal tips, 37. Arrow indicates 17.5 kDa, the mass of rHTXcat2. Note that the endogenous 17.5 kDa band is evident with serum depleted of Xcat2 antibodies (lanes 124).
Fig. 7. Xcat2 protein is present in PGCs of early embryos. Dissociated cells from stages 8-10 (A,B) or stages 11-13 (C,D) were treated with anti-Xcat2 antiserum. No staining was observed at these stages for other vegetal pole cells (B) or with preimmune serum (D). Scale bars, 100 um.
