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???displayArticle.abstract??? VegT is an essential maternal regulator of germ layer specification in Xenopus. The localization of VegT mRNA to the vegetal cortex of the oocyte during oogenesis ensures its inheritance by vegetal and not animal cells, and directs the differentiation of vegetal cells into endoderm. Similarly localized mRNAs, Vg1 and Bicaudal-C, are also inherited by vegetal cells, while germ plasm-associated mRNAs, such as Xcat2, become incorporated into vegetally derived primordial germ cells. Although mRNA localization is clearly important for tissue specification, the mechanism of mRNA anchoring to the oocyte vegetal cortex is not understood. Here, we examine the role of VegT in cortical localization. We report that depletion of VegT mRNA caused the release of Vg1 mRNA from the vegetal cortex and a reduction of Vg1 protein, without affecting the total amount of Vg1 transcript. Furthermore, we found that Bicaudal-C and Wnt11 mRNAs were also dispersed, but not degraded, by VegT depletion, while the localization of Xcat2 and Xotx1 mRNAs was unaffected. This effect was specific to the loss of VegT mRNA and not VegT protein, since a morpholino oligo against VegT, that blocked translation without degrading mRNA, did not disperse the vegetally localized mRNAs. Therefore, a subset of localized mRNAs is dependent on VegT mRNA for anchoring to the vegetal cortex, indicating a novel function for maternal VegT mRNA.
FIG. 3. VegT depletion disrupts vegetal localization of Vg1 mRNA. Vg1 mRNA localization was examined by whole-mount in situ
hybridization of uninjected oocytes (Control) and oocytes injected with 30 ng of a morpholino oligo (VegT Morpholino) or 7 ng of chimeric
phosphorothioate/phosphodiester oligo (VegT Thioate). The results shown are representative of three independent experiments (n 5 15â 20 per
sample in each experiment). (Aâ C) Vegetal localization of Vg1 mRNA was observed in 100% of uninjected and morpholino oligo-injected oocytes
and in 5% of chimeric oligo-injected oocytes. (Dâ F) Histological sections (20 mm) of stained oocytes showing localized Vg1 mRNA (arrowheads)
at the vegetal cortex of uninjected and morpholino oligo-injected oocytes, but not in chimeric oligo-injected oocytes. The staining adjacent to the
germinal vesicle (GV) is nonspecific and was also observed in sense probe controls (data not shown). (Gâ I) Sibling embryos cultured to the tailbud
stage displayed a complete loss of axis formation with VegT depletion by the morpholino or chimeric antisense oligos. Vegetal (Aâ C), Sagittal
(Dâ F), ventralâ anterior to right (G), and lateral views (H, I) are shown. Scale bar, 0.6 (Aâ C), 0.2 (Dâ F), and 0.4 mm (Gâ I).
FIG. 4. Mislocalization of multiple vegetal mRNAs with VegT depletion. Whole-mount in situ hybridization analysis of Bicaudal-C (A C;
BicC), Xotx1 (D F), Wnt11 (G I), and Xcat2 (J L) localization in uninjected oocytes (Control) and oocytes injected with 30 ng of morpholino
oligo (VegT Morpholino) or 7 ng of chimeric oligo (VegT Thioate). The results shown are representative of three independent experiments
(n 5 15 20 per sample in each experiment). Vegetal localization of each mRNA was observed in 100% of uninjected and morpholino
oligo-injected oocytes. While vegetal localization of Xotx1 and Xcat2 was observed in 95 100% of chimeric oligo-injected oocytes,
localization of Bicaudal-C and Wnt11 was observed in less than 10% of oocytes. Vegetal views are shown. Scale bar, 0.5 mm.
