XB-ART-27040Development January 1, 1989; 107 Suppl 31-6.
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The process of localizing a maternal messenger RNA in Xenopus oocytes.
The maternal mRNA Vg1 is localized to the vegetal pole during oogenesis in Xenopus. We have cultured oocytes in vitro to begin to understand how this localization occurs. Endogenous Vg1 mRNA undergoes localization when oocytes are cultured in vitro, and synthetic Vg1 mRNA injected into such oocytes is localized in the same fashion. Vg1 mRNA is associated with a detergent-insoluble fraction from homogenized oocytes, suggesting a possible cytoskeletal association. The use of cytoskeletal inhibitors reveals a two-step process for localizing Vg1 mRNA. Microtubule inhibitors such as nocodazole and colchicine inhibit the localization of Vg1 mRNA in late stage III/early stage IV oocytes, but have no effect on Vg1 mRNA once it is localized. The microfilament inhibitor cytochalasin B, however, has little effect on the translocation of Vg1 mRNA in middle-stage oocytes but causes a release of the message in late-stage oocytes. We propose a model for the localization of Vg1 mRNA in which translocation of the message to the vegetal cortex is achieved via cytoplasmic microtubules and the anchoring of the message at the cortex involves cortical microfilaments.
PubMed ID: 2636138
Species referenced: Xenopus
Genes referenced: fn1 gdf1
Article Images: [+] show captions
|Fig. 1. Localization of endogenous VgJ mRNA in cultured oocytes. Oocytes were grown in Leibowitz medium supplemented with vitellogenin-containing frog serum as previously described (Yisraeli and Melton, 1988). After the indicated time in culture, the oocytes were fixed and hybridized with a Vgl probe made to the coding region (Melton, 1987; Yisraeli and Melton, 1988). In these dark-field photographs, silver autoradiographic grains appear white.|
|Fig. 2. Localization of exogenous in vitro synthesized Vgl mRNA injected into oocytes. Capped, radioactively labeled Vgl and globin transcripts were synthesized as described (Krieg and-Melton, 1987). Oocytes injected with message were cultured as above for 5 days, time 0, the initial site of injection; J days, oocytes injected with the indicated message fixed after 5 days in culture.|
|Fig. 3. Attachment of Vgl mRNA to the detergent insoluble pellet of extracts. Stage V/VI oocytes were homogenized in the indicated buffer at room temperature. After removing an aliquot and extracting the RNA (total RNA), the homogenate was centrifuged and RNA was prepared separately from the supernatant (soluble) and insoluble (pellet) fractions and analyzed by Northern blot hybridization. The positions of fibronectin and Vgl messages on the blot are indicated at right. The RNA from two oocytes prepared in this way was run in each lane.|
|Fig. 4. Effect of cytoskeletal inhibitors on Vgl mRNA in late-stage oocytes. Stage V/VI oocytes were incubated in saline (untreated), cytochalasin B (25ug/mL, cvtochalasin B), or nocodazole (10ug/mL, nocodazole) overnight at 20°C and then analyzed by in situ hybridization for the localization of Vgl mRNA. In addition, detergent extracts of the oocytes were performed for each treatment and the corresponding Northern blots are shown below each section, p, pellet; s, soluble fraction.|
|Fig. 5. Effect of cytoskeletal inhibitors on Vgl mRNA in middle-stage oocytes. Late stage III oocytes were cultured for 5 days in the presence of medium and serum alone (+serum), medium and serum with cytochalasin B (+serum +cytochalasin 6), and medium and serum with nocodazole (+serum +nocodazole). In situ hybridization visualized the location of the Vgl mRNA in the oocyte sections. At left is a section from an oocyte cultured in medium alone for 5 days, showing no localization whatsoever (-serum).|
|Fig. 6. A two-step model for the localization of Vgl mRNA in oocytes. The horizontal arrows indicate the normal process of oogenesis, with the black dots and black shading indicating the location of Vgl mRNA and its progressive localization. The larger, vertical arrows show where the various cytoskeletal inhibitors are thought to interrupt this process and point to the results of treating oocytes with these drugs. Thus, translocation of the Vgl mRNA to the vegetal cortex is thought to be associated with microtubules and the anchoring of the message is thought to involve microfilaments.|