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Figure 1. CK2α transcripts levels and localization by WISH(A) Analysis of total CK2α transcripts in Xenopus oocytes by RT-qPCR. Oocytes were collected, RNA extracted and quantitative PCR was carried out with specific primers to the CK2α coding sequence. The transcript for CK2α increases during oogenesis. This experiment was repeated twice with similar results.(B,C) Whole mount in situ hybridization of oocytes at different stages with an antisense CK2α -digoxigenin-labeled probe. (B) Chromogenic staining, shown in purple, indicates transcript localization. (C) Representative no probe controls for the in situ hybridizations. Animal hemisphere is up in all panels. Scale bars 250μm or 500μm as indicated in picture. This experiment was repeated using oocytes from three different frogs with similar results.
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Figure 2. CK2α transcript localization by RT-qPCRRT-qPCR analysis of 10 pooled animal (A) or vegetal (V) halves at late stage III (A) and stage V (B) stages of oogenesis with specific primers for CK2α; XWnt11, a known vegetally localized transcript; XWnt5, an animally enriched transcript. From left to right, histograms represent transcript number normalized by Odc for CK2α, XWnt11 and XWnt5a. This experiment was repeated three times for CK2α and Odc, and twice for Wnts with similar results.
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Figure 3. CK2α protein is enriched in the animal hemisphere of stage VI Xenopus oocytes(A) Expression of CK2 proteins during Xenopus oogenesis. Extracts were prepared from oocytes stages I, II, III, IV, V, VI, and stage VI treated with progesterone (PG). The volume equivalent of 0.2 stage VI oocytes was subjected to immunoblotting. Thus, we loaded, 22 stage I oocytes, 6.8 stage II oocytes, 2.5 stage III oocytes, 0.7 stage IV oocytes, 0.3 stage V oocytes and 0.2 stage VI oocytes. All immunoblots were repeated three times. The asterisk (*) indicates previously identified non-specific cross-reactive proteins in Xenopus extracts [15].(B) Graph representing the data (band intensity) from the three independent experiments as in Figure 3A. Protein band intensity was divided by the number of oocytes loaded to obtain the band intensitity per oocyte, normalized to stage I oocyte (stage I =1), and represented as mean ±S.D.(C) Immunoblot analysis of CK2 protein levels in 10 pooled stage VI animal or vegetal halves. One fifth (1/5, 0.2 μl) and one fourth (1/4, 0.25 μl) of oocyte lysate were loaded for immunoblot analysis. The asterisk (*) indicates a non-specific cross-reactive protein in Xenopus extracts [15]. CK2α protein is animally enriched at stage VI of oogenesis, while other tested proteins are not. This is a representative experiment out of three with identical results.
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Figure 4. Analysis of CK2α transcripts during oogenesis and sequence of the XCK2α 3âUTR(A) Northern blot analysis of RNA extracted from different stages of Xenopus laevis oogenesis using a CK2α coding sequence probe (CK2α, left columns) or probe specific to the CK2α 3âUTR (3âUTR, right column). Analysis shows two major CK2α transcripts, estimated at 2.8 kb and 1.8 kb. The upper band was identified as containing the 3âUTR sequence. Position of the 28S and 18S rRNA bands is marked. The northern blots were performed twice with similar results. As younger oocytes express less RNA than late oocytes, we loaded RNA from 2.5 stage I oocytes, 1.3 stage II oocytes, 0.52 stage oocytes III, 0.08 stage IV oocytes, 0.03 stage V oocytes and 0.01 stage VI oocytes.(B) Graph representing the ratio of the 2.8kb to 1.8kb XCK2α transcripts quantified from the Northern blot in Figure 4B.(C) An EST clone containing a XCK2α and its 3âUTR was identified (IMAGE ID: 4971090, BC072167 / Genbank Accession #: CF289385/CF289386) and sequenced. The 3âUTR was defined as starting at the first base downstream of the stop codon.
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Figure 5. CK2α coding and 3âUTR transcript regions are necessary for ectopic mRNA localization(A) On the left, schematic representation of the constructs GFP-CK2α-3âUTR, GFP-CK2α and GFP-3âUTR. On the right, summary of the results presented in this figure.(B) Histograms depicting the animal to vegetal ratio of ectopic mRNAs. Oocytes were injected vegetally with 1 ng of in vitro transcribed mRNA from the constructs GFP-CK2α-3âUTR, GFP-CK2α and GFP-3âUTR and five animal and vegetal halves were processed for RT-qPCR to the GFP sequence. The animal to vegetal ratio of GFP copy number was calculated (Left histogram) 48 hrs after injection of mRNAs from all the constructs into stage IV, and (Right histogram) 3 and 24 hrs after injection of GFP-CK2α-3âUTR mRNA into stage III. The CK2α-3âUTR mRNA relocalized animally while CK2α and 3âUTR mRNAs did not relocalize. These experiments were repeated twice with similar results.(C) Localization of digoxin-labeled ectopic mRNAs twenty-four hours after vegetal injection. Stage IV oocytes were injected vegetally with 1 ng of in vitro transcribed digoxigenin-labeled sense mRNA from the GFP-CK2α-3âUTR and GFP- CK2α constructs (time 0). 24 hours after injection, oocytes were fixed, stained with anti-digoxigenin antibodies as whole-mounts, and bisected with a steel knife for photomicrography. Chromogenic staining is shown in purple. Uninjected oocytes showed no staining (control). * = animal. The CK2α-3âUTR mRNA relocalized animally while CK2α mRNA did not relocalize. This experiment was repeated twice with similar results.
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Figure 6. CK2α localization and dorsal axis formation. During oogenesis, CK2α mRNA is localized to the animal pole, in a process dependent on its coding and 3âUTR sequences, while the dorsal determinants are localized to the vegetal pole. Fertilized embryos will inherit the dorsal determinants and CK2 protein in non-overlapping regions that will come together after cortical rotation in the region where dorsal specification will be initiated.
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