Kataoka K et al. (2005), Identification of asymmetrically localized tran...

XB-ART-1103

Dev Growth Differ 2005 Oct 01;478:511-21. doi: 10.1111/j.1440-169X.2005.00826.x.

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Identification of asymmetrically localized transcripts along the animal-vegetal axis of the Xenopus egg.

Kataoka K , Tazaki A , Kitayama A , Ueno N , Watanabe K , Mochii M .

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In many organisms, proper embryo development depends on the asymmetrical distribution of mRNA in the cytoplasm of the egg. Here we report comprehensive screening of RNA localized in the animal or vegetal hemisphere of the Xenopus egg. Macroarrays including over 40,000 independent embryonic cDNA clones, representing at least 17,000 unigenes, were differentially hybridized with labeled probes synthesized from the mRNA of animal or vegetal blastomeres. After two rounds of screening, we identified 33 clones of transcripts that may be preferentially distributed in the vegetal region of the early stage embryo, but transcripts localized in the animal region were not found. To assess the array results, we performed northern blot and quantitative real-time reverse transcription-polymerase chain reaction analysis. As a result, 21 transcripts of the 33 were confirmed to be localized in the vegetal region of the early stage embryo. Whole-mount in situ hybridization analysis revealed that 11 transcripts, including 7 previously reported genes, were localized in the vegetal hemisphere of the egg. These 11 transcripts were categorized into three groups according to their expression patterns in the egg. The first group, which contained four transcripts, showed uniform expression in the vegetal hemisphere, similar to VegT. The second group, which contained three transcripts, showed gradual expression from the vegetal pole to the equator, similar to Vg1. The last group, which contained three transcripts, was expressed at the germ plasm, similar to Xdazl. One transcript, Xwnt11, showed both the second and the third expression patterns.

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Species referenced: Xenopus laevis
Genes referenced: acsbg2 acsl1 amd1 camk2g ctdspl dazl e2f1 gdf1 kremen1 ldlrap1 mapk12 mmp24 mrc2 neff1 neo1 otx1 pes1 pgat plekhg1 prpsap2 rnf38 rps6kb1 sf3a1 slc4a1 tob2 usp40 vegt wnt11b XB5814120

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	Fig. 1. A dissociated embryo. An 8-cell-stage wild-type embryo was cultured in calcium-free, magnesium-free medium, and vitelline membrane was removed. Four animal blastomeres are pigmented on the left, and the vegetal blastomeres are on the right. Animal view. Bar, approximately 400 μm.
	Fig. 2. Hybridized mini-array filter images. The amplified cDNA was blotted as duplicated spots on a nylon membrane (EF1-α cDNA was blotted as a control at the four corners), and hybridized with the probe prepared from RNA of animal (A) or vegetal (V) blastomeres in the 8-cell-stage embryo, as represented in the upper two panels. The lower left two panels (a) and (v), are magnifications of the spots in the upper panels. For each clone, a schematic representation of the orientation of that spot paired within the image is provided at the lower right.
	Fig. 3. Differential hybridization analysis by northern blots. To confirm the reliability of the array analysis, 20 selected genes out of 33 positive ones were subjected to the differential hybridization by northern blot. 10 μg of total RNA from animal or vegetal blastomeres was loaded at each membrane (animal, left lane; vegetal, right lane). Fourteen transcripts were abundant in the vegetal blastomeres of the 8-cell-stage embryo, while one, XL014k14, was not. Signals of five other genes were not detected in this analysis. The upper end of each image, except for EF1-α, coincides with the loading start level. The arrowheads represent 28S and 18S ribosomal RNA. XL087b08 and XL099d22 hybridized with transcripts of at least two different sizes. EF1-α was the internal control for these RNA.
	Fig. 4. In situ hybridization showing vegetally localized genes. Unfertilized eggs were obtained from albino (A–K) and wild-type (E′– K′) Xenopus laevis. XL002d04 (A), XL003i03 (B), XL099d22 (C), and XL105l17 (VegT) (D) were expressed uniformly in the vegetal hemisphere. XL037i04 (Xotx1) (E, E′), XL087b08 (XNIF) (F, F′), and XL081p07 (Vg1) (G, G′) were gradually expressed with the most condensed staining at the vegetal pole. XL13k14 (I, I′), XL026p20 (Xdazl) (J, J′), and XL096g22 (Xpat) (K, K′) were expressed in the germ plasm. XL010a22 (Xwnt11) (H, H′) was gradually expressed with the most condensed staining at the vegetal pole, and also expressed in the germ plasm (arrows). (A–G) Lateral view of unpigmented eggs with the animal pole on the top, and (H–K) Vegetal view. (E′–K′) Cut face of the egg, which was bisected along animal-vegetal axis, with the pigmented animal pole on the top. Lowest panels were higher magnification view of H′–K′. Scale bar in A–K, approximately 200 μm.