Nakaya M et al. (2000), Meiotic maturation induces animal-vegetal asymm...

XB-ART-10038

Development 2000 Dec 01;12723:5021-31. doi: 10.1242/dev.127.23.5021.

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Meiotic maturation induces animal-vegetal asymmetric distribution of aPKC and ASIP/PAR-3 in Xenopus oocytes.

Nakaya M , Fukui A , Izumi Y , Akimoto K , Asashima M , Ohno S .

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The asymmetric distribution of cellular components is an important clue for understanding cell fate decision during embryonic patterning and cell functioning after differentiation. In C. elegans embryos, PAR-3 and aPKC form a complex that colocalizes to the anterior periphery of the one-cell embryo, and are indispensable for anterior-posterior polarity that is formed prior to asymmetric cell division. In mammals, ASIP (PAR-3 homologue) and aPKCgamma form a complex and colocalize to the epithelial tight junctions, which play critical roles in epithelial cell polarity. Although the mechanism by which PAR-3/ASIP and aPKC regulate cell polarization remains to be clarified, evolutionary conservation of the PAR-3/ASIP-aPKC complex suggests their general role in cell polarity organization. Here, we show the presence of the protein complex in Xenopus laevis. In epithelial cells, XASIP and XaPKC colocalize to the cell-cell contact region. To our surprise, they also colocalize to the animal hemisphere of mature oocytes, whereas they localize uniformly in immature oocytes. Moreover, hormonal stimulation of immature oocytes results in a change in the distribution of XaPKC 2-3 hours after the completion of germinal vesicle breakdown, which requires the kinase activity of aPKC. These results suggest that meiotic maturation induces the animal-vegetal asymmetry of aPKC.

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Species referenced: Xenopus laevis
Genes referenced: asip mhc2-dab pard3 prkci prkcz
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	Fig. 1. Schematic structure of aPKC and ASIP/PAR-3/Bazooka and their intracellular localization. (A) Comparison of the C-terminal sequence of mouse aPKC isotypes and XaPKC. The antiaPKC antibodies used in the present study were raised against the C-terminal 20 aa sequence of mouse PKCz. The sequence shares significant sequence identity to that of mouse PKCl and XaPKCl. (B) Schematic structure of mammalian ASIP, C. elegans PAR-3 and Drosophila Bazooka. The N-terminal domain (CR1), three PDZ domains (CR-2) and the aPKC binding region (CR-3) are evolutionarily conserved. The polyclonal antibody against ASIP was raised against the aPKC binding region of ASIP. (C) Cellular localization of PKC-3 and PAR-3 in a C. elegans 1-cell embryo. The arrow indicates the anterior-posterior axis. (D) Cellular localization of aPKC and ASIP in polarized epithelial cells. The arrow indicates the apical-basal axis.
	Fig. 2. Identification of XaPKC and XASIP in Xenopus. (A) Western analysis of XaPKC in Xenopus oocytes, eggs and epithelial A6 cells. XaPKC was identified as an 80 kDa band by an anti-mouse PKCz antibody. XaPKC is expressed maternally throughout oogenesis, in unfertilized eggs and in epithelial A6 cells. Samples loaded as oocyte and egg extracts were estimated to contain 25 mg protein per lane. (B) Immunoprecipitation analysis of XaPKC and XASIP. XASIP is identified as a 180 kDa band in anti-ASIP immunoprecipitates of egg extracts (upper panel). The 80 kDa XaPKC was detected in the anti- ASIP immunoprecipitates as well as in the anti-aPKC immunoprecipitates. Samples loaded into each lane correspond to extracts of 30 eggs (anti-aPKC I.P.) or 50 eggs (anti-ASIP I.P.).
	Fig. 3. Immunofluorescence staining of XaPKC and XASIP in Xenopus epithelial A6 cells. Confluent A6 cells were fixed in cold methanol and incubated with the indicated first antibody. After washing, cells were incubated with the FITC-conjugated secondary antibody and images were captured with a digital camera (Princeton Instruments). (A) Control staining by normal rabbit IgG. (B) Staining by anti-aPKC antibody. XaPKC staining localizes at the cell-cell junction as well as in the cytoplasm. (C) Control staining by antigen preincubated anti-ASIP antibody. (D) Staining by anti-ASIP antibody. The ASIP staining at the cell-cell junction disappears in the presence of excess antigen. Scale bar, approximately 40 mm.
	Fig. 4. Immunohistochemical staining of an ovary section and isolated oocytes. (A,B) Albino Xenopus ovary was fixed by the freeze-substitution method. (A) Control, stained by anti-aPKC preincubated with excess antigen. (B) Distribution of XaPKC in the ovary, stained by anti-aPKC antibody. (C,D) Wholemount immunohistochemistry of albino Xenopus oocytes. (C) Control, stained by rabbit IgG. (D) Distribution of XaPKC in various stage oocytes, stained by anti-aPKC antibody. Signals were detected by DAB staining. Scale bars, 250 mm (A,B); 500 mm (C,D).
	Fig. 5. Whole-mount immunohistochemical staining of albino unfertilized eggs and 8-cell embryos. Albino eggs and embryos were fixed in Dent’s fixative and staining signals were detected by DAB. Antibodies used are anti-aPKC antibody (A-C,E-G) and anti- ASIP antibody (D,H). Scale bar, approximately 250 mm.
	Fig. 6. Redistribution of the XaPKC staining during in vitro maturation of oocytes. (A) Schematic events in the meiotic cell cycle. The schematic staining pattern of XaPKC is summarized below. (B) Germinal vesicle breakdown and the redistribution of XaPKC staining during oocyte maturation after stimulation with 4 mM progesterone. The line graph shows the percentage of mature oocytes showing GVBD, and the bar graph those showing a loss of vegetal pole staining by anti-aPKC antibody, at various times after progesterone stimulation.
	Fig. 7. Change in the distribution of the ectopic Tag-aPKCl fusion protein during hormone-induced maturation of oocytes. (A) Wild-type oocytes were injected with RNA synthesized in vitro, incubated with (+) or without (-) 4 mM progesterone for 16-18 hours, and stained with anti-Tag antibody after fixation and bleaching. (B) The ratio of staining in both hemisphere with anti-Tag antibody, indicating the Tag-aPKCl distribution in immature or mature oocytes. These experiments were reproduced more than three times. (C) Western analysis of the oocyte extracts prepared 18 hours after mRNA injection with anti-Tag antibody.
	Fig. 8. Albino unfertilized eggs and 8-cell embryos analyzed by whole-mount in situ hybridization. Eggs and embryos were fixed in MEMFA and hybridized with DIGlabeled sense (control) or anti-sense probes. (A,B,E,F) Animal views, (C,G) vegetal views and (D,H) equatorial views of eggs and embryos. Scale bar, approximately 250 mm.