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First published online 23 June 2004
doi: 10.1242/dev.01244

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zyg-11 and cul-2 regulate progression through meiosis II and polarity establishment in C. elegans

ISREC (Swiss Institute for Experimental Cancer Research), 155, chemin des Boveresses, CH-1066 Epalinges/Lausanne, Switzerland

View larger version (64K): [in a new window]   Fig. 7. Evolution of polarity in zyg-11(RNAi) embryos. (A-C) Images from time-lapse sequences of zyg-11(RNAi) embryos expressing GFPPAR-2 and GFP-HIS, as well as GFP-TUB, shown during the meoisis II delay (left panel), the first mitotic division (middle panel) and the twoto four-cell stage transition (right panel) (see also movies 8-10 at http://dev.biologists.org/supplemental/). Recordings were started during the meiosis II delay. Arrowheads point to first polar body, arrows indicate condensed sperm DNA; stippled arrowhead and arrows indicate that these positions can be determined only from the movies. Scale bar: 10 µm. (D) Schematic representation of two-cell stage embryos (n=19) corresponding to recordings A (6 embryos), B (10 embryos) or C (3 embryos). See text for details.

View larger version (58K): [in a new window]   Fig. 1. zyg-11, cul-2 and cyb-3 are required for progression through meiosis II. (A) Images from time-lapse sequences of embryos expressing GFP-HIS (see also Movies 1-7 at http://dev.biologists.org/supplemental/). Metaphase views are: polar (P), the spindle is viewed from the pole and five homologues (meiosis I) or sister chromatids (meiosis II) are observed in a circle around the sixth; lateral (L), the spindle is viewed from the side and up to three pairs of homologues (meiosis I) or sister chromatids (meiosis II) are observed; or intermediate (I), between the polar and lateral views. In this and other figures, embryos are oriented with polar bodies to the left and all panels of a kind are at the same magnification. Scale bar: 5 µm. (B) Timing of events in embryos from fertilization (t=0) until the end of meiosis II. Average duration of each stage, defined in Table 1, is indicated. Letters in the time-lines indicate the time at which the corresponding images in (A) are taken. Vertical white lines indicate the time at which GFP-PAR-2 first becomes enriched at the cell cortex in the vicinity of oocyte-derived chromosomes in zyg-11(RNAi) (n=4) or zyg-11(mn40) (n=3) embryos imaged starting at the end of meiosis I.

View larger version (74K): [in a new window]   Fig. 2. zyg-11 is required for timely metaphase to anaphase transition and M phase exit at meiosis II. (A-N) Lateral views of wild-type or zyg-11(RNAi) embryos at the indicated stages, stained with antibodies against -tubulin (A-C,G-J), or -tubulin and phosphorylated histone H3 (D-F,K-N). Insets below panels A-C and G-J, as well as the entire panels D-F and K-N are magnified views of a meiotic spindle and have a width of 7 µm. Insets below panels A-C and G-J show -tubulin (green) and DNA (blue) on the left, and DNA alone on the right. Panels D-F and K-N show -tubulin (green), phosphorylated histone H3 (red) and DNA (blue) on the left, and phosphorylated histone H3 alone on the right. To view polar bodies and sperm chromosomes, the DNA signal in the low magnification images is a projection of several 1-µm confocal optical sections. Arrowheads point to the first polar body, arrows indicate condensed sperm DNA. Scale bar: 10 µm. Note that the focus of phosphorylated H3 lies between homologues at metaphase I and sister chromatids at metaphase II in both wild-type and zyg-11(RNAi) embryos. (O,P) Wild-type (O) and zyg-11(RNAi) (P) anaphase II embryos expressing GFP-CYB-3 stained with antibodies against -tubulin (not shown) and GFP (red); DNA is shown in blue. (Q) Quantification of signal intensities in wild-type (n=6), zyg-11(RNAi) (n=14) and cul-2(RNAi) (n=19) embryos in metaphase II or anaphase II stained as in (O); embryos were staged using the anti--tubulin and DNA signals. The difference between wild type and zyg-11(RNAi) or cul-2(RNAi) is statistically significant (Student's t-test: P=6x10-5 and P=4x10-6, respectively).

View larger version (69K): [in a new window]   Fig. 3. cul-2 destabilizes GFP-ZYG-11; zyg-11 does not regulate GFP-ZIF-1. Images of paralyzed animals (A-C,H-J) or of fixed embryos stained with anti-GFP antibodies (D-F,K-M) from strains expressing GFP-ZYG-11 (A-F) or GFPZIF-1 (H-M). Plain outlines indicate oocytes, stippled outlines embryos. Note also the gut autofluorescence outside of embryos in live specimens. Scale bars: 50 µm. (G,N) Quantification of GFPZYG-11 (G) and GFP-ZIF-1 (N) signal intensities in fixed early embryos (i.e. <10 cell stage) of the indicated genotypes stained as in (D-F,K-M). Increase of GFP-ZYG-11 in cul-2(RNAi) is 16.4±3.0-fold, of GFP-ZYG-11 in elc-1(RNAi) is 17.3±3.2-fold, and of GFPZIF-1 in cul-2(RNAi) is 5.8±3.3-fold.

View larger version (63K): [in a new window]   Fig. 4. zyg-11 and cul-2 function redundantly with APC at meiosis I. Meiotic figures from embryos of the indicated genotypes stained with antibodies against -tubulin (green in merge) are shown; DNA is shown in blue in merged image and alone on the right. Width of panel represents 7 µm. NA, not available.

View larger version (107K): [in a new window]   Fig. 5. Inverted AP polarity during meiosis II delay in the absence of zyg-11 or cul-2 function. Wild-type (A-D), zyg-11(RNAi) (E-H), cul-2(RNAi) (I-L), and cyb-3(RNAi) (M-P) embryos during meiosis II, and wild-type embryos during the first mitotic cell cycle (Q-T), stained with antibodies against -tubulin (green in merged image) and a polarity marker as indicated (PAR-1, PAR-2, PAR-3 or PAR-1+PGL-1 to detect P granules; red in merged image and viewed alone in the top panel); DNA is shown in blue in merged image. Scale bar: 10 µm. Note that enrichment of PAR proteins and P granules in the absence of zyg-11 or cul-2 function is somewhat variable during meiosis II, and is less pronounced, both in intensity and in lateral extent, than in wild-type embryos during the first mitotic cell cycle.

View larger version (65K): [in a new window]   Fig. 6. zyg-11 can prevent polarity establishment independently of promoting meiosis II cell cycle progression. (A-D) 25°C mat-1(ax161) or 25°C mat-1(ax161) zyg-11(RNAi) embryos stained with antibodies against -tubulin (green) and PGL-1 to detect P granules (red); DNA is shown in blue. Depicted are embryos from the metaphase I (A,C) and the metaphase I (late) (B,D) categories (see Fig. 4). Insets show magnified views of meiotic chromosomes. Scale bar: 10 µm; width of inset represents 7 µm. (E,F) Live ncc-1(RNAi) [heterozygous for zyg-11(mn40)] or ncc-1(RNAi) zyg-11(mn40) embryos during meiosis expressing GFP-PAR-2 (green) and stained with Hoechst 33342 to view DNA (blue in merged image). Insets show magnified views of meiotic chromosomes; width of inset represents 8 µm. Left panels show a DIC image of the same embryo. (G-J) Live mat-1(RNAi) [heterozygous for zyg-11(mn40)] or mat-1(RNAi) zyg-11(mn40) embryos expressing GFP-PAR-2 (green) and stained with Hoechst 33342 to view DNA (blue in merged image). Insets show magnified views of meiotic chromosomes; width of inset represents 8 µm. Scale bar: 10 µm.

View larger version (119K): [in a new window]   Fig. 8. Establishment of AP polarity occurs independently of astral microtubules. zyg-11(mn40) (A) or zyg-11(mn40) tba-2(RNAi) (B) meiosis II embryos, wild-type (E,G,I,K) or tba-2(RNAi) embryos (F,H,J,L) during the first mitotic cell cycle, and tba-2(RNAi) embryos during the second mitotic cell cycle (D) are shown. All embryos are stained with antibodies against -tubulin (green in merged image) and a centriolar or polarity marker as indicated (SAS-4, PAR-1, GFP-PAR-2, GFP-PAR-6, PGL-1; red in merged image); DNA is shown in blue in the merged image. A dozen 1 µm confocal optical sections were imaged. The top panels show a projection of all slices for the -tubulin channel. Insets show magnified views of centrosomes; width of inset represents 5 µm. The merged images show a single section for -tubulin and the centriolar or polarity marker, along with a projection of all slices for the DNA signal. Scale bar: 10 µm. (C) Plots of position along the circumference in zyg-11(mn40) tba-2(RNAi) embryos (x-axis, degrees, with 0 degrees being posterior-most) as a function of the distance separating oocyte chromosomes from the cortex (y-axis, µm). Red dots indicate cortical locations where GFP-PAR-2 levels are at least five times higher than in the cytoplasm (determined with Metamorph software). Top plot, ten embryos; bottom plot, embryo shown in B. Note that GFP-PAR-2 is present in the cortical region closest to oocyte chromosomes. (E-L) Pairs of wild-type and tba-2(RNAi) embryos, from approximately the same stage, during prophase of the first cell cycle. In all tba-2(RNAi) embryos lacking detectable astral microtubules, including those with only two tiny dots of -tubulin presumably corresponding to paternally contributed centrioles (F,L, see also D), polarity markers were distributed as in wild type [number of embryos examined for each polarity marker (the number of embryos that only have two tiny dots is given in parentheses): PAR-1, 10 (5); GFP-PAR-2, 6 (1); GFP-PAR-6, 6 (0); PGL-1, 10 (5)].

View larger version (21K): [in a new window]   Fig. 9. Working model of polarity establishment in C. elegans embryos. (A) In wild-type meiosis II, a CUL-2-based E3 ligase that also requires zyg-11 function ubiquitinates a substrate (X) that can induce polarity when triggered by a surrogate polarity cue, thus targeting the substrate for degradation by the proteasome. After exit from the meiotic cell cycle, E3 ligase activity is downregulated, allowing substrate accumulation and polarity establishment in response to the bona fide centrosome polarity cue. Green oval, oocyte chromosomes; green disk, centrosomes; blue crescent, cortical PAR-2; orange disks, P granules. Ubiquitin (Ub, blue circles) moieties are also shown. (B) In the absence of zyg-11 or cul-2, substrate accumulation occurs during the meiotic cell cycle, resulting in polarity establishment in response to a surrogate polarity cue that correlates with the position of oocyte chromosomes. A related outcome may ensue when the APC is inactivated at meiosis I (not depicted here). After exit from the meiotic cell cycle in the absence of zyg-11 or cul-2, a second focus of polarity is established in response to the centrosome polarity cue.