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First published online 23 June 2004
doi: 10.1242/dev.01245

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CUL-2 and ZYG-11 promote meiotic anaphase II and the proper placement of the anterior-posterior axis in C. elegans

Ji Liu^*, Srividya Vasudevan^* and Edward T. Kipreos

Department of Cellular Biology, University of Georgia, Athens, GA 30602-2607, USA

View larger version (53K): [in a new window]   Fig. 1. CUL-2 complex components and ZYG-11 are required for progression into meiotic anaphase II. (A) Histone H2B::GFP epifluorescence movie sequences of live zygotes of the indicated genotype. Time post-fertilization is indicated in the upper left corner (hours:minutes). Interphase denotes the initiation of chromosome decondensation after meiotic exit. The metaphase images are generally transverse views to show the pentagonal array except for the elc-1 (00:01) and zyg-11(mn40) (00:25), which are side views showing paired homologous chromosomes and sister chromatids, respectively. Arrowheads denote polar bodies. (B) The duration of meiosis I post-fertilization (left) and meiosis II (right) derived from histone H2B::GFP movies. See Materials and methods for the number of embryos analyzed. (C) Histone H2B::GFP images of separated metaphase II chromosomes in cul-2(RNAi) and cul-2(ek1) embryos. Scale bars: 5 µm.

View larger version (75K): [in a new window]   Fig. 2. The cul-2 meiotic phenotypes are not caused by a failure to remove the cohesin REC-8 from sister chromatids. (A) Immunofluorescence images of REC-8 in wild-type and cul-2(RNAi) embryos. Images of wild-type (top) and cul-2(RNAi) zygotes (bottom) stained with DAPI and anti-REC-8 antibody at the indicated meiotic stages. Note that in late metaphase II cul-2(RNAi) zygotes, REC-8 is not detected but chromosomes fail to segregate. Arrowheads indicate polar bodies. (B) Depletion of rec-8 by RNAi does not rescue the meiotic delay in cul-2(RNAi) zygotes. Histone H2B::GFP fluorescence movie sequences in rec-8(RNAi) or cul-2(RNAi); rec-8(RNAi) double RNAi embryos. In the rec-8(RNAi) zygote, chromosomes compress and rapidly move to the cell cortex between time points 00:17 and 00:22. Time from fertilization (hours:minutes) is shown in the upper left corner of images. Stars denote meiotic chromosomes or interphase pronuclei. Dashed lines denote the egg boundary; arrowheads denote polar bodies. Scale bars: 5 µm.

View larger version (65K): [in a new window]   Fig. 3. Cyclin B1 is not degraded during meiosis in cul-2(RNAi) embryos. (A) DIC (top) and epifluorescence (bottom) images of wild-type, cul-2(RNAi) and zyg-11(RNAi) gravid adults expressing a cyb-1::GFP transgene. Embryo stages are labeled. Note that CYB-1::GFP signal is absent in the wild-type pronuclei stage embryo (immediately after meiosis) but perdures in cul-2(RNAi) and zyg-11(RNAi) embryos. Intestine autofluorescence is observed above the oocyte and eggs. Scale bar: 10 µm. (B) Graph of CYB-1::GFP epifluorescence intensity at times post-fertilization. Data was collected under the same conditions for each embryo and is presented in equivalent arbitrary units. The completion of meiosis is denoted by a thick striped bar. (C) Meiosis II timing for individual embryos of the strains listed, derived from histone H2B::GFP movies.

View larger version (64K): [in a new window]   Fig. 4. The meiosis II spindle in cul-2(RNAi) zygotes maintains a normal morphology for an extended period. (A) ß-tubulin::GFP epifluorescence movie sequences of live wild-type (upper) and cul-2(RNAi) (lower) zygotes to show meiotic II spindle dynamics. Time post-anaphase I is indicated in the upper left corner of images (hours:minutes). (B) Overlaid epifluorescence images of anti--tubulin (green) and DAPI (red) staining of meiosis II spindles for wild type (right) and cul-2(RNAi) (left) zygotes. The stage of meiosis is indicated above the image. Scale bars: 5 µm.

View larger version (60K): [in a new window]   Fig. 5. Polarity defects in cul-2, elc-1, rbx-1 and zyg-11 RNAi embryos. (A) Overlaid epifluorescence images of anti-PAR-2 (green, upper panel), anti-PAR-6 (green, middle panel), anti-PAR-3 (green, lower panel), anti--tubulin (red), and DAPI (blue) staining in wild-type (left) and cul-2(RNAi) (middle and right) zygotes. The wild-type zygotes are in interphase, whereas the cul-2(RNAi) zygotes are in meiosis II. Red stars denote meiotic spindles; red arrows, sperm asters; o, maternal pronuclei; s, sperm DNA; white arrowheads, polar bodies; and white arrows, regions of the cortex lacking PAR-6 or PAR-3 staining. (B) Matched DIC and PAR-2::GFP images of two-cell stage wild-type, cul-2(RNAi), elc-1(RNAi) and rbx-1(RNAi) embryos. (C) Overlaid epifluorescence images of anti-PAR-2 (green), anti--tubulin (red) and DAPI (blue) staining in zyg-11(RNAi) one-cell (left) and two-cell (right) stage embryos. (D) P granule localization in mitotic cul-2(RNAi) embryos: PAR-2::GFP (green), anti-P granule (red) and DAPI (blue). White arrowheads denote polar bodies. (E) Diagram of migration of spindles and pronuclei in wild-type (left) and cul-2(RNAi) zygotes (middle and right) derived from ß-tubulin::GFP/DIC movies. Timing begins with the initial observation of sperm asters and ends at pronuclei meeting. In five out of five cul-2(RNAi) embryos, the sperm asters formed during meiosis, although they were very small relative to their size after meiosis. Anterior is to the left. Scale bars: 10 µm.

View larger version (84K): [in a new window]   Fig. 6. Ectopic lateral PAR-2 in cul-2(RNAi) embryos. (A) The initial localization of lateral PAR-2, as monitored in epifluorescence movies of cul-2(RNAi) embryos expressing PAR-2::GFP and ß-tubulin::GFP (left), and PAR-2::GFP and histone H2B::GFP (right). Green arrows denote cortical PAR-2. Cortical PAR-2::GFP signals were observed in regions that were distal to the meiotic spindle (left), or to oocyte and sperm DNA (right), during the entire time period post-fertilization. (B) Epifluorescence (top) and confocal three-dimensional reconstruction of a cul-2(RNAi) embryo showing a circular PAR-2 pattern: PAR-2::GFP (green), anti--tubulin (red) and DAPI (blue). Note that the confocal reconstruction is only of the embryo and excludes the surrounding germ cell DAPI signal. Middle image shows a side view; bottom image, anterior is to the front. (C) Simultaneous localization of PAR-2 and PAR-6 in meiosis II-stage cul-2(RNAi) embryos. (Left) Anterior and lateral PAR-2::GFP (top) with uniform anti-PAR-6 staining (bottom). (Right) Anterior anti-PAR-2 staining with anterior exclusion of PAR-6::GFP. (D) PAR-2::GFP localization in tbb-2(RNAi) and tbb-2; cul-2 double RNAi meiotic embryos. Epifluorescence images of PAR-2::GFP (green, top), DAPI (blue, top) and anti--tubulin (red, bottom). o, location of maternal DNA; s, location of sperm DNA. The left tbb-2; cul-2 double RNAi embryo has a lateral PAR-2::GFP patch, whereas the right embryo has PAR-2::GFP on the end of the embryo near both the maternal and condensed sperm DNA. (E) emb-30(tn377) (left) and emb-30(tn377); cul-2(RNAi) (right) zygotes were stained with anti--tubulin (red) and either anti-PAR-2 (green, upper panel) or anti-PAR-6 (green, lower panel). For each genotype, left panels show anterior PAR-2 localization (top) or PAR-6 exclusion (bottom), and right panels show lateral/posterior PAR-2 localization (top) or PAR-6 exclusion (bottom). White arrows denote regions of the cortex lacking PAR-6 staining. Anterior is to the left. Scale bars: 10 µm.