We are interested in further exploring how polarity is set up along the antero-posterior (AP) axis of one-cell stage C.elegans embryos. A sperm component, which most likely correspond to astral microtubules nucleated from the sperm-inherited centrosome, initially confers posterior character to one side of the embryo. Downstream of the sperm component, a set of maternal genes (pars,
pkc-2,
cdc-42) is required for establishing AP polarity cues. In response to these polarity cues, the mitotic spindle is displaced towards the posterior and factors such as PIE-1 or P-granules are segregated towards the posterior of one-cell stage embryos. Consequently, the first division results in two daughter cells that differ in size and fate. We have become interested in the
zyg-11 locus because
zyg-11 (
mn40) mutant embryos have defective first divisions and misegregation of P-granules, suggesting that AP polarity cues are aberrant (Kemphues et al; Dev Biol, 1986). In addition,
zyg-11 hypomorphic mutant embryos have a delay in meiosis II cell cycle progression. ZYG-11 belongs to an evolutionary conserved family of proteins with no assigned function (Carter et al; MGG, 1990). We initiated a study of
zyg-11 to better characterize the meiotic defect and begin addressing whether the meiosis II delay is responsible for the polarity defects. We utilized in utero time- lapse fluorescent microscopy of GFP-histone and GFP-tubulin expressing worms to characterize the kinetics of cell cycle progression in
zyg-11(RNAi) embryos. We found that the first deviation from the wt is a striking delay at meiosis II. Instead of the normal 9 minutes,
zyg-11(RNAi) embryos spend 33 minutes in metaphase of meiosis II. Thus,
zyg-11 is required for proper metaphase to anaphase transition at meiosis II. We next examined the distribution of PAR-2 and P-Granules in
zyg-11(RNAi) embryos and found them to be affected starting at the prolonged meiosis II. PAR-2 localized to the anterior cortex and P-granules were enriched in the anterior cytoplasm in 58% of embryos examined. Therefore,
zyg-11 appears to be required upstream of the par genes to set up proper AP polarity. Interestingly, using time lapse fluorescent microscopy with GFP-histone and GFP-tubulin transgenic animals, we found that inactivation of the cullin
cul-2 by RNAi results in a meiotic phenotype indistinguishable from that of
zyg-11(RNAi) embryos. Moreover we found that PAR-2 and P-granules are distributed abnormally as in
zyg-11(RNAi) embryos. These results taken together suggest that a persisting spindle at meiosis II is sufficient to generate inverted polarity much like the situation described with an arrested meiosis I spindle (Wallenfang et al; Nature, 2000). However,
zyg-11 and
cul-2 may be both independently required for progression through meiosis II and establishment of AP polarity. We sought to determine the distribution of ZYG-11 by generating transgenic animals expressing an integrated
zyg-11::gfp transgene. We found that ZYG-11::GFP is cytoplasmic at meiosis and disappears before the first interphase, consistent with temperature shift experiments showing that
zyg-11 is required in a short time period after fertilization (Kemphues et al; Dev Biol, 1986). We are in the process of investigating the link between
zyg-11 and
cul-2 and plan to present data pertaining to their mechanism of action in the context of meiotic cell cycle progression and setting up of AP polarity.