To identify the primary defect in the spe-11 mutant embryos, we have undertaken a detailed phenotypic analysis. Given that the spe-11 terminal phenotype is a round, fragile 1-cell embryo, we have examined two diagnostics of eggshell integrity. First, we have shown that spe-11(hc90) mutant embryos are osmotically sensitive, indicating a disruption in the inner layer of the eggshell, which confers the osmotic barrier. Second, the chitin layer of the eggshell is defective in these mutants. Chitin is observed only in a restricted crescent at the surface of the embryo in the null spe-11(hc90) embryos, in contrast to wild type embryos where chitin is present around the periphery of the embryo. As an additional marker of early embryogenesis, we have investigated the trafficking of intracellular vesicles called cortical granules, which undergo a characteristic translocation during egg activation. The spe-11 mutants are not compromised in the cell cycle dependent process of cortical granule movement because CAV-1::GFP, a marker of cortical granules, exhibits normal trafficking in spe-11 mutants. We also asked if the localization of other egg activation genes was normal in the absence of SPE-11. EGG-3::GFP is normally localized following fertilization in spe-11(hc90) mutants. Thus, the earliest defects we have detected in spe-11 mutants are in eggshell formation. In the past year, we performed a non-complementation screen in order to recover a strong temperature-sensitive allele of spe-11 that could be used for a genetic suppressor screen. Thus far, we have recovered a new allele of spe-11, spe-11(av33). It was found to be a non-conditional allele that produces a protein product two amino acids longer than the reference allele of spe-11, spe-11(hc90). This new allele behaves like the reference allele and thus we believe it too is a null allele. We have also initiated a genetic suppressor screen with a truncation allele of spe-11, spe-11(bn65). This allele of spe-11 encodes 80% of the full protein length. We anticipate that this screen will isolate bypass suppressors of spe-11, perhaps that will make the oocyte more sensitive to activation or that facilitate SPE-11 localization or function. We have also generated a transgenic line that expresses GFP:SPE-11 under the endogenous spe-11 promoter and 3UTR. This line expresses GFP:SPE-11 very brightly in the mature sperm and will be used to perform microscopic studies to determine the fate of the SPE-11 protein following fertilization.
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