The role of SPE-11 in C. elegans egg activation

为了确定sp11突变胚胎的主要缺陷，我们进行了详细的表型分析。鉴于spe-11末端表型是一个圆形的，脆弱的1细胞胚胎，我们检查了两种蛋壳完整性的诊断。首先，我们已经证明了spe-11（hc90）突变胚胎具有渗透敏感性，这表明蛋壳内层（赋予渗透屏障）发生了破坏。其次，在这些突变体中，蛋壳的几丁质层有缺陷。在零胚11型（hc90）胚胎中，几丁质仅在胚胎表面呈有限的新月形，而野生型胚胎的几丁质则在胚胎周围存在。作为早期胚胎发生的另一个标志，我们研究了称为皮质颗粒的细胞内囊泡的运输，这些囊泡在卵子激活期间经历了特征性的易位。sp11突变体在依赖细胞周期的皮质颗粒运动过程中没有受到损害，因为CAV-1::GFP（皮质颗粒的标记物）在sp11突变体中表现出正常的运输。我们还询问在没有SPE-11的情况下，其他卵子激活基因的定位是否正常。在spe-11（hc90）突变体中，EGG-3::GFP通常在受精后定位。因此，我们在spe-11突变体中发现的最早缺陷是在蛋壳形成中。我们正在继续使用与GFP或mCherry融合的额外皮质和蛋壳标记进行这种类型的分析。这种分析应该有助于确定早期开发的哪些方面受到缺乏SPE-11的影响，哪些方面独立于SPE-11发挥作用。

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. We are continuing this type of analysis using additional cortical and eggshell markers fused to GFP or mCherry. This analysis should help determine which aspects of early development are perturbed by the lack of SPE-11 and which function independently of SPE-11. In the past, 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 two new alleles of spe-11, spe-11(av33 and av34). Both were found to be a non-conditional alleles that produce truncated protein products, like most of the existing spe-11 alleles. These new alleles behave like the reference allele and thus we believe they too are null alleles. We are continuing this screen in hopes of identifying a temperature-sensitive allele of spe-11 that can be used in a suppressor screen. Alternatively, we hope to at least identify a missense allele, which would also be a good candidate for a suppressor screen. Because this screen has suggested the existence of non-allelic non-complementing alleles, we are planning to isolate these enhancer alleles in the future as they may be quite informative as to the genetic pathways in which spe-11 functions. We have also initiated a genetic suppressor screen using 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 dominant and recessive 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. In addition, we are constructing transgenic animals expressing fluorescent (GFP) in vivo markers of the eggshell using cbd-1 and cpg-1 translational fusions. These transgenics will provide important tools for evaluating the subtle alterations of the eggshell, which will be of great utility in our investigations of the role of SPE-11 in eggshell formation. In addition to the above strategies, we are also performing yeast two hybrid screens to find SPE-11 interactors and are performing structure-function assays on spe-11 variants by asking which variants are capable of rescuing a null.