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

为了确定spe-11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的末端表型是一个圆形的、脆弱的1-细胞胚胎，我们已经检查了两种蛋壳完整性的诊断方法。首先，我们已经证明了spe-11(Hc90)突变胚胎是渗透敏感的，这表明蛋壳内层发生了破坏，从而提供了渗透屏障。其次，在这些突变体中，蛋壳的几丁质层是有缺陷的。在SPE-11(Hc90)缺失型胚胎中，甲壳素仅在胚胎表面的限制性新月体中观察到，这与野生型胚胎不同，在野生型胚胎中，甲壳素存在于胚胎周围。作为早期胚胎发生的另一个标志，我们研究了被称为皮质颗粒的细胞内小泡的运输，这些小泡在卵子激活过程中经历了一种特征性的易位。SPE-11突变体在依赖细胞周期的皮质颗粒运动过程中不受影响，因为皮质颗粒的标志物CAV-1：：GFP在SPE-11突变体中表现出正常的运输。我们还询问了在没有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)突变体中受精后，Egg-3：：GFP通常被定位。因此，我们在spe-11突变体中检测到的最早缺陷是蛋壳形成。 在过去，我们进行了非互补筛选，以恢复Spe-11的一个强温度敏感等位基因，该等位基因可用于遗传抑制筛选。到目前为止，我们发现了Spe-11的一个新等位基因Spe-11(Av33)。它被发现是一个非条件等位基因，它产生的蛋白质产物比spe-11的参考等位基因spe-11(Hc90)长两个氨基酸。这个新等位基因的行为与参考等位基因相似，因此我们认为它也是一个空等位基因。我们还使用spe-11、spe-11(Bn65)的截断等位基因启动了两个遗传抑制基因筛选。Spe-11的这个等位基因编码80%的全长蛋白质。我们预计，这些筛选将分离SPE-11的显性和隐性旁路抑制因子，这可能会使卵母细胞对激活更加敏感，或者促进SPE-11的定位或功能。 我们已经建立了一个在内源spe-11启动子和3UTR下表达GFP：spe-11的转基因株系。这条线在成熟精子中非常明亮地表达GFP：SPE-11，并将被用于进行显微镜研究，以确定受精后SPE-11蛋白的命运。此外，我们正在使用CBD-1和CPG-1的翻译融合构建在体内表达蛋壳荧光(GFP)标记的转基因动物。这些转基因将为评估蛋壳的细微变化提供重要的工具，这将对我们研究SPE-11在蛋壳形成中的作用非常有用。

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. 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 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 two genetic suppressor screens 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 these screens 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 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.