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，我们已经检查了蛋壳完整性的两个诊断。首先，我们已经证明spe-11（hc 90）突变体胚胎对渗透敏感，这表明蛋壳内层的破坏，这赋予了渗透屏障。第二，这些突变体的蛋壳几丁质层是有缺陷的。在spe-11（hc 90）基因缺失的胚胎中，几丁质仅在胚胎表面的限制性新月体中被观察到，而野生型胚胎中几丁质存在于胚胎周围。作为早期胚胎发生的额外标记，我们研究了称为皮质颗粒的细胞内囊泡的运输，其在卵激活期间经历特征性易位。spe-11突变体在皮质颗粒运动的细胞周期依赖性过程中不受损害，因为CAV-1：：GFP，皮质颗粒的标记物，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，其他卵激活基因的定位是否正常。EGG-3：：GFP通常在受精后定位于spe-11（hc 90）突变体中。因此，我们在spe-11突变体中检测到的最早的缺陷是在蛋壳形成中。 在过去，我们进行了非互补筛选，以恢复一个强大的温度敏感的等位基因spe-11，可用于遗传抑制筛选。到目前为止，我们已经恢复了一个新的spe-11等位基因，spe-11（av 33）。发现它是一个非条件等位基因，产生的蛋白质产物比spe-11的参考等位基因spe-11（hc 90）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无效等位基因。我们还启动了两个遗传抑制筛选使用的截断等位基因spe-11，spe-11（bn 65）。spe-11的这个等位基因编码全长蛋白质的80%。我们预计，这些屏幕将分离显性和隐性旁路抑制SPE-11，也许这将使卵母细胞更敏感的激活或促进SPE-11的定位或功能。 我们已经产生了在内源性spe-11启动子和3UTR下表达GFP：SPE-11的转基因株系。该细胞系在成熟精子中非常明亮地表达GFP：SPE-11，并将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翻译融合构建表达蛋壳荧光（GFP）体内标记的转基因动物。这些转基因将提供重要的工具，用于评估蛋壳的细微变化，这将是非常有用的，在我们的调查中的SPE-11在蛋壳形成的作用。

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. 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.