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

SPE-11 在秀丽隐杆线虫卵激活中的作用

• 批准号: 7734295
• 负责人: Andy Golden
• 金额: $ 24.89万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7734295
• 关键词: Alleles; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Cell Cycle; Characteristics; Chitin; Cytoplasmic Granules; Defect; Development; Diagnostic; Disruption; Embryo; Embryonic Development; Ensure; Event; Exhibits; Fertilization; Gene Activation; Genetic; Goals; Injection of therapeutic agent; Life; Localized; Movement; Oocytes; Partner in relationship; Phenotype; Process; Proteins; Role; Series; Surface; Temperature; Transgenes; Vesicle; egg; embryo cell; exhaust; male; mutant; novel; sperm cell; trafficking

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. It has been shown that injection of a spe-11 transgene into the hermaphrodite germline is sufficient to rescue the loss of spe-11 in sperm (Browning and Strome, 1996). We have investigated if a stable spe-11::GFP transgene is able to perform the same function. spe-11(hc90) hermaphrodites expressing this transgene are fertile, indicating rescue of the egg activation defect. In addition, hermaphrodites that have exhausted their sperm supply produce live progeny when mated to spe-11(hc90) males. Thus, the spe-11::GFP transgene expressed in oocytes or sperm is competent to replace endogenous SPE-11, pointing to the existence of a regulatory mechanism to ensure proper activation of SPE-11 activity following fertilization. We are currently performing a non-complementation screen in order to recover a strong temperature sensitive allele of spe-11 that can be used for a genetic suppressor screen.

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。 已经表明，将spe-11转基因注射到两性生殖系中足以挽救精子中spe-11的损失（布朗宁和Strome，1996）。 我们已经研究了稳定的spe-11：：GFP转基因是否能够执行相同的功能。表达该转基因的SPE-11（HC 90）雌雄同体是可育的，表明挽救了卵活化缺陷。 此外，当雄性spe-11（hc 90）交配时，已经耗尽精子供应的雌雄同体产生活的后代。因此，在卵母细胞或精子中表达的spe-11：：GFP转基因能够取代内源性SPE-11，这表明存在一种调节机制，以确保受精后SPE-11活性的适当激活。 我们目前正在进行一个非互补筛选，以恢复一个强大的温度敏感等位基因spe-11，可用于遗传抑制筛选。