Reactive oxygen species (ROS) are signalling molecules whose study in intact organisms has been hampered by their potential toxicity. This has prevented a full understanding of their role in organismal processes such as development, aging and disease. In Caenorhabditis elegans, the development of the vulva is regulated by a signalling cascade that includes LET-60ras (homologue of mammalian Ras), MPK-1 (ERK1/2) and LIN-1 (an ETS transcription factor). We show that both mitochondrial and cytoplasmic ROS act on a gain-of-function (gf) mutant of the LET-60ras protein through a redox-sensitive cysteine (C118) previously identified in mammals. We show that the prooxidant paraquat as well as isp-1, nuo-6 and sod-2 mutants, which increase mitochondrial ROS, inhibit the activity of LET-60rasgf on vulval development. In contrast, the antioxidant NAC and loss of sod-1, both of which decrease cytoplasmic H₂0₂, enhance the activity of LET-60rasgf. CRISPR replacement of C118 with a non-oxidizable serine (C118S) stimulates LET-60rasgf activity, whereas replacement of C118 with aspartate (C118D), which mimics a strongly oxidised cysteine, inhibits LET-60rasgf. These data strongly suggest that C118 is oxidized by cytoplasmic H₂0₂ generated from dismutation of mitochondrial and/or cytoplasmic superoxide, and that this oxidation inhibits LET-60ras. This contrasts with results in cultured mammalian cells where it is mostly nitric oxide, which is not found in worms, that oxidizes C118 and activates Ras. Interestingly, PQ, NAC and the C118S mutation do not act on the phosphorylation of MPK-1, suggesting that oxidation of LET-60ras acts on an as yet uncharacterized MPK-1-independent pathway. We also show that elevated cytoplasmic superoxide promotes vulva formation independently of C118 of LET-60ras and downstream of LIN-1. Finally, we uncover a role for the NADPH oxidases (BLI-3 and DUOX-2) and their redox-sensitive activator CED-10rac in stimulating vulva development. Thus, there are at least three genetically separable pathways by which ROS regulates vulval development.

活性氧（ROS）是信号分子，其在完整生物中的研究受到其潜在毒性的阻碍。这使人们无法完全了解它们在生物过程中的作用，例如发育，衰老和疾病。在秀丽隐杆线虫中，外阴的发育受包括LET-60ras（哺乳动物Ras的同源物），MPK-1（ERK1 / 2）和LIN-1（ETS转录因子）的信号级联调控。我们显示线粒体和细胞质的ROS通过先前在哺乳动物中确定的氧化还原敏感的半胱氨酸（C118）作用于LET-60ras蛋白的功能获得（gf）突变。我们表明百草枯的抗氧化剂以及isp-1，nuo-6和sod-2增加线粒体ROS的突变体抑制了LET-60rasgf对外阴发育的活性。相比之下，抗氧化剂NAC和sod-1的缺失（两者均会降低细胞质H ₂ 0 ₂）会增强LET-60rasgf的活性。用不可氧化的丝氨酸（C118S）的CRISPR替代C118刺激了LET-60rasgf的活性，而模仿强氧化半胱氨酸的天冬氨酸（C118D）的CRISPR替代C118抑制了LET-60rasgf。这些数据强烈表明C118被细胞质H ₂ 0 ₂氧化。线粒体和/或细胞质超氧化物歧化产生的这种氧化，并且这种氧化抑制了LET-60ras。这与培养的哺乳动物细胞中的结果相反，在哺乳动物细胞中，大部分氧化氮（在蠕虫中没有发现）氧化C118并激活Ras。有趣的是，PQ，NAC和C118S突变并不作用于MPK-1的磷酸化，这表明LET-60ras的氧化作用仍是尚未表征的MPK-1独立途径。我们还表明，升高的细胞质超氧化物能独立于LET-60ras的C118和LIN-1的下游促进外阴形成。最后，我们揭示了NADPH氧化酶（BLI-3和DUOX-2）及其氧化还原敏感激活剂CED-10rac在刺激外阴发育中的作用。因此，ROS至少通过三种遗传上可分离的途径调节外阴发育。