Redox Regulation of Germline and Vulval Development in Caenorhabditis elegans

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Science  05 Dec 2003:
Vol. 302, Issue 5651, pp. 1779-1782
DOI: 10.1126/science.1087167


In vitro studies have indicated that reactive oxygen species (ROS) and the oxidation of signaling molecules are important mediators of signal transduction. We have identified two pathways by which the altered redox chemistry of the clk-1 mutants of Caenorhabditis elegans acts in vivo on germline development. One pathway depends on the oxidation of an analog of vertebrate low density lipoprotein (LDL) and acts on the germline through the Ack-related tyrosine kinase (ARK-1) kinase and inositol trisphosphate (IP3) signaling. The other pathway is the oncogenic ras signaling pathway, whose action on germline as well as vulval development appears to be modulated by cytoplasmic ROS.

Reactive oxygen species (ROS) are short-lived reactive molecules that can modify cellular components including nucleic acids, proteins, and lipids. For example, the oxidation of LDLs by ROS is one of the causative factors of atherosclerosis (1). ROS are toxic but the oxidation of macromolecules can also serve as a signaling device (2). Moreover, in vitro studies have shown that ROS act as intracellular messengers in signal transduction pathways, such as ras signaling (3, 4). However, little is known about the effect of ROS on signal transduction in intact animals (5).

Ubiquinone (UQ or coenzyme Q) is a redox-active lipid that has numerous biochemical roles and is involved in the production of ROS. However, UQ is also an antioxidant that prevents the initiation and/or propagation of lipid peroxidation in cellular membranes (6). The Caenorhabditis elegans clk-1 gene encodes a conserved enzyme that is necessary for UQ biosynthesis (7). In the absence of CLK-1, mutants accumulate demethoxyubiquinone (DMQ) (8, 9), which can partially replace UQ as an electron carrier (8). However, clk-1 mutants require dietary UQ for their survival (9, 10).

clk-1 mutants show a highly pleiotropic phenotype that includes an average slowing down and deregulation of a number of physiological processes, including aging (11). Presumably, given that clk-1 mutants obtain significant amounts of UQ from their diet (12), these phenotypes result from the presence of DMQ, which might be a better anti-oxidant than UQ (13). Thus, the clk-1 mutant phenotypes could be the consequence of altered redox signaling.

In addition to the previously described phenotypes, we find that somatic and germline development are desynchronized in clk-1 mutants. In wild-type hermaphrodites, primary spermatocytes and sperm are observed at the late fourth larval stage (L4), and oogenesis commences shortly after the adult molt (Fig. 1). However, the majority of clk-1 mutants are either before or in the process of spermatogenesis at the adult molt (fig. S1A), and only 3% of the anterior gonads have initiated oogenesis at 6 hours after the adult molt (Fig. 1, B and C). Although the development of the germline of the posterior gonad of clk-1 mutants is also delayed compared with that of the wild type, it is less affected than the anterior gonad. The delay in germline development also produces a delay in egg-laying (fig. S2A).

Fig. 1.

The effects of clk-1 and dsc-4 on germline development. (A) Schematic representation of the proximal portion of the gonad and germline of late L4 and young adult hermaphrodites. The gonad normally consists of two U-shaped arms (anterior and posterior) that join the centrally located uterus. Left is proximal (relative to the vulva) and right is distal. (B) The proximal end of the posterior germline at 6 hours after the adult molt. Left is anterior and top is dorsal. Asterisks indicate the nucleus of the most proximal oocyte; arrows indicate the proximal end of the germline; “e” indicates a fertilized egg; the dotted line indicates a region where spermatogenesis is taking place, and the solid line indicates a region containing primary sperm atocytes. The bar is 10 μm. (C) The percentage of germlines at each of four different developmental stages is shown for each genotype at 6 hours after the adult molt (n ≥ 30). The anterior (A) and posterior (P) gonad arms are presented separately.

We find that the dsc-4(qm182) mutation, which was isolated as a suppressor of the slow defecation phenotype of clk-1 mutants (14), also suppresses the germline phenotypes. Both dsc-4(qm182) and dsc-4(RNAi) (RNAi, for RNA interference) suppress the delayed germline development (Fig. 1, B and C, and Fig. 2A) and egg-laying (fig. S2A), but do not affect the rate of postembryonic development (fig. S3), which indicates that dsc-4 acts by suppressing the slow development of the germline and not by slowing down somatic development.

Fig. 2.

Genetic analysis of the mechanism of action of dsc-4 on the germline development of clk-1. The percentage of germlines observed to be at each of three different developmental stages at 1.5 hours after the adult molt is shown. The stages of development of the anterior (A) and posterior (P) gonad arms are presented separately. All RNAi experiments were carried out by the feeding method; controls were fed the HT115 bacteria containing the RNAi feeding vector, pPD129.36. (A to C) The effect of RNAi against various genes in the clk-1(qm30) or clk-1(qm30); sid-1(qt2) mutant backgrounds (n ≥ 30). The sid-1 mutants were used to test whether the site of action of the RNAi is the intestine. As RNAi is not systemic in sid-1 mutants (31), RNAi by feeding is confined to the intestine (15). (D) The effect of the itr-1(sy328) mutation on germline development (n ≥ 20). All genotypes include the unc-24(e138) mutation, which is linked to itr-1. (E) The effect of the let-60(n1046) gain-of-function mutation on germline development (n ≥ 30). (F) The effect of dsc-4(RNAi) on the germline development of clk-1 and clk-1; let-60 mutants (n ≥ 30).

We cloned dsc-4 and found that it encodes an 892-residue protein similar, and probably orthologous, to the large subunit of the microsomal triglyceride transfer protein (MTP) (fig. S4) (15). MTP is an endoplasmic reticulum (ER) protein that is necessary for the secretion of apolipoprotein B (apoB)–containing lipoproteins, in particular LDLs (16). Lipoproteins consist of a high-molecular-weight protein complexed to various lipids, including triglycerides, cholesteryl esters, cholesterol, and phospholipids (17). apoB is the core component of LDL. In humans, mutations in the large subunit of MTP cause abetalipoproteinemia, a severe deficiency in LDL secretion (18). dsc-4::gfp transcriptional and rescuing translational reporter genes are expressed in the intestine from early embryogenesis, just after the beginning of elongation, throughout larval stages and adulthood (19). In the worm, the intestine is the digestive organ and the major secretory organ; in particular, it secretes the vitellogenins, which are apoB homologs.

The genome of C. elegans contains five apoB-like genes (vit-2 to vit-6) (20). To determine whether altered secretion of apoB-dependent lipoproteins is responsible for the effect of dsc-4 on C. elegans germline development we carried out RNAi against vit-2, -5, and -6. Because the coding region of the vit-5 RNAi clone used is 98% identical to both vit-3 and -4, the vit-5 RNAi treatment is expected to disrupt all three genes (fig. S5A). The effect of vit-5(RNAi) is as strong as that of dsc-4(RNAi) (Fig. 2A), but not additive to it (fig. S5B). The effect of RNAi against vit-2 and -6 is extremely weak, and RNAi against any of the vit genes did not prevent egg production (21). The vit genes were originally isolated as genes that encode yolk proteins. However, if dsc-4 was required for yolk production, mutation of dsc-4 should drastically reduce brood size, as do mutations in rme-2, which encodes a receptor for yolk proteins (22), but it does not. Thus, our results indicate that, in addition to their roles as yolk proteins, at least some of the VITs are also part of lipoprotein particles that resemble the apoB-dependent LDL particles found in vertebrates.

In mammals, cholesterol is a major constituent of lipoproteins, and reducing its intake or synthesis leads to reduced levels of LDL. Cholesterol depletion completely suppresses the slow germline development of clk-1 mutants but has only a mild effect on other genotypes, such as dsc-4 and clk-1; dsc-4 (fig. S1B). This suggests that, as in mammals, reducing cholesterol intake reduces the secretion of LDL-like particles.

To test whether the germline phenotype of clk-1 is caused by lower levels of ROS owing to the presence of DMQ, we performed RNAi against the C. elegans superoxide dismutases, sod-1 to sod-4. Reducing the activity of these detoxifying enzymes will result in an elevation of the level of ROS. We found that sod-1(RNAi), but not RNAi against any of the other sod genes, suppresses the delayed egg production of clk-1 mutants (fig. S2C), but has no effect on the wild type (fig. S2B). Moreover, sod-1(RNAi) is as efficient as dsc-4(RNAi) and vit-5(RNAi) in suppressing slow germline development (Fig. 2A). As SOD-1 is a cytoplasmic Cu/Zn superoxide dismutase, these results indicate that the slow germline development of clk-1 mutants is suppressed by an increase of cytoplasmic superoxide, which suggests that this phenotype results from low levels of ROS. The effect of sod-1(RNAi) is not additive to that of dsc-4(qm182) (fig. S2D), which suggests that the oxidation of LDL-like lipoproteins affects germline development in C. elegans. In addition, sod-1(RNAi) appears to act in the intestine (Fig. 2B), the site of lipoprotein formation and secretion.

In conclusion, our results indicate that decreasing the oxidation of LDL-like lipoproteins slows down germline development (as observed in clk-1 mutants), whereas decreasing the production of native LDL-like lipoproteins (as observed in clk-1; dsc-4 mutants) can restore a normal rate of development. Thus, native lipoproteins inhibit, and oxidized lipoproteins stimulate, germline development (Fig. 3A). However, it is not clear yet whether oxidized LDL itself has an effect on the germline, as envisioned in Fig. 3A, or whether the oxidation of LDL acts simply by reducing the level of native LDL because oxidized LDL is recognized as damaged and is removed.

Fig. 3.

Genetic interactions and schematic representations of pathways affecting germline development. (A) Factors affecting the relative abundance of native and oxidized LDL-like lipoproteins and their effects on germline development. (B) A comparison of the components in the LDL formation and oxidation pathways in vertebrates and worms. (C) A model of genetic interactions affecting germline development in clk-1 mutants.

Our studies provide an invertebrate model system to explore the biology of LDL-like lipoproteins, including their oxidation (Fig. 3B). Many of the constituents and processes identified in vertebrate studies have been found to be present: assembly in the ER and its requirement for MTP activity, an apoB-like protein core for the particle, the importance of cholesterol in determining lipoprotein levels, oxidation, and the involvement of UQ and SOD activity in regulating the level of oxidation (23, 24).

To identify the mechanism of action of lipoproteins on the germline, we focused on the ARK-1 kinase, which has been shown to affect the activity of the epidermal growth factor receptor (EGFR)–like receptor LET-23 on ovulation (25) and is specifically expressed in the germline at high levels (26). We find that ark-1(RNAi) suppresses the slow germline development of clk-1 mutants by acting in the germline (Fig. 2C) and that its effect is not additive to that of dsc-4(qm182) (table S1). Together these findings suggest that ARK-1 is normally activated by lipoproteins and inhibits germline development.

ARK-1 acts on LET-23, which stimulates both the inositol trisphosphate (IP3) and ras signal transduction pathways (27). We find that both a gain-of-function allele of the IP3 receptor [itr-1(sy328)] and the ras gain-of-function allele [let-60(n1046)] moderately suppress the slow germline development of clk-1 (Fig. 2, D and E). However, the gonad function of let-23 is believed to be independent of let-60ras (25). We also find that let-60 acts independently of the dsc-4/ark-1 pathway in our system: the let-60(gf) mutation enhances the effect of dsc-4(RNAi), and this combination suppresses more strongly than any other condition (Fig. 2F). This suggests that germline development is affected independently by both the let-60ras and dsc-4/ark-1 pathways (Fig. 3C).

The effect of let-60(gf) on the germline of clk-1 mutants suggests that the ras pathway is down-regulated by clk-1 mutations, which is consistent with the redox-sensitivity of ras signaling in in vitro cellular models (4). To test this further, we examined the effect of clk-1 on the activity of let-60 for vulva formation. In this pathway, ras activation inhibits transcription factors such as LIN-1, which itself inhibits vulva formation (28). Thus, let-60(gf) and lin-1(lf) mutants have multiple vulvae instead of only one (29). We found that clk-1 strongly suppresses let-60(gf) but has no effect on lin-1(e1026), which suggests that clk-1 acts on the ras pathway upstream of lin-1 (Fig. 4, A and B). We found that the effect of clk-1 on the ras pathway is due to reduced redox signaling. Indeed, RNAi against sod-1 and, to a lesser extent, sod-4, the extracellular Cu/Zn SOD, substantially restores vulva formation in clk-1; let-60(gf) mutants (Fig. 4C). Neither the sods on clk-1(+) nor dsc-4 on clk-1;let-60 had a significant effect on vulva formation (30). That the cytoplasmic and extracellular SODs have an effect is consistent with the cytoplasmic and membrane-associated localization of ras.

Fig. 4.

clk-1 affects ras signaling during vulva formation. (A) A let-60(n1046gf) mutant with multiple vulvae (indicated by asterisks) and a clk-1(qm30); let-60(n1046gf) mutant with a single vulva. (B) The multivulva phenotype (Muv) of let-60(gf) but not lin-1(e1026) is suppressed by clk-1 (n ≥ 30). (C) The effect of RNAi against the four sod genes on the Muv phenotype of clk-1; let-60(gf) mutants (n ≥ 750).

The phenotype of clk-1 mutants is extremely pleiotropic, with most aspects of development, behavior, and reproduction being slowed on average (11). We have found that at least one of these phenotypes could be explained by a reduction of the level of oxidation of LDL-like lipoproteins and that clk-1 affects the ras pathway by lowering cytoplasmic ROS levels. These findings provide a unique model to study the effect of redox signal transduction on the development of whole organisms and suggest a model for the clk-1 pleiotropy, in which the complexity of the phenotype is due to the multiplicity of signaling roles that are carried out by the oxidative modification of cellular constituents.

Supporting Online Material

Materials and Methods

Table S1

Figs. S1 to S6


References and Notes

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