Temperature-sensitive developmental mutants of Caenorhabditis elegans

About 200 temperature-sensitive mutants of the nematode Caenorhabditis elegans have been isolated. At restrictive temperature, the mutants are blocked in the reproductive life cycle. They have been placed into six broad categories based on their defective phenotypes. The six categories are: (1) mutants blocked in embryogenesis; (2) mutants defective in gonadogenesis; (3) mutants defective in spermatogenesis; (4) mutants that accumulate at an intermediate growth stage; (5) mutants that produce sterile adult progeny; (6) mutants that have a temperature-sensitive morphological defect that interrupts the reproductive life cycle. The critical times of temperature sensitivity have been measured using temperature-shift experiments. Most of the gonadogenesis and spermatogenesis mutants are temperature sensitive during the period of cellular differentiation rather than proliferation. The temperature responses of the gonadogenesis and zygote-defective mutants indicate a common association between functions in gonadogenesis and early embryogenesis. Many of the mutants placed in different categories share other temperature-sensitive phenotypes upon close examination. This implies that many of the functions required for development are general metabolic reactions under increased demand during differentiation and embryogenesis.     

Genome evolution and developmental constraint in Caenorhabditis elegans

It has been hypothesized that evolutionary changes will be more frequent in later ontogeny than early ontogeny because of developmental constraint. To test this hypothesis, a genomewide examination of molecular evolution through ontogeny was carried out using comparative genomic data in Caenorhabditis elegans and Caenorhabditis briggsae. We found that the mean rate of amino acid replacement is not significantly different between genes expressed during and after embryogenesis. However, synonymous substitution rates differed significantly between these two classes. A genomewide survey of correlation between codon bias and expression level found codon bias to be significantly correlated with mRNA expression (r(s) = -0.30 and P < 10(-131)) but does not alone explain differences in dS between classes. Surprisingly, it was found that genes expressed after embryogenesis have a significantly greater number of duplicates in both the C. elegans and C. briggsae genomes (P < 10(-20) and P < 10(-13)) when compared with early-expressed and nonmodulated genes. A similarity in the distribution of duplicates of nonmodulated and early-expressed genes, as well as a disproportionately higher number of early pseudogenes, lend support to the hypothesis that this difference in duplicate number is caused by selection against gene duplicates of early-expressed genes, reflecting developmental constraint. Developmental constraint at the level of gene duplication may have important implications for macroevolutionary change.     

Multiple mechanisms are involved in regulating the expression of the developmental timing regulator lin-28 in Caenorhabditis elegans

The timing of postembryonic developmental programs in Caenorhabditis elegans is regulated by a set of so-called heterochronic genes, including lin-28 that specifies second larval programs. lin-66 mutations described herein cause delays in vulval and seam cell differentiation, indicating a role for lin-66 in timing regulation. A mutation in daf-12/nuclear receptor or alg-1/argonaute dramatically enhances the retarded phenotypes of the lin-66 mutants, and these phenotypes are suppressed by a lin-28 null allele. We further show that the LIN-28 protein level is upregulated in the lin-66 mutants and that this regulation is mediated by the 3'UTR of lin-28. We have also identified a potential daf-12-response element within lin-28 3'UTR and show that two microRNA (miRNA) (lin-4 and let-7)-binding sites mediate redundant inhibitory activities that are likely lin-66-independent. Quantitative PCR data suggest that the lin-28 mRNA level is affected by lin-14 and miRNA regulation, but not by daf-12 and lin-66 regulation. These results suggest that lin-28 expression is regulated by multiple independent mechanisms including LIN-14-mediated upregulation of mRNA level, miRNAs-mediated RNA degradation, LIN-66-mediated translational inhibition and DAF-12-involved translation promotion.     

Longevity determined by developmental arrest genes in Caenorhabditis elegans

The antagonistic pleiotropy theory of aging proposes that aging takes place because natural selection favors genes that confer benefit early on life at the cost of deterioration later in life. This theory predicts that genes that impact development would play a key role in shaping adult lifespan. To better understand the link between development and adult lifespan, we examined the genes previously known to be essential for development. From a pool of 57 genes that cause developmental arrest after inhibition using RNA interference, we have identified 24 genes that extend lifespan in Caenorhabditis elegans when inactivated during adulthood. Many of these genes are involved in regulation of mRNA translation and mitochondrial functions. Genetic epistasis experiments indicate that the mechanisms of lifespan extension by inactivating the identified genes may be different from those of the insulin/insulin-like growth factor 1 (IGF-1) and dietary restriction pathways. Inhibition of many of these genes also results in increased stress resistance and decreased fecundity, suggesting that they may mediate the trade-offs between somatic maintenance and reproduction. We have isolated novel lifespan-extension genes, which may help understand the intrinsic link between organism development and adult lifespan.     

N-Glycans of Caenorhabditis elegans are specific to developmental stages

We have examined the N-glycans present during the developmental stages of Caenorhabditis elegans using two approaches, 1) a combination of permethylation followed by MALDI-TOF mass spectrometry (MS) and 2) derivatization with 2-aminobenzamide followed by separation by high-performance liquid chromatography and analyses by MALDI-TOF MS, post source decay (PSD) MS, and MALDI-QoTOF MS/MS. The N-glycan profile of each developmental stage (Larva 1, Larva 2, Larva 3, Larva 4, and Dauer and adult) appears to be unique. The pattern of complex N-glycans was stage-specific with the general trend of number and abundance of glycans being Dauer approximately = L1 > adult approximately = L4 > L3 approximately = L2. Dauer larvae contained complex N-glycans with higher molecular masses than those seen in other stages. MALDI-QoTOF MS/MS of Hex4HexNAc4 showed an N-acetyllac-tosamine substitution not previously observed in C. elegans. Phosphorylcholine (Pc)-substituted glycans were also found to be stage-specific. Higher molecular weight Pc-containing glycans, including fucose-containing ones such as difucosyl Pc-glycan (Pc1dHex2Hex5HexNAc6) seen in Dauer larvae, have not been observed in any organism. Pc2Hex4HexNAc3, from Dauer larvae, when subjected to PSD MS analyses, showed Pc may substitute both core and terminally linked GlcNAc; no such structure has previously been reported in any organism. C. elegans-specific fucosyl and native methylated glycans were found in all developmental stages. Taken together, the above results demonstrate that in-depth investigation of the role of the above N-glycans during C. elegans development should lead to a better understanding of their significance and the ways that they may govern interactions, both within the organism during development and between the mobile nematode and its pathogens.     

gon-4, a cell lineage regulator required for gonadogenesis in Caenorhabditis elegans

The gon-4 gene is required for gonadogenesis in the nematode Caenorhabditis elegans. Normally, two precursor cells, Z1 and Z4, follow a reproducible pattern of cell divisions to generate the mature somatic gonadal structures (e.g., uterus in hermaphrodites, vas deferens in males). In contrast, in gon-4 mutants, the Z1/Z4 cell lineages are variably aborted in both hermaphrodites and males: Z1 and Z4 divide much later than normal and subsequent divisions are either absent or severely delayed. In gon-4 adults, normal somatic gonadal structures are never observed, and germ-line and vulval tissues, which depend on somatic gonadal cues for their development, are also aberrant. In contrast, nongonadal tissues and the timing of other developmental events (e.g., molts) appear to be normal in gon-4 mutants. The gon-4 alleles are predicted to be strong loss-of-function or null alleles by both genetic and molecular criteria. We have cloned gon-4 in an attempt to learn how it regulates gonadogenesis. The gon-4 gene encodes a novel, acidic protein. A GON-4::GFP fusion protein, which rescues a gon-4 mutant to fertility, is expressed in somatic gonadal cells during early gonadal development. Furthermore, this fusion protein is nuclear. We conclude that gon-4 is a regulator of the early lineage of Z1 and Z4 and suggest that it is a part of a genetic program common to the regulation of both hermaphrodite and male gonadogenesis.     

Glypican LON-2 is a conserved negative regulator of BMP-like signaling in Caenorhabditis elegans

Bone morphogenetic protein (BMP) pathways are required for a wide variety of developmental and homeostatic decisions, and mutations in signaling components are associated with several diseases. An important aspect of BMP control is the extracellular regulation of these pathways. We show that LON-2 negatively regulates a BMP-like signaling pathway that controls body length in C. elegans. lon-2 acts genetically upstream of the BMP-like gene dbl-1, and loss of lon-2 function results in animals that are longer than normal. LON-2 is a conserved member of the glypican family of heparan sulfate proteoglycans, a family with several members known to regulate growth-factor signaling in many organisms. LON-2 is functionally conserved because the Drosophila glypican gene dally rescues the lon-2(lf) body-size defect. We show that the LON-2 protein binds BMP2 in vitro, and a mutant variation of LON-2 found in lon-2(e2140) animals diminishes this interaction. We propose that LON-2 binding to DBL-1 negatively regulates this pathway in C. elegans by attenuating ligand-receptor interactions. This is the first report of a glypican directly interacting with a growth-factor pathway in C. elegans and provides a mechanistic model for glypican regulation of growth-factor pathways.     

Role of pleiotropy in the evolution of a cryptic developmental variation in Caenorhabditis elegans

Robust biological systems are expected to accumulate cryptic genetic variation that does not affect the system output in standard conditions yet may play an evolutionary role once phenotypically expressed under a strong perturbation. Genetic variation that is cryptic relative to a robust trait may accumulate neutrally as it does not change the phenotype, yet it could also evolve under selection if it affects traits related to fitness in addition to its cryptic effect. Cryptic variation affecting the vulval intercellular signaling network was previously uncovered among wild isolates of Caenorhabditis elegans. Using a quantitative genetic approach, we identify a non-synonymous polymorphism of the previously uncharacterized nath-10 gene that affects the vulval phenotype when the system is sensitized with different mutations, but not in wild-type strains. nath-10 is an essential protein acetyltransferase gene and the homolog of human NAT10. The nath-10 polymorphism also presents non-cryptic effects on life history traits. The nath-10 allele carried by the N2 reference strain leads to a subtle increase in the egg laying rate and in the total number of sperm, a trait affecting the trade-off between fertility and minimal generation time in hermaphrodite individuals. We show that this allele appeared during early laboratory culture of N2, which allowed us to test whether it may have evolved under selection in this novel environment. The derived allele indeed strongly outcompetes the ancestral allele in laboratory conditions. In conclusion, we identified the molecular nature of a cryptic genetic variation and characterized its evolutionary history. These results show that cryptic genetic variation does not necessarily accumulate neutrally at the whole-organism level, but may evolve through selection for pleiotropic effects that alter fitness. In addition, cultivation in the laboratory has led to adaptive evolution of the reference strain N2 to the laboratory environment, which may modify other phenotypes of interest.     

The dauerlarva, a post-embryonic developmental variant of the nematode Caenorhabditis elegans.

In the postembryological development of the free-living nematode Caenorhabditis elegans, a morphologically recognizable, nongrowing stage, called the dauerlarva, may arise. Using synchronous populations and following growth and molting, it has been shown that the dauerlarva is formed by a facultative, reversible arrest at a specific point in the life cycle, the second of four cuticle molts, in response to external conditions.At each molt a normal animal passes through “lethargus,” a stage in which feeding and locomotion are transiently arrested. In the dauerlarva stage, feeding is arrested indefinitely and locomotion is markedly reduced. A simple quantitative assay, based on the exceptional resistance of dauerlarvae to sodium dodecyl sulfate (SDS), has been developed to study dauerlarva formation and its reversal. The SDS resistance of dauerlarvae requires both non-feeding and an especially impermeable cuticle. Dauerlarva formation can be efficiently induced by limiting the concentration of bacteria (the food supply), but not by complete starvation. Quantitative recovery to normal development can be induced by transfer to fresh medium with excess bacteria. Simpler stimuli can elicit recovery at slower rates, the principal factors besides nutrition being nutrition being optimal ionic and osmotic conditions and a noninhibitory concentration of animals. There are identifiable stages in recovery, beginning with a resumption of feeding. The cuticle, ultrastructurally very different from normal cuticle, is shed at the next molt, after which development appears normal. A temperature-sensitive mutant, which forms dauerlarvae at high temperature despite the presence of abundant food, is described, and the use of dauerlarvae for further mutant isolation is discussed.     

Different Mi-2 complexes for various developmental functions in Caenorhabditis elegans

Biochemical purifications from mammalian cells and Xenopus oocytes revealed that vertebrate Mi-2 proteins reside in multisubunit NuRD (Nucleosome Remodeling and Deacetylase) complexes. Since all NuRD subunits are highly conserved in the genomes of C. elegans and Drosophila, it was suggested that NuRD complexes also exist in invertebrates. Recently, a novel dMec complex, composed of dMi-2 and dMEP-1 was identified in Drosophila. The genome of C. elegans encodes two highly homologous Mi-2 orthologues, LET-418 and CHD-3. Here we demonstrate that these proteins define at least three different protein complexes, two distinct NuRD complexes and one MEC complex. The two canonical NuRD complexes share the same core subunits HDA-1/HDAC, LIN-53/RbAp and LIN-40/MTA, but differ in their Mi-2 orthologues LET-418 or CHD-3. LET-418 but not CHD-3, interacts with the Krüppel-like protein MEP-1 in a distinct complex, the MEC complex. Based on microarrays analyses, we propose that MEC constitutes an important LET-418 containing regulatory complex during C. elegans embryonic and early larval development. It is required for the repression of germline potential in somatic cells and acts when blastomeres are still dividing and differentiating. The two NuRD complexes may not be important for the early development, but may act later during postembryonic development. Altogether, our data suggest a considerable complexity in the composition, the developmental function and the tissue-specificity of the different C. elegans Mi-2 complexes.     

A hierarchy of regulatory genes controls a larva-to-adult developmental switch in C. elegans

The heterochronic genes lin-4, lin-14, lin-28, and lin-29 control the timing of specific postembryonic developmental events in C. elegans. The experiments described here examine how these four genes interact to control a particular stage-specific event of the lateral hypodermal cell lineages. This event, termed the "larva-to-adult switch" (L/A switch), involves several coordinate changes in the behavior of hypodermal cells at the fourth molt: cessation of cell division, formation of adult (instead of larval) cuticle, cell fusion, and cessation of the molting cycle. The phenotypes of multiply mutant strains suggest a model wherein the L/A switch is controlled by the stage-specific activity of a regulatory hierarchy: At early stages of wild-type development, lin-14 and lin-28 inhibit lin-29 and thus prevent switching. Later, lin-4 inhibits lin-14 and lin-28, allowing activation of lin-29, which in turn triggers the switch in the L4 stage. lin-29 may activate the L/A switch by regulating genes that control cell division, differentiation, and stage-specific gene expression in hypodermal cells.     

Caenorhabditis elegans RME-6 is a novel regulator of RAB-5 at the clathrin-coated pit

Here we identify a new regulator of endocytosis called RME-6. RME-6 is evolutionarily conserved among metazoans and contains Ras-GAP (GTPase-activating protein)-like and Vps9 domains. Consistent with the known catalytic function of Vps9 domains in Rab5 GDP/GTP exchange, we found that RME-6 binds specifically to Caenorhabditis elegans RAB-5 in the GDP-bound conformation, and rme-6 mutants have phenotypes that indicate low RAB-5 activity. However, unlike other Rab5-associated proteins, a rescuing green fluorescent protein (GFP)-RME-6 fusion protein primarily localizes to clathrin-coated pits, physically interacts with alpha-adaptin, a clathrin adaptor protein, and requires clathrin to achieve its cortical localization. In rme-6 mutants, transport from the plasma membrane to endosomes is defective, and small 110-nm endocytic vesicles accumulate just below the plasma membrane. These results suggest a mechanism for the activation of Rab5 in clathrin-coated pits or clathrin-coated vesicles that is essential for the delivery of endocytic cargo to early endosomes.     

Diet-dependent depletion of queuosine in tRNAs in Caenorhabditis elegans does not lead to a developmental block

Queuosine (Q), a hypermodified nucleoside, occurs at the wobble position of transfer RNAs (tRNAs) with GUN anticodons. In eubacteria, absence of Q affects messenger RNA (mRNA) translation and reduces the virulence of certain pathogenic strains. In animal cells, changes in the abundance of Q have been shown to correlate with diverse phenomena including stress tolerance, cell proliferation and tumour growth but the function of Q in animals is poorly understood. Animals are thought to obtain Q (or its analogues) as a micronutrient from dietary sources such as gut microflora. However, the difficulty of maintaining animals under bacteria-free conditions on Q-deficient diets has severely hampered the study of Q metabolism and function in animals. In this study, we show that as in higher animals, tRNAs in the nematode Caenorhabditis elegans are modified by Q and its sugar derivatives. When the worms were fed on Q-deficient Escherichia coli, Q modification was absent from the worm tRNAs suggesting that C. elegans lacks a de novo pathway of Q biosynthesis. The inherent advantages of C. elegans as a model organism, and the simplicity of conferring a Q-deficient phenotype on it make it an ideal system to investigate the function of Q modification in tRNA.