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.     

Temperature-sensitive zygote defective mutants of Caenorhabditis elegans

Three genetically complementing temperature-sensitive mutants of Caenorhabditis elegans have been studied. Each of the three mutants has two critical times of temperature sensitivity and two distinctive corresponding phenotypes. Exposure to high temperature during gonadogenesis blocks the production of zygotes. Exposure of adults to high temperature interrupts embryogenesis of the zygotes being produced. Each of the mutants carries an autosomal mutation with a maternal effect. These mutants indicate that the individual temperature-sensitive functions are required at least twice during development and that early embryogenesis is dependent on the contribution of these functions from the maternal gonad.     

A test for epistasis among induced mutations in Caenorhabditis elegans

Synergistic epistasis, in which deleterious mutations tend to magnify each other's effects, is a necessary component of the mutational deterministic hypothesis for the maintenance of sexual production. We tested for epistasis for life-history traits in the soil nematode Caenorhabditis elegans by inducing mutations in two genetic backgrounds: a wild-type strain and a set of genetically loaded lines that contain large numbers of independent mildly detrimental mutations. There was no significant difference between the effect of new mutations on the wild-type background and the genetically loaded background for four out of five fitness correlates. In these four cases, the maximum level of epistasis compatible with the data was very low. The fifth trait, late productivity, is not likely to be an important component of fitness. This suggests either that specific environmental conditions are required to cause epistasis or that synergistic epistasis is not a general phenomenon. We also suggest a new mechanism by which deleterious mutations may provide an advantage to sexual reproduction under low selection coefficients.     

Maternal effects and temperature-sensitive period of mutations affecting embryogenesis in Caenorhabditis elegans

We have used standard tests to investigate the nature of gene expression of a new set of temperature-sensitive mutants defining 30 emb genes (essential for embryogenesis) in the nematode Caenorhabditis elegans. The mode of gene expression as determined by progeny tests for parental effects divides the genes into four classes. For 18 genes maternal gene expression is necessary and sufficient for normal embryogenesis; for 2 genes zygotic expression is necessary and sufficient; for 7 genes either maternal or zygotic expression is sufficient; for 3 genes both maternal and zygotic expression are necessary. One mutant displayed partial paternal sufficiency. The results of temperature-shift experiments define two “execution stages,” corresponding to the limits of the temperature-sensitive period (TSP), and indicate the nature and the time of action or synthesis of the gene products. Most of the maternally expressed genes have very early execution stages indicating translation before fertilization, but some are temperature sensitive late in embryogenesis. Early execution stages for 2 zygotically necessary genes demonstrate that the zygotic genome can be active in the earliest stages of embryogenesis. All taken together, the mode of gene expression, TSP, and arrest stage (terminal phenotype) allow us to classify functionally and begin to order the genes essential for embryogenesis. The results indicate a preeminent role for maternal genes and gene products in embryogenesis, in agreement with the results of others.     

Frequent germline mutations and somatic repeat instability in DNA mismatch-repair-deficient Caenorhabditis elegans

Mismatch-repair-deficient mutants were initially recognized as mutation-prone derivatives of bacteria, and later mismatch repair deficiency was found to predispose humans to colon cancers (HNPCC). We generated mismatch-repair-deficient Caenorhabditis elegans by deleting the msh-6 gene and analyzed the fidelity of transmission of genetic information to subsequent generations. msh-6-defective animals show an elevated level of spontaneous mutants in both the male and female germline; also repeated DNA tracts are unstable. To monitor DNA repeat instability in somatic tissue, we developed a sensitive system, making use of heat-shock promoter-driven lacZ transgenes, but with a repeat that puts this reporter gene out of frame. In genetic msh-6-deficient animals lacZ+ patches are observed as a result of somatic repeat instability. RNA interference by feeding wild-type animals dsRNA homologous to msh-2 or msh-6 also resulted in somatic DNA instability, as well as in germline mutagenesis, indicating that one can use C. elegans as a model system to discover genes involved in maintaining DNA stability by large-scale RNAi screens.     

Globins in Caenorhabditis elegans

Extensive in silico search of the genome of Caenorhabditis elegans revealed the presence of 33 genes coding for globins that are all transcribed. These globins are very diverse in gene and protein structure and are localized in a variety of cells, mostly neurons. The large number of C. elegans globin genes is assumed to be the result of multiple evolutionary duplication and radiation events. Processes of subfunctionalization and diversification probably led to their cell-specific expression patterns and fixation into the genome. To date, four globins (GLB-1, GLB-5, GLB-6, and GLB-26) have been partially characterized physicochemically, and the crystallographic structure of two of them (GLB-1 and GLB-6) was solved. In this article, a three-dimensional model was designed for the other two globins (GLB-5 and GLB-26), and overlays of the globins were constructed to highlight the structural diversity among them. It is clear that although they all share the globin fold, small variations in the three-dimensional structure have major implications on their ligand-binding properties and possibly their function. We also review here all the information available so far on the globin family of C. elegans and suggest potential functions.     

Mechanotransduction in Caenorhabditis elegans

One of the looming mysteries in signal transduction today is the question of how mechanical signals, such as pressure or mechanical force delivered to a cell, are interpreted to direct biological responses. All living organisms, and probably all cells, have the ability to sense and respond to mechanical stimuli. At the single-cell level, mechanical signaling underlies cell-volume control and specialized responses such as the prevention of poly-spermy in fertilization. At the level of the whole organism, mechanotransduction underlies processes as diverse as stretch-activated reflexes in vascular epithelium and smooth muscle; gravitaxis and turgor control in plants; tissue development and morphogenesis; and the senses of touch, hearing, and balance. Intense genetic, molecular, and elecrophysiological studies in organisms ranging from nematodes to mammals have highlighted members of the recently discovered DEG/ENaC family of ion channels as strong candidates for the elusive metazoan mechanotransducer. Here, we discuss the evidence that links DEG/ENaC ion channels to mechanotransduction and review the function of Caenorhabiditis elegans members of this family called degenerins and their role in mediating mechanosensitive behaviors in the worm.     

Genetic suppression of phenotypes arising from mutations in dystrophin-related genes in Caenorhabditis elegans

BACKGROUND: Dystrophin is the product of the gene that is mutated in Duchenne muscular dystrophy (DMD), a progressive neuromuscular disease for which no treatment is available. Mice carrying a mutation in the gene for dystrophin (mdx mice) display only a mild phenotype, but it is aggravated when combined with a mutation in the MyoD gene. The nematode worm Caenorhabditis elegans has a dystrophin homologue (dys-1), but null mutations in dys-1 do not result in muscle degeneration.RESULTS: We generated worms carrying both the dys-1 null mutation cx18, and a weak mutation, cc561ts, of the C. elegans MyoD homologue hlh-1. The double mutants displayed a time-dependent impairment of locomotion and egg laying, a phenotype not seen in the single mutants, and extensive muscle degeneration. This result allowed us to look for genes that, when misexpressed, could suppress the dys-1; hlh-1 phenotype. When overexpressed, the dyc-1 gene - whose loss-of-function phenotype resembles that of dys-1 - partially suppressed the dys-1; hlh-1 phenotype. The dyc-1 gene encodes a novel protein sharing similarities with the mammalian neural nitric oxide synthase (nNOS)-binding protein CAPON, and is expressed in the muscles of the worm. CONCLUSIONS: As a C. elegans model for dystrophin-dependent myopathy, the dys-1; hlh-1 worms should permit the identification of genes, and ultimately drugs, that would reverse the muscle degeneration in this model.     

Most ultraviolet irradiation induced mutations in the nematode Caenorhabditis elegans are chromosomal rearrangements

In this study we have determined the utility of 254-nm ultraviolet light (UV) as a mutagenic tool in C. elegans. We have demonstrated that irradiation of adult hermaphrodites provides a simple method for the induction of heritable chromosomal rearrangements. A screening protocol was employed that identifies either recessive lethal mutations in the 40 map unit region balanced by the translocation eT1(III;V), or unc-36(III) duplications. Mutations were recovered in 3% of the chromosomes screened after a dose of 120 J/m2. This rate resembles that for 1500 R gamma-ray-induced mutations selected in a similar manner. The mutations were classified either as lethals [mapping to Linkage Group (LG)III or LGV] or as putative unc-36 duplications. In contrast to the majority of UV-induced mutations analysed in microorganisms, we found that a large fraction of the C. elegans UV-induced mutations are not simple intragenic lesions, but are deficiencies for more than one adjacent gene or more complex events. Preliminary evidence for this conclusion came from the high frequency of mutations that had a dominant effect causing reduced numbers of adult progeny. Subsequently 6 out of 9 analysed LGV mutations were found to be deficiencies. Other specific rearrangements also identified were: one translocation, sT5(II;III), and two unc-36 duplications, sDp8 and sDp9. It was concluded that UV irradiation can easily be used as an additional tool for the analysis of C. elegans chromosomes, and that C. elegans should prove to be a useful organism in which to study the mechanisms whereby UV acts as a mutagen in cells of complex eukaryotes.     

Resistance to volatile anesthetics by mutations enhancing excitatory neurotransmitter release in Caenorhabditis elegans

The molecular mechanisms whereby volatile general anesthetics (VAs) disrupt behavior remain undefined. In Caenorhabditis elegans mutations in the gene unc-64, which encodes the presynaptic protein syntaxin 1A, produce large allele-specific differences in VA sensitivity. UNC-64 syntaxin normally functions to mediate fusion of neurotransmitter vesicles with the presynaptic membrane. The precise role of syntaxin in the VA mechanism is as yet unclear, but a variety of results suggests that a protein interacting with syntaxin to regulate neurotransmitter release is essential for VA action in C. elegans. To identify additional proteins that function with syntaxin to control neurotransmitter release and VA action, we screened for suppressors of the phenotypes produced by unc-64 reduction of function. Loss-of-function mutations in slo-1, which encodes a Ca(2+)-activated K+ channel, and in unc-43, which encodes CaM-kinase II, and a gain-of-function mutation in egl-30, which encodes Gqalpha, were isolated as syntaxin suppressors. The slo-1 and egl-30 mutations conferred resistance to VAs, but unc-43 mutations did not. The effects of slo-1 and egl-30 on VA sensitivity can be explained by their actions upstream or parallel to syntaxin to increase the level of excitatory neurotransmitter release. These results strengthen the link between transmitter release and VA action.     

Isoform-specific mutations in the Caenorhabditis elegans heterochronic gene lin-14 affect stage-specific patterning

The Caenorhabditis elegans heterochronic gene lin-14 specifies the temporal sequence of postembryonic developmental events. lin-14, which encodes differentially spliced LIN-14A and LIN-14B1/B2 protein isoforms, acts at distinct times during the first larval stage to specify first and second larval stage-specific cell lineages. Proposed models for the molecular basis of these two lin-14 gene activities have included the production of functionally distinct isoforms and the generation of a temporal gradient of LIN-14 protein. We report here that loss of the LIN-14B1/B2 isoforms alone affects one of the two lin-14 temporal patterning functions, the specification of second larval stage lineages. A temporal expression difference between LIN-14A and LIN-14B1/B2 is not responsible for the stage-specific phenotype: protein levels of all LIN-14 isoforms are high in early first larval stage animals and decrease during the first larval stage. However, LIN-14A can partially substitute for LIN-14B1/B2 when expressed at a higher-than-normal level in the late L1 stage. These data indicate that LIN-14B1/B2 isoforms do not provide a distinct function of the lin-14 locus in developmental timing but rather may contribute to an overall level of LIN-14 protein that is the critical determinant of temporal cell fate.     

Molecular analysis of mutations in the Caenorhabditis elegans collagen gene dpy-7.

Collagens are a family of proteins contributing to the body structure of eukaryotes. They are encoded by a large and diverse gene family in the nematode Caenorhabditis elegans but by only a few genes in vertebrates. We have studied mutant alleles of the C. elegans dpy-7 gene, one of a large group of genes whose mutant phenotype is altered body form and several of which have previously been shown to encode cuticular collagens. We made use of the C. elegans physical map to screen specifically for collagen genes in the region of the X chromosome to which dpy-7 maps. This yielded a wild-type collagen gene clone which we showed, by micro-injection, could repair the dpy-7 mutant phenotype in transgenic animals. We cloned the homologous sequence from four dpy-7 mutant strains and by sequence analysis identified a single mutation in each case. All four mutations result in the substitution of a glycine with a larger residue in the conserved Gly-X-Y collagen domains. Similar substitutions in vertebrate collagens cause the heritable brittle bone disorder osteogenesis imperfecta. Whereas the human mutations are dominant, the dpy-7 mutations are recessive, and this may reflect different levels of complexity of collagenous macromolecular structures in the two organisms.     

Identification of mutations that delay somatic or reproductive aging of Caenorhabditis elegans

Aging is an important feature of animal biology characterized by progressive, degenerative changes in somatic and reproductive tissues. The rate of age-related degeneration is genetically controlled, since genes that influence lifespan have been identified. However, little is known about genes that affect reproductive aging or aging of specific somatic tissues. To identify genes that are important for controlling these degenerative changes, we used chemical mutagenesis to perform forward genetic screens in Caenorhabditis elegans. By conducting a screen focused on somatic aging, we identified mutant hermaphrodites that displayed extended periods of pharyngeal pumping, body movement, or survival. One of these mutations is a novel allele of the age-1 gene. age-1 encodes a phosphatidylinositol-3-kinase (PI3K) that functions in the insulin/insulin-like growth factor-1 (IGF-1) signaling pathway. age-1(am88) creates a missense change in the conserved PIK domain and causes dramatic extensions of the pharyngeal pumping and body movement spans, as well as a twofold extension of the lifespan. By conducting screens focused on reproductive aging in mated hermaphrodites, we identified mutants that displayed increased progeny production late in life. To characterize these mutations, we developed quantitative measurements of age-related morphological changes in the gonad. The am117 mutation delayed age-related declines in progeny production and morphological changes in the gonad. These studies provide new insights into the genetic regulation of age-related degenerative changes in somatic and reproductive tissues.     

Species differentiation in Caenorhabditis briggsae and Caenorhabditis elegans

Identification of five laboratory strains (1-5) of putative Caenorhabditis briggsae was undertaken. Examination of the male bursal ray arrangement, mating tests with males of Caenorhabditis elegans, malate dehydrogenase zymograms, and SDS polyacrylamide electrophoresis demonstrated that strain 4 was C. briggsae and the others were C. elegans.