Suppressors of a Lin-12 Hypomorph Define Genes That Interact with Both Lin-12 and Glp-1 in Caenorhabditis Elegans

The lin-12 gene of Caenorhabditis elegans is thought to encode a receptor which mediates cell-cell interactions required to specify certain cell fates. Reversion of the egg-laying defective phenotype caused by a hypomorphic lin-12 allele identified rare extragenic suppressor mutations in five genes, sel-1, sel-9, sel-10, sel-11 and sel(ar40) (sel = suppressor and/or enhancer of lin-12). Mutations in each of these sel genes suppress defects associated with reduced lin-12 activity, and enhance at least one defect associated with elevated lin-12 activity. None of the sel mutations cause any obvious phenotype in a wild-type background. Gene dosage experiments suggest that sel-1 and sel(ar40) mutations are reduction-of-function mutations, while sel-9 and sel-11 mutations are gain-of-function mutations. sel-1, sel-9, sel-11 and sel(ar40) mutations do not suppress amorphic lin-12 alleles, while sel-10 mutations are able to bypass partially the requirement for lin-12 activity in at least one cell fate decision. sel-1, sel-9, sel-10, sel-11 and sel(ar40) mutations are also able to suppress the maternal-effect lethality caused by a partial loss-of-function allele of glp-1, a gene that is both structurally and functionally related to lin-12. These sel genes may therefore function in both lin-12 and glp-1 mediated cell fate decisions.     

Genetic and Phenotypic Studies of Hypomorphic Lin-12 Mutants in Caenorhabditis Elegans

The lin-12 gene of Caenorhabditis elegans is thought to encode a receptor for intercellular signals that specify certain cell fates during development. We describe several alleles of lin-12 that reduce but do not eliminate lin-12 activity (hypomorphic alleles). These alleles cause a novel egg-laying defective (Egl) phenotype in hermaphrodites as well as incompletely penetrant cell fate transformations seen with high penetrance in lin-12 null mutants. Characterization of the Egl phenotype revealed additional roles of lin-12 in the development of the egg-laying system that were not apparent from studying lin-12 null mutants: lin-12 activity is required for proper early vulval morphogenesis as well as for some unknown later aspect of egg-laying system development. Reversion of the Egl phenotype caused by one lin-12 hypomorphic allele was used to identify potential interacting genes as described in the accompanying paper.     

Genes That Regulate Both Development and Longevity in Caenorhabditis Elegans

The nematode Caenorhabditis elegans responds to conditions of overcrowing and limited food by arresting development as a dauer larva. Genetic analysis of mutations that alter dauer larva formation (daf mutations) is presented along with an updated genetic pathway for dauer vs. nondauer development. Mutations in the daf-2 and daf-23 genes double adult life span, whereas mutations in four other dauer-constitutive genes positioned in a separate branch of this pathway (daf-1, daf-4, daf-7 and daf-8) do not. The increased life spans are suppressed completely by a daf-16 mutation and partially in a daf-2; daf-18 double mutant. A genetic pathway for determination of adult life span is presented based on the same strains and growth conditions used to characterize Daf phenotypes. Both dauer larva formation and adult life span are affected in daf-2; daf-12 double mutants in an allele-specific manner. Mutations in daf-12 do not extend adult life span, but certain combinations of daf-2 and daf-12 mutant alleles nearly quadruple it. This synergistic effect, which does not equivalently extend the fertile period, is the largest genetic extension of life span yet observed in a metazoan.     

Identification and Characterization of Autosomal Genes That Interact with Glass in the Developing Drosophila Eye

The glass gene encodes a zinc finger, DNA-binding protein that is required for photoreceptor cell development in Drosophila melanogaster. In the developing compound eye, glass function is regulated at two points: (1) the protein is expressed in all cells' nuclei posterior to the morphogenetic furrow and (2) the ability of the Glass protein to regulate downstream genes is largely limited to the developing photoreceptor cells. We conducted a series of genetic screens for autosomal dominant second-site modifiers of the weak allele glass(3), to discover genes with products that may regulate glass function at either of these levels. Seventy-six dominant enhancer mutations were recovered (and no dominant suppressors). Most of these dominant mutations are in essential genes and are associated with recessive lethality. We have assigned these mutations to 23 complementation groups that include multiple alleles of Star and hedgehog as well as single alleles of Delta, roughened eye, glass and hairy. Mutations in 18 of the complementation groups are embryonic lethals, and of these, 13 show abnormal adult retinal phenotypes in homozygous clones, usually with altered numbers of photoreceptor cells in some of the ommatidia.     

Genes That Implement the Hermaphrodite Mode of Dosage Compensation in Caenorhabditis Elegans

We report a genetic characterization of several essential components of the dosage compensation process in Caenorhabditis elegans. Mutations in the genes dpy-26, dpy-27, dpy-28, and the newly identified gene dpy-29 disrupt dosage compensation, resulting in elevated X-linked gene expression in XX animals and an incompletely penetrant maternal-effect XX-specific lethality. These dpy mutations appear to cause XX animals to express each set of X-linked genes at a level appropriate for XO animals. XO dpy animals are essentially wild type. Both the viability and the level of X-linked gene expression in XX animals carrying mutations in two or more dpy genes are the same as in animals carrying only a single mutation, consistent with the view that these genes act together in a single process (dosage compensation). To define a potential time of action for the gene dpd-28 we performed reciprocal temperature-shift experiments with a heat sensitive allele. The temperature-sensitive period for lethality begins 5 hr after fertilization at the 300-cell stage and extends to about 9 hr, a point well beyond the end of cell proliferation. This temperature-sensitive period suggests that dosage compensation is functioning in XX animals by mid-embryogenesis, when many zygotically transcribed genes are active. While mutations in the dpy genes have no effect on the sexual phenotype of otherwise wild-type XX or XO animals, they do have a slight feminizing effect on animals whose sex-determination process is already genetically perturbed. The opposite directions of the feminizing effects on sex determination and the masculinizing effects on dosage compensation caused by the dpy mutations are inconsistent with the wild-type dpy genes acting to coordinately control both processes. Instead, the feminizing effects are most likely an indirect consequence of disruptions in dosage compensation caused by the dpy mutations. Based on the cumulative evidence, the likely mechanism of dosage compensation in C. elegans involves reducing X-linked gene expression in XX animals to equal that in XO animals via the action of the dpy genes.     

Molecular Genetics of the Caenorhabditis Elegans Heterochronic Gene Lin-14

We describe a general strategy for the genetic mapping in parallel of multiple restriction fragment length polymorphism (RFLP) loci. This approach allows the systematic identification for cloning of physical genetic loci within about 100 kb of any gene in Caenorhabditis elegans. We have used this strategy of parallel RFLP mapping to clone the heterochronic gene lin-14, which controls the timing and sequence of many C. elegans postembryonic developmental events. We found that of about 400 polymorphic loci in the C. elegans genome associated with the Tc1 family of repetitive elements, six are within 0.3 map unit of lin-14. The three closest lin-14-linked Tc1-containing restriction fragments were cloned and used to identify by hybridization an 830-kb region of contiguous cloned DNA fragments assembled from cosmid and yeast artificial chromosome libraries. A lin-14 intragenic recombinant that separated a previously cryptic lin-14 semidominant mutation from a cis-acting lin-14 suppressor mutation was used to map the location of the lin-14 gene to a 25-kb region of this 830-kb contig. DNA probes from this region detected lin-14 allele-specific DNA alterations and a lin-14 mRNA. Two lin-14 semi-dominant alleles, which cause temporally inappropriate lin-14 gene activity and lead to the reiterated expression of specific early developmental events, were shown to delete sequences from the lin-14 gene and mRNA. These deletions may define cis-acting sequences responsible for the temporal regulation of lin-14.     

Identification of X Chromosome Regions in Caenorhabditis Elegans That Contain Sex-Determination Signal Elements

The primary sex-determination signal of Caenorhabditis elegans is the ratio of X chromosomes to sets of autosomes (X/A ratio). This signal coordinately controls both sex determination and X chromosome dosage compensation. To delineate regions of X that contain counted signal elements, we examined the effect on the X/A ratio of changing the dose of specific regions of X, using duplications in XO animals and deficiencies in XX animals. Based on the mutant phenotypes of genes that are controlled by the signal, we expected that increases (in males) or decreases (in hermaphrodites) in the dose of X chromosome elements could cause sex-specific lethality. We isolated duplications and deficiencies of specific X chromosome regions, using strategies that would permit their recovery regardless of whether they affect the signal. We identified a dose-sensitive region at the left end of X that contains X chromosome signal elements. XX hermaphrodites with only one dose of this region have sex determination and dosage compensation defects, and XO males with two doses are more severely affected and die. The hermaphrodite defects are suppressed by a downstream mutation that forces all animals into the XX mode of sex determination and dosage compensation. The male lethality is suppressed by mutations that force all animals into the XO mode of both processes. We were able to subdivide this region into three smaller regions, each of which contains at least one signal element. We propose that the X chromosome component of the sex-determination signal is the dose of a relatively small number of genes.     

Genes Affecting Sensitivity to Serotonin in Caenorhabditis Elegans

Regulating the response of a postsynaptic cell to neurotransmitter is an important mechanism for controlling synaptic strength, a process critical to learning. We have begun to define and characterize genes that may control sensitivity to the neurotransmitter serotonin in the nematode Caenorhabditis elegans by identifying serotonin-hypersensitive mutants. We reported previously that mutations in the gene unc-2, which encodes a putative calcium channel subunit, result in hypersensitivity to serotonin. Here we report that mutants defective in the unc-36 gene, which encodes a homologue of a calcium channel auxiliary subunit, are also serotonin-hypersensitive. Moreover, the unc-36 gene appears to be required in the same cells as unc-2 for control of the same behaviors. Mutations in several other genes, including unc-8, unc-10, unc-20, unc-35, unc-75, unc-77, and snt-1 also result in hypersensitivity to serotonin. Several of these mutations have previously been shown to confer resistance to acetylcholinesterase inhibitors, suggesting that they may affect acetylcholine release. Moreover, we found that mutations that decrease acetylcholine synthesis cause defective egg-laying and serotonin hypersensitivity. Thus, acetylcholine appears to negatively regulate the response to serotonin and may participate in the process of serotonin desensitization.     

Characterization of Caenorhabditis Elegans Lectin-Binding Mutants

We have identified 45 mutants of Caenorhabditis elegans that show ectopic surface binding of the lectins wheat germ agglutinin (WGA) and soybean agglutinin (SBA). These mutations are all recessive and define six genes: srf-2, srf-3, srf-4, srf-5, srf-8 and srf-9. Mutations in these genes fall into two phenotypic classes: srf-2, -3, -5 mutants are grossly wild-type, except for their lectin-binding phenotype; srf-4, -8, -9 mutants have a suite of defects, including uncoordinated movement, abnormal egg laying, and defective copulatory bursae morphogenesis. Characterization of these pleiotropic mutants at the cellular level reveals defects in the migration of the gonadal distal tip cell and in axon morphology. Unexpectedly, the pleiotropic mutations also interact with mutations in the lin-12 gene, which encodes a putative cell surface receptor involved in the control of cell fate. We propose that the underlying defect in the pleiotropic mutations may be in the general processing or secretion of extracellular proteins.     

The Caenorhabditis Elegans Sel-1 Gene, a Negative Regulator of Lin-12 and Glp-1, Encodes a Predicted Extracellular Protein

The Caenorhabditis elegans lin-12 and glp-1 genes encode members of the LIN-12/NOTCH family of receptors. The sel-1 gene was identified as an extragenic suppressor of a lin-12 hypomorphic mutant. We show in this report that the sel-1 null phenotype is wild type, except for an apparent elevation in lin-12 and glp-1 activity in sensitized genetic backgrounds, and that this genetic interaction seems to be lin-12 and glp-1 specific. We also find that sel-1 encodes a predicted extracellular protein, with a domain sharing sequence similarity to predicted proteins from humans and yeast. SEL-1 may interact with the LIN-12 and GLP-1 receptors and/or their respective ligands to down-regulate signaling.     

A Genetic Mosaic Screen of Essential Zygotic Genes in Caenorhabditis Elegans

We have devised a simple genetic mosaic screen, which circumvents the difficulties posed by phenotypic analysis of early lethal mutants, to analyze essential zygotic genes in Caenorhabditis elegans. The screen attempts to distinguish genes involved in cell type and/or lineage specific processes such as determination, differentiation or morphogenesis from genes involved in general processes such as intermediary metabolism by using the pattern of gene function to classify genes: genes required in one or a subset of early blastomeres may have specific functions, whereas genes required in all early blastomeres may have general functions. We found that 12 of 17 genes examined function in specific early blastomeres, suggesting that many zygotic genes contribute to specific early processes. We discuss the advantages and limitations of this screen, which is applicable to other regions of the C. elegans genome.     

Longevity-Determining Genes in Caenorhabditis Elegans: Chromosomal Mapping of Multiple Noninteractive Loci

We have used chromosome mapping with polymorphic markers to define genetic components governing life span in the nematode Caenorhabditis elegans. A complex recombinant-inbred population was derived from an interstrain cross, yielding >1000 genotypes, each a composite of homozygous segments from the two parental strains. Genotypes were analyzed for the last-surviving 1-5% of worms in aging cohorts, and for young controls, by multiplex polymerase chain reaction using polymorphic markers to distinguish the parental alleles. We identified five regions of the genome at which one parental allele was significantly enriched in long-lived subpopulations. At four of five loci, the same alleles were selected in aging cohorts maintained under two different conditions, implying that these genes determine life span in differing environments.     

Viable Maternal-Effect Mutations That Affect the Development of the Nematode Caenorhabditis Elegans

We carried out a genetic screen for viable maternal-effect mutants to identify genes with a critical function relatively early in development. This type of mutation would not have been identified readily in previous screens for viable mutants and therefore could define previously unidentified genes. We screened 30,000 genomes and identified 41 mutations falling into 24 complementation groups. We genetically mapped these 24 loci; only two of them appear to correspond to previously identified genes. We present a partial phenotypic characterization of the mutants and a quantitative analysis of the degree to which they can be maternally or zygotically rescued.     

Isolation, Characterization and Epistasis of Fluoride-Resistant Mutants of Caenorhabditis Elegans

We have isolated 13 fluoride-resistant mutants of the nematode Caenorhabditis elegans. All the mutations are recessive and mapped to five genes. Mutants in three of the genes (class 1 genes: flr-1 X, flr-3 IV, and flr-4 X) are resistant to 400 μg/ml Naf. Furthermore, they grow twice as slowly as and have smaller brood size than wild-type worms even in the absence of fluoride ion. In contrast, mutants in the other two genes (class 2 genes: flr-2 V and flr-5 V) are only partially resistant to 400 μg/ml NaF, and they have almost normal growth rates and brood sizes in the absence of fluoride ion. Studies on the phenotypes of double mutants showed that class 2 mutations are epistatic to class 1 mutations concerning growth rate and brood size but hypostatic with respect to fluoride resistance. We propose two models that can explain the epistasis. Since fluoride ion depletes calcium ion, inhibits some protein phosphatases and activates trimeric G-proteins, studies on these mutants may lead to discovery of a new signal transduction system that controls the growth of C. elegans.     

Identification of Mutations in Three Genes That Interact with Zeste in the Control of White Gene Expression in Drosophila Melanogaster

Three previously described genes, enhancer of yellow, 1, 2 and 3, are shown to cooperate with the zeste gene in the control of white gene expression. The mutations e(y)1(u1), e(y)3(u1), and to a lesser extent e(y)2(u1), enhance the effect of the zeste null allele z(v77h). Different combinations of e(y)1(u1), e(y)2(u1) and e(y)3(u1) mutations with several other z alleles also enhance the white mutant phenotype, but only to levels characteristic of white alleles containing a deletion of the upstream eye enhancer. Loss of zeste protein binding sites from the white locus does not eliminate the effect of e(y)1(u1) and e(y)3(u1) mutations, suggesting that the products of these genes interact with some other nucleotide sequences. Combinations of either e(y)1(u1) or e(y)2(u1) mutations with e(y)3(u1) are lethal. The products of these three genes may represent, together with zeste, a group of proteins involved in the organization of long-distance interactions between DNA sequences.     

A Cis-Acting Locus That Promotes Crossing over between X Chromosomes in Caenorhabditis Elegans

This study reports the characterization of a cis-acting locus on the Caenorhabditis elegans X chromosome that is crucial for promoting normal levels of crossing over specifically between the X homologs and for ensuring their proper disjunction at meiosis I. The function of this locus is disrupted by the mutation me8, which maps to the extreme left end of the X chromosome within the region previously implicated by studies of X;A translocations and X duplications to contain a meiotic pairing site. Hermaphrodites homozygous for a deletion of the locus (Df/Df) or heterozygous for a deletion and the me8 mutation (me8/Df) exhibit extremely high levels of X chromosome nondisjunction at the reductional division; this is correlated with a sharp decrease in crossing over between the X homologs as evidenced both by reductions in genetic map distances and by the presence of achiasmate chromosomes in cytological preparations of oocyte nuclei. Duplications of the wild-type region that are unlinked to the X chromosome cannot complement the recombination and disjunction defects in trans, indicating that this region must be present in cis to the X chromosome to ensure normal levels of crossing over and proper homolog disjunction. me8 homozygotes exhibit an altered distribution of crossovers along the X chromosome that suggests a defect in processivity along the X chromosome of an event that initiates at the chromosome end. Models are discussed in which the cis-acting locus deleted by the Dfs functions as a meiotic pairing center that recruits trans-acting factors onto the chromosomes to nucleate assembly of a crossover-competent complex between the X homologs. This pairing center might function in the process of homolog recognition, or in the initiation of homologous synapsis.     

Properties of a Class of Genes Required for Ray Morphogenesis in Caenorhabditis Elegans

We have identified eight mutations that define at least five terminal differentiation genes (ram genes) whose products are required during the extension of the male-specific ray sensilla in Caenorhabditis elegans. ram gene mutations result in morphological abnormalities in the sensory rays but do not appear to interfere with ray functions. A similar ray morphology phenotype was observed in males harboring mutations in three previously defined genes, dpy-11, dpy-18 and sqt-1, that also affect body shape. One of these genes, sqt-1, is known to encode a collagen. Mutations in different ram genes failed to complement, from which we infer that their gene products functionally interact. For one ram gene, failure to complement was shown to result from haploinsufficiency. Intergenic noncomplementation did not extend to the body morphology genes. The temperature-sensitive periods of both ram and body morphology mutations corresponded to the period of development in which ray extension occurs. We propose that ram gene products act together in a critical interaction between the rays and the cuticle required for wild-type ray morphology.     

Essential Genes in the Hdf6 Region of Chromosome I in Caenorhabditis Elegans

In this paper we describe the analysis of essential genes in the hDf6 region of chromosome I of Caenorhabditis elegans. Nineteen complementation groups have been identified which are required for the growth, survival or fertility of the organism (essential genes). Since ten of these genes were represented by more than one allele, a Poisson calculation predicts a minimum estimate of 25 essential genes in hDf6. The most mutable gene in this region was let-354 with seventeen alleles. An average mutation rate of 5 x 10(-5) mutations/gene/chromosome screened was calculated for an ethyl methanesulfonate dose of 15 mM. Mutations were recovered by screening for lethal mutations using the duplication sDp2 for recovery. Our analysis shows that duplications are very effective for maintenance and mapping of large numbers of lethal mutations. Approximately 600 lethal mutations were mapped in order to identify the 54 that are in the deficiency hDf6. The hDf6 region appears to have a lower proportion of early arresting mutations than other comparably sized regions of the genome.