The lin-3/let-23 pathway mediates inductive signalling during male spicule development in Caenorhabditis elegans

During Caenorhabditis elegans male spicule development, four pairs of precursor cells respond to multiple positional cues and establish a pattern of fates that correlates with relative anterior-posterior cell position. One of the extracellular cues is provided by the F and U cells, which promote anterior fates. We show that the genes in the lin-3/let-23 signalling pathway required for hermaphrodite vulval induction also mediate this F/U signal. Reduction-of-function mutations in lin-3, let-23, sem-5, let-60 or lin-45 disrupt the fate of anterior cells. Likewise, activation of the pathway with ubiquitously produced signal results in posterior cells inappropriately adopting the anterior fates even in the absence of F and U. We have further used this genetic pathway to begin to understand how multiple positional cues are integrated to specify cell fate. We demonstrate that lin-15 acts in spicule development as it does in vulval induction, as a negative regulator of let-23 receptor activity. A second extracellular cue, from Y.p, also acts antagonistically to the lin-3/let-23 pathway. However, this signal is apparently integrated into the lin-3/let-23 pathway at some step after lin-45 raf and is thus functionally distinct from lin-15. We have also investigated the role of lin-12 in forming the anterior/posterior pattern of fates. A lin-12 gain-of-function defect is masked by redundant positional information from F and U

Developmental patterning in the Caenorhabditis elegans hindgut

AbstractDevelopmental pattern formation allows cells within a tissue or organ to coordinate their development and establish cell types in relationship to one another. To better characterize the developmental patterning events within one organ, the C. elegans hindgut, we have analyzed the expression pattern of several genes using green fluorescent protein-based reporter transgenes. In wild-type animals, these genes are expressed in subsets of hindgut cells rather than in individual cell types. In mutant animals, we find that some, but not all, genes expressed in cells with altered development exhibit a corresponding alteration of gene expression. The results are consistent with a model where a combination of factors contribute to each cell's fate, and address how developmental information converges to specify cell types

Copulation defective mutants of C. elegans

To identify genes involved in male copulatory behavior, we carried out an F2 clonal screen in a him-5 mutant background. We identified 20 mutations that affect male mating behavior without causing gross defects in morphology. ​ Male mating in C. elegans comprises at least five steps (Liu and Sternberg, 1995). (l) The male responds to the hermaphrodite by backing his tail along the length of the hermaphrodite, (2) he turns over or under her body before reaching the head or tail, (3) he locates the vulva with his tail, at which point he stops backing, (4) he inserts his spicules into the vulva, and (5) he transfers sperm. To study the genetic basis for male mating behavior, we are isolating and characterizing Copulation Defective (Cod) mutations. We screened for mutant strains defective in this behavior using the screen described by Hodgkin (1983). him-5(e1490) worms are mutagenized with ethyl methane sulfonate (EMS); 20 P0 L4 hermaphrodites are picked singly to Petri plates; ten F1 worms are picked per mutagenized P0; and ten F2 L4 hermaphrodites are singled per P0 and their male progeny tested for mating efficiency via a qualitative mating test (six males crossed with six unc-52(e444) hermaphrodites, which are paralyzed at adulthood (Brenner, 1974). Mutants with phenotypes that are likely to reduce mating efficiency in a non-specific manner (such as those causing an Unc, Dpy, or Lon phenotype) were discarded. Those strains that appear morphologically normal under the dissecting microscope yet fail to mate or mate at a very low efficiency (1-5% cross progeny compared to wild type) were screened under Nomarski optics for defects in male reproductive structures. We screened over 3000 haploid genomes, and picked over 100 strains with reproduction defects. Nineteen strains were successfully backcrossed, which represents about 25% of the total strains attempted. This result suggests that most strains harbor two or more mutations that contribute to the mating-deficiency defect. Preliminary analysis of behavior suggests that every major step in the wild-type mating pathway has at least one corresponding Cod mutation blocking the behavior, with several mutations blocking at the spicule insertion step. The screen also yielded morphological mutants, whose phenotypes include crumpled spicules, abnormal rays, and a gonad migration defect; some of these will be described elsewhere (Chamberlin & Sternberg; micropublication in preparation)

A toolkit for rapid gene mapping in the nematode Caenorhabditis briggsae

<p>Abstract</p> <p>Background</p> <p>The nematode <it>C. briggsae </it>serves as a useful model organism for comparative analysis of developmental and behavioral processes. The amenability of <it>C. briggsae </it>to genetic manipulations and the availability of its genome sequence have prompted researchers to study evolutionary changes in gene function and signaling pathways. These studies rely on the availability of forward genetic tools such as mutants and mapping markers.</p> <p>Results</p> <p>We have computationally identified more than 30,000 polymorphisms (SNPs and indels) in <it>C. briggsae </it>strains AF16 and HK104. These include 1,363 SNPs that change restriction enzyme recognition sites (snip-SNPs) and 638 indels that range between 7 bp and 2 kb. We established bulk segregant and single animal-based PCR assay conditions and used these to test 107 polymorphisms. A total of 75 polymorphisms, consisting of 14 snip-SNPs and 61 indels, were experimentally confirmed with an overall success rate of 83%. The utility of polymorphisms in genetic studies was demonstrated by successful mapping of 12 mutations, including 5 that were localized to sub-chromosomal regions. Our mapping experiments have also revealed one case of a misassembled contig on chromosome 3.</p> <p>Conclusions</p> <p>We report a comprehensive set of polymorphisms in <it>C. briggsae </it>wild-type strains and demonstrate their use in mapping mutations. We also show that molecular markers can be useful tools to improve the <it>C. briggsae </it>genome sequence assembly. Our polymorphism resource promises to accelerate genetic and functional studies of <it>C. briggsae </it>genes.</p

Caenorhabditis briggsae Recombinant Inbred Line Genotypes Reveal Inter-Strain Incompatibility and the Evolution of Recombination

The nematode Caenorhabditis briggsae is an emerging model organism that allows evolutionary comparisons with C. elegans and exploration of its own unique biological attributes. To produce a high-resolution C. briggsae recombination map, recombinant inbred lines were generated from reciprocal crosses between two strains and genotyped at over 1,000 loci. A second set of recombinant inbred lines involving a third strain was also genotyped at lower resolution. The resulting recombination maps exhibit discrete domains of high and low recombination, as in C. elegans, indicating these are a general feature of Caenorhabditis species. The proportion of a chromosome's physical size occupied by the central, low-recombination domain is highly correlated between species. However, the C. briggsae intra-species comparison reveals striking variation in the distribution of recombination between domains. Hybrid lines made with the more divergent pair of strains also exhibit pervasive marker transmission ratio distortion, evidence of selection acting on hybrid genotypes. The strongest effect, on chromosome III, is explained by a developmental delay phenotype exhibited by some hybrid F2 animals. In addition, on chromosomes IV and V, cross direction-specific biases towards one parental genotype suggest the existence of cytonuclear epistatic interactions. These interactions are discussed in relation to surprising mitochondrial genome polymorphism in C. briggsae, evidence that the two strains diverged in allopatry, the potential for local adaptation, and the evolution of Dobzhansky-Muller incompatibilities. The genetic and genomic resources resulting from this work will support future efforts to understand inter-strain divergence as well as facilitate studies of gene function, natural variation, and the evolution of recombination in Caenorhabditis nematodes

Caenorhabditis briggsae Methods

Caenorhabditis briggsae is being developed in parallel to C. elegans as a model system, primarily for the study of evolution. Like C. elegans, C. briggsae is a protandrous hermaphrodite and like C. elegans, its genome has been sequenced. From this point, these two model systems diverge. The development, behavior, and physiology of C. elegans have been characterized through tens of thousands of genetic and molecular studies. Genetic and molecular characterizations of C. briggsae are relatively few. Experimental resources in C. elegans include a high density recombination map that is well integrated with the genome sequence. The C. briggsae recombination map has yet to be published and attempts to integrate it with the genome sequence are in their infancy. Despite these deficiencies, C. briggsae is attractive for several reasons. First as a parallel system, it can be used to test the generality of results obtained in C. elegans. Second, it appears that the structure of the C. briggsae world-wide population is qualitatively different from that of C. elegans and that C. briggsae may be more amenable to studies of gene flow, genome evolution, and speciation. Finally, C. briggsae and C. remanei are sister species with C. elegans as an outgroup, making the C. briggsae–C. remanei species better for some aspects of comparative genomics

Multiple regulatory changes contribute to the evolution of the Caenorhabditis lin-48 ovo gene

Recent work points to the importance of changes in gene expression patterns in species-specific differences. Here, we investigate the evolution of the nematode lin-48 ovo gene. lin-48 is expressed in several cells in both Caenorhabditis elegans and Caenorhabditis briggsae, but acts in the excretory duct cell only in C. elegans. We find the differences result both from alterations in the cis-regulatory sequences and in proteins that mediate lin-48 expression. One factor that contributes to the species differences is the bZip protein CES-2. Our results indicate the accumulation of several regulatory changes affecting one gene can contribute to evolutionary change

Caenorhabditis briggsae Methods

Caenorhabditis briggsae is being developed in parallel to C. elegans as a model system, primarily for the study of evolution. Like C. elegans, C. briggsae is a protandrous hermaphrodite and like C. elegans, its genome has been sequenced. From this point, these two model systems diverge. The development, behavior, and physiology of C. elegans have been characterized through tens of thousands of genetic and molecular studies. Genetic and molecular characterizations of C. briggsae are relatively few. Experimental resources in C. elegans include a high density recombination map that is well integrated with the genome sequence. The C. briggsae recombination map has yet to be published and attempts to integrate it with the genome sequence are in their infancy. Despite these deficiencies, C. briggsae is attractive for several reasons. First as a parallel system, it can be used to test the generality of results obtained in C. elegans. Second, it appears that the structure of the C. briggsae world-wide population is qualitatively different from that of C. elegans and that C. briggsae may be more amenable to studies of gene flow, genome evolution, and speciation. Finally, C. briggsae and C. remanei are sister species with C. elegans as an outgroup, making the C. briggsae–C. remanei species better for some aspects of comparative genomics