Natural genetic variation as a tool for discovery in Caenorhabditis nematodes

Over the last 20 years, studies of Caenorhabditis elegans natural diversity have demonstrated the power of quantitative genetic approaches to reveal the evolutionary, ecological, and genetic factors that shape traits. These studies complement the use of the laboratory-adapted strain N2 and enable additional discoveries not possible using only one genetic background. In this chapter, we describe how to perform quantitative genetic studies in Caenorhabditis, with an emphasis on C. elegans. These approaches use correlations between genotype and phenotype across populations of genetically diverse individuals to discover the genetic causes of phenotypic variation. We present methods that use linkage, near-isogenic lines, association, and bulk-segregant mapping, and we describe the advantages and disadvantages of each approach. The power of C. elegans quantitative genetic mapping is best shown in the ability to connect phenotypic differences to specific genes and variants. We will present methods to narrow genomic regions to candidate genes and then tests to identify the gene or variant involved in a quantitative trait. The same features that make C. elegans a preeminent experimental model animal contribute to its exceptional value as a tool to understand natural phenotypic variation.     

Evolution of susceptibility to ingested double-stranded RNAs in Caenorhabditis nematodes

Background

The nematode Caenorhabditis elegans is able to take up external double-stranded RNAs (dsRNAs) and mount an RNA interference response, leading to the inactivation of specific gene expression. The uptake of ingested dsRNAs into intestinal cells has been shown to require the SID-2 transmembrane protein in C. elegans. By contrast, C. briggsae was shown to be naturally insensitive to ingested dsRNAs, yet could be rendered sensitive by transgenesis with the C. elegans sid-2 gene. Here we aimed to elucidate the evolution of the susceptibility to external RNAi in the Caenorhabditis genus.

Principal Findings

We study the sensitivity of many new species of Caenorhabditis to ingested dsRNAs matching a conserved actin gene sequence from the nematode Oscheius tipulae. We find ample variation in the Caenorhabditis genus in the ability to mount an RNAi response. We map this sensitivity onto a phylogenetic tree, and show that sensitivity or insensitivity have evolved convergently several times. We uncover several evolutionary losses in sensitivity, which may have occurred through distinct mechanisms. We could render C. remanei and C. briggsae sensitive to ingested dsRNAs by transgenesis of the Cel-sid-2 gene. We thus provide tools for RNA interference studies in these species. We also show that transgenesis by injection is possible in many Caenorhabditis species.

Conclusions

The ability of animals to take up dsRNAs or to respond to them by gene inactivation is under rapid evolution in the Caenorhabditis genus. This study provides a framework and tools to use RNA interference and transgenesis in various Caenorhabditis species for further comparative and evolutionary studies.     

WormScan: a technique for high-throughput phenotypic analysis of Caenorhabditis elegans

Background

There are four main phenotypes that are assessed in whole organism studies of Caenorhabditis elegans; mortality, movement, fecundity and size. Procedures have been developed that focus on the digital analysis of some, but not all of these phenotypes and may be limited by expense and limited throughput. We have developed WormScan, an automated image acquisition system that allows quantitative analysis of each of these four phenotypes on standard NGM plates seeded with E. coli. This system is very easy to implement and has the capacity to be used in high-throughput analysis.

Methodology/Principal Findings

Our system employs a readily available consumer grade flatbed scanner. The method uses light stimulus from the scanner rather than physical stimulus to induce movement. With two sequential scans it is possible to quantify the induced phototactic response. To demonstrate the utility of the method, we measured the phenotypic response of C. elegans to phosphine gas exposure. We found that stimulation of movement by the light of the scanner was equivalent to physical stimulation for the determination of mortality. WormScan also provided a quantitative assessment of health for the survivors. Habituation from light stimulation of continuous scans was similar to habituation caused by physical stimulus.

Conclusions/Significance

There are existing systems for the automated phenotypic data collection of C. elegans. The specific advantages of our method over existing systems are high-throughput assessment of a greater range of phenotypic endpoints including determination of mortality and quantification of the mobility of survivors. Our system is also inexpensive and very easy to implement. Even though we have focused on demonstrating the usefulness of WormScan in toxicology, it can be used in a wide range of additional C. elegans studies including lifespan determination, development, pathology and behavior. Moreover, we have even adapted the method to study other species of similar dimensions.     

hlh-12, a gene that is necessary and sufficient to promote migration of gonadal regulatory cells in Caenorhabditis elegans,evolved within the Caenorhabditis clade

Specialized cells of the somatic gonad primordium of nematodes play important roles in the final form and function of the mature gonad. Caenorhabditis elegans hermaphrodites are somatic females that have a two-armed, U-shaped gonad that connects to the vulva at the midbody. The outgrowth of each gonad arm from the somatic gonad primordium is led by two female distal tip cells (fDTCs), while the anchor cell (AC) remains stationary and central to coordinate uterine and vulval development. The bHLH protein HLH-2 and its dimerization partners LIN-32 and HLH-12 had previously been shown to be required for fDTC specification. Here, we show that ectopic expression of both HLH-12 and LIN-32 in cells with AC potential transiently transforms them into fDTC-like cells. Furthermore, hlh-12 was known to be required for the fDTCs to sustain gonad arm outgrowth. Here, we show that ectopic expression of HLH-12 in the normally stationary AC causes displacement from its normal position and that displacement likely results from activation of the leader program of fDTCs because it requires genes necessary for gonad arm outgrowth. Thus, HLH-12 is both necessary and sufficient to promote gonadal regulatory cell migration. As differences in female gonadal morphology of different nematode species reflect differences in the fate or migratory properties of the fDTCs or of the AC, we hypothesized that evolutionary changes in the expression of hlh-12 may underlie the evolution of such morphological diversity. However, we were unable to identify an hlh-12 ortholog outside of Caenorhabditis. Instead, by performing a comprehensive phylogenetic analysis of all Class II bHLH proteins in multiple nematode species, we found that hlh-12 evolved within the Caenorhabditis clade, possibly by duplicative transposition of hlh-10. Our analysis suggests that control of gene regulatory hierarchies for gonadogenesis can be remarkably plastic during evolution without adverse phenotypic consequence.     

Evolution of early embryogenesis in rhabditid nematodes

The cell-biological events that guide early-embryonic development occur with great precision within species but can be quite diverse across species. How these cellular processes evolve and which molecular components underlie evolutionary changes is poorly understood. To begin to address these questions, we systematically investigated early embryogenesis, from the one- to the four-cell embryo, in 34 nematode species related to C. elegans. We found 40 cell-biological characters that captured the phenotypic differences between these species. By tracing the evolutionary changes on a molecular phylogeny, we found that these characters evolved multiple times and independently of one another. Strikingly, all these phenotypes are mimicked by single-gene RNAi experiments in C. elegans. We use these comparisons to hypothesize the molecular mechanisms underlying the evolutionary changes. For example, we predict that a cell polarity module was altered during the evolution of the Protorhabditis group and show that PAR-1, a kinase localized asymmetrically in C. elegans early embryos, is symmetrically localized in the one-cell stage of Protorhabditis group species. Our genome-wide approach identifies candidate molecules—and thereby modules—associated with evolutionary changes in cell-biological phenotypes.     

Evolution of dnmt-2 and mbd-2-like genes in the free-living nematodes Pristionchus pacificus, Caenorhabditis elegans and Caenorhabditis briggsae

Whole genome sequencing of several metazoan model organisms provides a platform for studying genome evolution. How representative are the genomes of these model organisms for their respective phyla? Within nematodes, for example, the free-living soil nematode Caenorhabditis elegans is a highly derived species with unusual genomic characters, such as a reduced Hox cluster (Curr. Biol., 13, 37–40) and the absence of a Hedgehog signaling system. Here, we describe the recent loss of a DNA methyltransferase-2 gene (dnmt-2) in C.elegans. A dnmt-2-like gene is present in the satellite model organism Pristionchus pacificus, another free-living nematode that diverged from C.elegans 200–300 million years ago. In contrast, C.elegans, Caenorhabditis briggsae and P.pacificus all contain an mbd-2-like gene, which encodes another essential component of the methylation system of higher animals and fungi. Cel-mbd-2 is expressed throughout development and RNA interference (RNAi) experiments result in variable phenotypes. In contrast, Cbr-mbd-2 RNAi results in paralyzed larval or adult worms suggesting recent changes of gene function within the genus Caenorhabditis. We speculate that both genes were part of an ancestral DNA methylation system in nematodes and that gene loss and sequence divergence have abolished DNA methylation in C.elegans.     

Molecular population genetics and phenotypic sensitivity to ethanol for a globally diverse sample of the nematode Caenorhabditis briggsae

New genomic resources and genetic tools of the past few years have advanced the nematode genus Caenorhabditis as a model for comparative biology. However, understanding of natural genetic variation at molecular and phenotypic levels remains rudimentary for most species in this genus, and for C. briggsae in particular. Here we characterize phenotypic variation in C. briggsae’s sensitivity to the potentially important and variable environmental toxin, ethanol, for globally diverse strains. We also quantify nucleotide variation in a new sample of 32 strains from four continents, including small islands, and for the closest‐known relative of this species (C. sp. 9). We demonstrate that C. briggsae exhibits little heritable variation for the effects of ethanol on the norm of reaction for survival and reproduction. Moreover, C. briggsae does not differ significantly from C. elegans in our assays of its response to this substance that both species likely encounter regularly in habitats of rotting fruit and vegetation. However, we uncover drastically more molecular genetic variation than was known previously for this species, despite most strains, including all island strains, conforming to the broad biogeographic patterns described previously. Using patterns of sequence divergence between populations and between species, we estimate that the self‐fertilizing mode of reproduction by hermaphrodites in C. briggsae likely evolved sometime between 0.9 and 10 million generations ago. These insights into C. briggsae’s natural history and natural genetic variation greatly expand the potential of this organism as an emerging model for studies in molecular and quantitative genetics, the evolution of development, and ecological genetics.     

Functional constraint and divergence in the G protein family in <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis> and <Emphasis Type="Italic">Caenorhabditis briggsae</Emphasis>

Part of the challenge of the post-genomic world is to identify functional elements within the wide array of information generated by genome sequencing. Although cross-species comparisons and investigation of rates of sequence divergence are an efficient approach, the relationship between sequence divergence and functional conservation is not clear. Here, we use a comparative approach to examine questions of evolutionary rates and conserved function within the guanine nucleotide-binding protein (G protein) gene family in nematodes of the genus Caenorhabditis. In particular, we show that, in cases where the Caenorhabditis elegans ortholog shows a loss-of-function phenotype, G protein genes of C. elegans and Caenorhabditis briggsae diverge on average three times more slowly than G protein genes that do not exhibit any phenotype when mutated in C. elegans, suggesting that genes with loss of function phenotypes are subject to stronger selective constraints in relation to their function in both species. Our results also indicate that selection is as strong on G proteins involved in environmental perception as it is on those controlling other important processes. Finally, using phylogenetic footprinting, we identify a conserved non-coding motif present in multiple copies in the genomes of four species of Caenorhabditis. The presence of this motif in the same intron in the gpa-1 genes of C. elegans, C. briggsae and Caenorhabditis remanei suggests that it plays a role in the regulation of gpa-1, as well as other loci.Electronic Supplementary Material Supplementary material is available for this article at     

Several Grassland Soil Nematode Species Are Insensitive to RNA-Mediated Interference

Phenotypic analysis of defects caused by RNA mediated interference (RNAi) in Caenorhabditis elegans has proven to be a powerful tool for determining gene function. In this study we investigated the effectiveness of RNAi in four non-model grassland soil nematodes, Oscheius sp FVV-2., Rhabditis sp, Mesorhabditis sp., and Acrobeloides sp. In contrast to reference experiments performed using C. elegans and Caenorhabditis briggsae, feeding bacteria expressing dsRNA and injecting dsRNA into the gonad did not produce the expected RNAi knockdown phenotypes in any of the grassland nematodes. Quantitative reverse-transcribed PCR (qRT-PCR) assays did not detect a statistically significant reduction in the mRNA levels of endogenous genes targeted by RNAi in Oscheius sp., and Mesorhabditis sp. From these studies we conclude that due to low effectiveness and inconsistent reproducibility, RNAi knockdown phenotypes in non-Caenorhabditis nematodes should be interpreted cautiously.     

Mitochondrial DNA Sequence Divergence among Meloidogyne incognita,Romanomermis culicivorax,Ascaris suum,and Caenorhabditis elegans

Mitochondrial DNA sequences were obtained from the NADH dehydrogenase subunit 3 (ND3), large rRNA, and cytochrome b genes from Meloidogyne incognita and Romanomermis culicivorax. Both species show considerable genetic distance within these same genes when compared with Caenorhabditis elegans or Ascaris suum, two species previously analyzed. Caenorhabditis, Ascaris, and Meloidogyne were selected as representatives of three subclasses in the nematode class Secernentea: Rhabditia, Spiruria, and Diplogasteria, respectively. Romanomermis served as a representative out-group of the class Adenophorea. The divergence between the phytoparasitic lineage (represented by Meloidogyne) and the three other species is so great that virtually every variable position in these genes appears to have accumulated multiple mutations, obscuring the phylogenetic information obtainable from these comparisons. The 39 and 42% amino acid similarity between the M. incognita and C. elegans ND3 and cytochrome b coding sequences, respectively, are approximately the same as those of C. elegans-mouse comparisons for the same genes (26 and 44%). This discovery calls into question the feasibility of employing cloned C. elegans probes as reagents to isolate phytoparasitic nematode genes. The genetic distance between the phytoparasitic nematode lineage and C. elegans markedly contrasts with the 79% amino acid similarity between C. elegans and A. suum for the same sequences. The molecular data suggest that Caenorhabditis and Ascaris belong to the same subclass.     

Dissection of lin-11 enhancer regions in Caenorhabditis elegans and other nematodes

The Caenorhabditis elegans LIM homeobox gene lin-11 plays crucial roles in the morphogenesis of the reproductive system and differentiation of several neurons. The expression of lin-11 in different tissues is regulated by enhancer regions located upstream as well as within lin-11 introns. These regions are functionally separable suggesting that multiple regulatory inputs operate to control the spatiotemporal pattern of lin-11 expression. To further dissect apart the nature of lin-11 regulation we focused on three Caenorhabditis species C. briggsae, C. remanei, and C. brenneri that are substantially diverged from C. elegans but share almost identical vulval morphology. We show that, in these species, the 5′ region of lin-11 possesses conserved sequences to activate lin-11 expression in the reproductive system. Analysis of the in vivo role of these sequences in C. elegans has led to the identification of three functionally distinct enhancers for the vulva, VC neurons, and uterine π lineage cells. We found that the π enhancer is regulated by FOS homolog FOS-1 and LIN-12/Notch pathway effectors, LAG-1 (Su(H)/CBF1 family) and EGL-43 (EVI1 family). These results indicate that multiple factors cooperate to regulate π-specific expression of lin-11 and together with other findings suggest that the mechanism of lin-11 regulation by LIN-12/Notch signaling is evolutionarily conserved in Caenorhabditis species. Our work demonstrates that 4-way comparison is a powerful tool to study conserved mechanisms of gene regulation in C. elegans and other nematodes.     

Pervasive Divergence of Transcriptional Gene Regulation in Caenorhabditis Nematodes

Because there is considerable variation in gene expression even between closely related species, it is clear that gene regulatory mechanisms evolve relatively rapidly. Because primary sequence conservation is an unreliable proxy for functional conservation of cis-regulatory elements, their assessment must be carried out in vivo. We conducted a survey of cis-regulatory conservation between C. elegans and closely related species C. briggsae, C. remanei, C. brenneri, and C. japonica. We tested enhancers of eight genes from these species by introducing them into C. elegans and analyzing the expression patterns they drove. Our results support several notable conclusions. Most exogenous cis elements direct expression in the same cells as their C. elegans orthologs, confirming gross conservation of regulatory mechanisms. However, the majority of exogenous elements, when placed in C. elegans, also directed expression in cells outside endogenous patterns, suggesting functional divergence. Recurrent ectopic expression of different promoters in the same C. elegans cells may reflect biases in the directions in which expression patterns can evolve due to shared regulatory logic of coexpressed genes. The fact that, despite differences between individual genes, several patterns repeatedly emerged from our survey, encourages us to think that general rules governing regulatory evolution may exist and be discoverable.     

The Molecular and Structural Characterization of Two Vitellogenins from the Free-Living Nematode Oscheius tipulae

This paper describes the purification of yolk proteins, which are important for the reproduction of egg-laying animals, and the structural characterization of two vitellogenins, VT1 and OTI-VIT-6, of the nematode Oscheius tipulae. O. tipulae is an alternative model organism to its relative, the widely used Caenorhabditis elegans, and is a good model to understand reproduction in insect parasitic nematodes of the genus Heterorhabditis. The native purified O. tipulae vitellogenin is composed of three polypeptides (VT1, VT2 and VT3), whereas in C. elegans, vitellogenin is composed of four polypeptides. The gene (Oti-vit-1) encoding yolk polypeptide VT1 has been recently identified in the genome of O. tipulae. Immunoblotting and N-terminal sequencing confirmed that VT1 is indeed coded by Oti-vit-1. Utilizing the same experimental approaches, we showed that the polypeptides VT2 and VT3 are derived from the proteolytic processing of the C- and N-terminal portions of the precursor OTI-VIT-6, respectively. We also showed that the recombinant polypeptide (P40), corresponding to the N-terminal sequence of OTI-VIT-6, preferentially interacts with a 100-kDa polypeptide found in adult worm extracts, as we have previously shown for the native vitellins of O. tipulae. Using the putative nematode vitellogenin amino acid sequences available in the UniProtKB database, we constructed a phylogenetic tree and showed that the O. tipulae vitellogenins characterized in this study are orthologous to those of the Caenorhabditis spp. Together, these results represent the first structural and functional comparative study of nematode yolk proteins outside the Caenorhabditis genus and provide insight into the evolution of these lipoproteins within the Nematode Phylum.