The microRNAs of Caenorhabditis elegans

The soil nematode, Caenorhabditis elegans, occupies a central place in the short history of microRNA (miRNA) research. The converse is also true: miRNAs have emerged as key regulatory components in the life cycle of the worm, as well as numerous other organisms. Since the landmark discovery in 1993 of the first miRNA gene, lin-4, several other miRNAs have been characterized in detail in C. elegans and shown to participate in diverse biological processes. Moreover, the worm has provided, by virtue of its ease of genetic manipulation and amenability to high-throughput methods, an ideal platform for elucidating many general and conserved aspects of miRNA biology, namely mechanisms of biogenesis, target recognition, gene silencing, and regulation thereof. In this review, we summarize both the contribution of miRNAs to C. elegans physiology and development, as well as the contribution of C. elegans research to our understanding of general features of miRNA biology.     

Whole-Genome Profiling of Mutagenesis in Caenorhabditis elegans

Deep sequencing offers an unprecedented view of an organism''s genome. We describe the spectrum of mutations induced by three commonly used mutagens: ethyl methanesulfonate (EMS), N-ethyl-N-nitrosourea (ENU), and ultraviolet trimethylpsoralen (UV/TMP) in the nematode Caenorhabditis elegans. Our analysis confirms the strong GC to AT transition bias of EMS. We found that ENU mainly produces A to T and T to A transversions, but also all possible transitions. We found no bias for any specific transition or transversion in the spectrum of UV/TMP-induced mutations. In 10 mutagenized strains we identified 2723 variants, of which 508 are expected to alter or disrupt gene function, including 21 nonsense mutations and 10 mutations predicted to affect mRNA splicing. This translates to an average of 50 informative mutations per strain. We also present evidence of genetic drift among laboratory wild-type strains derived from the Bristol N2 strain. We make several suggestions for best practice using massively parallel short read sequencing to ensure mutation detection.MUTAGENESIS and the screening for mutants have long been a key tool of the practicing geneticist. The early work of T. H. Morgan and his colleagues relied on recovery of spontaneous mutations, which was limiting for the study of inheritance due to their infrequent occurrence (Morganet al. 1922; also see Sturtevant 1965). The discovery by H. J. Muller and others that X rays cause mutations ushered in the era of inducing mutations (Muller 1927). There is a long history of studies on mutagen specificity, both in prokaryotes and in eukaryotes, and today many mutagens are utilized in a variety of model organisms. In this article we use whole-genome deep sequencing in the model organism Caenorhabditis elegans to explore the types and frequencies of mutations induced by various mutagens and to document the feasibility of global identification of mutations.The mutagenic properties of ethyl methanesulfonate (EMS) were first demonstrated using the T4 viral system (Loveless 1959). Soon after, Lewis and Bacher (1968) demonstrated how to administer EMS to Drosophila melanogaster to generate mutations, and later Sydney Brenner did the same for the nematode C. elegans (Brenner 1974). The now classic article by Coulondre and Miller (1977) demonstrated the types of nucleotide substitutions generated by EMS and confirmed earlier observations (Bautz and Freese 1960) concerning the strong bias for GC to AT transitions. Today, EMS is still the most powerful and popular mutagen used by researchers studying D. melanogaster and C. elegans. Purely on the basis of genetic inference, when used at a concentration of 50 mm, EMS is calculated to induce ∼20 function-affecting variant alleles in C. elegans strains derived using this mutagen (Greenwald and Horvitz 1982; Anderson 1995).The chemical N-ethyl-N-nitrosourea (ENU) has been used as a mutagen since the 1970s but came to prominence when it was demonstrated to be the most effective chemical mutagen in mice (Russell et al. 1979). Today it is still the chemical mutagen of choice for this organism (Anderson 2000; Acevedo-Arozena et al. 2008). ENU has also been used for C. elegans mutagenesis (De Stasio et al. 1997). Although it appears to have different biases with regard to gene targets and base changes relative to EMS, the background mutational load after ENU mutagenesis has not been fully characterized (De Stasio and Dorman 2001).The chemical 4,5′,8-trimethylpsoralen is a crosslinking agent that is activated by near ultraviolet light. Studies in Escherichia coli have shown that it causes both single-base changes and deletions (Piette et al. 1985; Sladek et al. 1989). C. elegans researchers became interested in the potential of ultraviolet trimethylpsoralen (UV/TMP) to generate deletions in worms after the first deletions in this organism were isolated using this mutagen (Yandell et al. 1994). UV/TMP is now a major reagent in the arsenal of the C. elegans knockout consortium laboratories (Barstead and Moerman 2006). As a tool for generating deletions in eukaryotes it is quite useful but, outside of studies on prokaryotes, little else is known about the spectrum of mutagenic effects caused by UV/TMP.Massively parallel short read sequencing technologies offer unprecedented opportunities to study the complete genetic complement of an individual organism (Hillier et al. 2008). For genetic model systems the impact of this technology extends to the identification and correlation of induced mutations with selected phenotypes (Sarin et al. 2008). Several of the technological and bioinformatic issues that arise with next generation sequencing have already been addressed for the nematode C. elegans (Hillier et al. 2008; Sarin et al. 2008; Shen et al. 2008; Rose et al. 2010). Still, it is not clear how deeply one must sequence to confidently identify a relevant variant allele in a target mutant strain. Also of importance are questions concerning mutagen choice and dosage as they relate to the rate of induction of new mutations and background mutational load. We have undertaken the following study on mutagenesis and mutation detection to establish the parameters necessary to exploit next generation sequencing technologies for C. elegans genetics. For the first time we offer a whole-genome direct measure of mutation spectrum and background load for EMS, ENU, and UV/TMP. Readers interested in whole-genome sequencing of EMS mutagenized strains in C. elegans should also see the accompanying article in this issue by Sarin et al. (2010). In our study we also measured the single-nucleotide variation among currently used wild-type strains. In addition, we measured sequence read depth of all sequence and coding sequence and from this we make a recommendation of average genome coverage to ensure the correct identification of the causative mutation. We also examined the issue of false positive and false negative calls and make recommendations to eliminate most false positives without losing bona fide mutations.     

Uncovering new functions for microRNAs in Caenorhabditis elegans

In the nematode Caenorhabditis elegans, microRNA (miRNA) regulation of development was first observed in the striking abnormalities of lin-4 and let-7 loss of function mutants. However, after these first two miRNA mutant phenotypes were described, progress on the identification of miRNA functions in worms slowed considerably. Recent advances reveal new functions for miRNAs in embryonic and larval development as well as in the regulation of lifespan and stress response. Results from a combination of?computational, biochemical, and genetic approaches have deepened our understanding of miRNA regulation of target mRNAs and support the hypothesis that miRNAs have an important role in ensuring the robustness of developmental and physiological pathways.     

A High-Throughput Method for the Analysis of Larval Developmental Phenotypes in Caenorhabditis elegans

Caenorhabditis elegans postembryonic development consists of four discrete larval stages separated by molts. Typically, the speed of progression through these larval stages is investigated by visual inspection of the molting process. Here, we describe an automated method to monitor the timing of these discrete phases of C. elegans maturation, from the first larval stage through adulthood, using bioluminescence. The method was validated with a lin-42 mutant strain that shows delayed development relative to wild-type animals and with a daf-2 mutant that shows an extended second larval stage. This new method is inherently high-throughput and will finally allow dissecting the molecular machinery governing the speed of the developmental clock, which has so far been hampered by the lack of a method suitable for genetic screens.     

A growing molecular toolbox for the functional analysis of microRNAs in Caenorhabditis elegans

With the growing number of microRNAs (miRNAs) being identified each year, more innovative molecular tools are required to efficiently characterize these small RNAs in living animal systems. Caenorhabditis elegans is a powerful model to study how miRNAs regulate gene expression and control diverse biological processes during development and in the adult. Genetic strategies such as large-scale miRNA deletion studies in nematodes have been used with limited success since the majority of miRNA genes do not exhibit phenotypes when individually mutated. Recent work has indicated that miRNAs function in complex regulatory networks with other small RNAs and protein-coding genes, and therefore the challenge will be to uncover these functional redundancies. The use of miRNA inhibitors such as synthetic antisense 2'-O-methyl oligoribonucleotides is emerging as a promising in vivo approach to dissect out the intricacies of miRNA regulation.     

Expression Profiling of MAP Kinase–Mediated Meiotic Progression in Caenorhabditis elegans

The LET-60 (Ras)/LIN-45 (Raf)/MPK-1 (MAP kinase) signaling pathway plays a key role in the development of multiple tissues in Caenorhabditis elegans. For the most part, the identities of the downstream genes that act as the ultimate effectors of MPK-1 signaling have remained elusive. A unique allele of mpk-1, ga111, displays a reversible, temperature-sensitive, tissue-specific defect in progression through meiotic prophase I. We performed gene expression profiling on mpk-1(ga111) animals to identify candidate downstream effectors of MPK-1 signaling in the germ line. This analysis delineated a cohort of genes whose expression requires MPK-1 signaling in germ cells in the pachytene stage of meiosis I. RNA in situ hybridization analysis shows that these genes are expressed in the germ line in an MPK-1-dependent manner and have a spatial expression pattern consistent with the location of activated MPK-1. We found that one MPK-1 signaling-responsive gene encoding a C₂H₂ zinc finger protein plays a role in meiotic chromosome segregation downstream of MPK-1. Additionally, discovery of genes responsive to MPK-1 signaling permitted us to order MPK-1 signaling relative to several events occurring in pachytene, including EFL-1/DPL-1 gene regulation and X chromosome reactivation. This study highlights the utility of applying global gene expression methods to investigate genes downstream of commonly used signaling pathways in vivo.     

Identification of Antifungal Compounds Active against Candida albicans Using an Improved High-Throughput Caenorhabditis elegans Assay

Candida albicans, the most common human pathogenic fungus, can establish a persistent lethal infection in the intestine of the microscopic nematode Caenorhabditis elegans. The C. elegans–C. albicans infection model was previously adapted to screen for antifungal compounds. Modifications to this screen have been made to facilitate a high-throughput assay including co-inoculation of nematodes with C. albicans and instrumentation allowing precise dispensing of worms into assay wells, eliminating two labor-intensive steps. This high-throughput method was utilized to screen a library of 3,228 compounds represented by 1,948 bioactive compounds and 1,280 small molecules derived via diversity-oriented synthesis. Nineteen compounds were identified that conferred an increase in C. elegans survival, including most known antifungal compounds within the chemical library. In addition to seven clinically used antifungal compounds, twelve compounds were identified which are not primarily used as antifungal agents, including three immunosuppressive drugs. This assay also allowed the assessment of the relative minimal inhibitory concentration, the effective concentration in vivo, and the toxicity of the compound in a single assay.     

The mir-51 family of microRNAs functions in diverse regulatory pathways in Caenorhabditis elegans

The mir-51 family of microRNAs (miRNAs) in C. elegans are part of the deeply conserved miR-99/100 family. While loss of all six family members (mir-51-56) in C. elegans results in embryonic lethality, loss of individual mir-51 family members results in a suppression of retarded developmental timing defects associated with the loss of alg-1. The mechanism of this suppression of developmental timing defects is unknown. To address this, we characterized the function of the mir-51 family in the developmental timing pathway. We performed genetic analysis and determined that mir-51 family members regulate the developmental timing pathway in the L2 stage upstream of hbl-1. Loss of the mir-51 family member, mir-52, suppressed retarded developmental timing defects associated with the loss of let-7 family members and lin-46. Enhancement of precocious defects was observed for mutations in lin-14, hbl-1, and mir-48(ve33), but not later acting developmental timing genes. Interestingly, mir-51 family members showed genetic interactions with additional miRNA-regulated pathways, which are regulated by the let-7 and mir-35 family miRNAs, lsy-6, miR-240/786, and miR-1. Loss of mir-52 likely does not suppress miRNA-regulated pathways through an increase in miRNA biogenesis or miRNA activity. We found no increase in the levels of four mature miRNAs, let-7, miR-58, miR-62 or miR-244, in mir-52 or mir-52/53/54/55/56 mutant worms. In addition, we observed no increase in the activity of ectopic lsy-6 in the repression of a downstream target in uterine cells in worms that lack mir-52. We propose that the mir-51 family functions broadly through the regulation of multiple targets, which have not yet been identified, in diverse regulatory pathways in C. elegans.     

Paramyosin of Caenorhabditis elegans

Paramyosin has been isolated from the nematode, Caenorhabditis elegans. Its identity has been established by a variety of criteria, including purification, molecular weight, immunological cross reactivity with known paramyosin and formation of characteristic paracrystals. The presence of paramyosin in both pharyngeal and body-wall musculature was shown by a technique that allows analysis by sodium dodecyl sulphate gels of the protein in a single worm. The possibility of defining the role of paramyosin in the structure and function of the invertebrate muscle through the isolation of mutants in this protein is discussed.     

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.     

Straightening Caenorhabditis elegans images

MOTIVATION: Caenorhabditis elegans, a roundworm found in soil, is a widely studied model organism with about 1000 cells in the adult. Producing high-resolution fluorescence images of C.elegans to reveal biological insights is becoming routine, motivating the development of advanced computational tools for analyzing the resulting image stacks. For example, worm bodies usually curve significantly in images. Thus one must 'straighten' the worms if they are to be compared under a canonical coordinate system. RESULTS: We develop a worm straightening algorithm (WSA) that restacks cutting planes orthogonal to a 'backbone' that models the anterior-posterior axis of the worm. We formulate the backbone as a parametric cubic spline defined by a series of control points. We develop two methods for automatically determining the locations of the control points. Our experimental methods show that our approaches effectively straighten both 2D and 3D worm images.