What has Caenorhabditis elegans ever done for us?

Abstract

The nematode Caenorhabditis elegans has many features that make it an excellent model for biological and medical research. This article discusses how the worm is facilitating drug discovery, reducing the number of mammals used in research and helping to cut research costs.

In your compost heap or on the fruit rotting at the bottom of your garden lives a tiny worm called Caenorhabditis elegans.¹ Since the worm's discovery by the French zoologist Émile Maupas in 1897, studies of C. elegans have transformed biological science and led to three Nobel prizes (see Box 1).¹ “This small worm is one of the most popular model organisms in biological studies,” says Dr Nadine Saul, a Biochemist at the Humboldt University of Berlin. “Biologists use C. elegans for, among others, pharmacological screenings, basic medical research, ecological studies and to uncover genetic principles.”

Box 1. From Bristol compost to Stockholm gold

Biologist Sidney Brenner discovered the strain of C. elegans originally used in the laboratory in compost collected from a Bristol mushroom farm.^1 His discovery led to three Nobel prizes and countless other biological advances. “Brenner was interested primarily in finding an organism that would allow him to study the molecular basis of animal development and behaviour. He saw the great potential of C. elegans due to its short life cycle, small size and ease of culturing in the laboratory without major concerns related to biosafety or space,” comments Professor De Rosa. “In addition, its anatomical simplicity, in particular in the nervous system, and its transparency allowing visualisation of the internal organs in living behaving animals made C. elegans an excellent animal for Brenner's goal of determining the role of each gene involved in neural development and function.” “Many of the genes and pathways important in apoptosis were discovered in C. elegans,” Professor Rayes adds. “For these discoveries, Brenner, Sulston and Horvitz were awarded the Nobel Prize in Physiology or Medicine in 2002. C. elegans allowed the development of a key tool in biomedical research, gene silencing by RNA interference,^5 which earned Fire and Mello the Nobel Prize in Physiology or Medicine in 2006. The transparency allowed expression of green fluorescent protein in a living organism. Chalfie, Shimomura and Tsien were awarded the 2008 Nobel Prize in Chemistry for this breakthrough. Obviously, I am being unfair to other great discoveries using C. elegans that I am not including.”

Box 2. The worm community

Professor De Rosa notes that researchers using C. elegans have a tradition of sharing ideas and reagents. “As a representative example, mutants generated in a particular laboratory are placed in an open depository. So, these animals are accessible for the rest of the labs,” she says. “Other examples are the access to free resources such as the WormBase, WormAtlas and WormBook where you can obtain information about, among other topics, previous research, essential methods and genetic data. All these features give a very simple message to community members and researchers more generally that ‘sharing is always better’.”

Studies of C. elegans, which is just 1–2mm long as an adult, led to several biological firsts.¹ In 1998, C. elegans became the first multicellular organism with a complete published genome sequence.² An adult hermaphrodite C. elegans expresses 302 neurones and 56 glial cells, which typically show the same cell-cell connections between individual worms.³ C. elegans remains the only organism in which researchers have established the complete neural wiring.⁴

“C. elegans also enabled the understanding of several fundamental biological processes, including many of the genes and pathways important in apoptosis [controlled cell death],” says Professor María José De Rosa from the Universidad Nacional del Sur, Argentina and an independent researcher at INIBIBB-CONICET, Buenos Aires. “C. elegans are transparent. So, it was possible to express, for the first time, green fluorescent protein [GFP] in a living organism.” GFP allows researchers to identify cells containing tagged recombinant DNA and to localise specific proteins.

Apart from apoptosis, research using C. elegans helped unravel the genetic basis of pathways involved in, for example, development, ageing and RNA-mediated interference.⁵ The worm was pivotal to the discovery of microRNAs, which regulate protein transcription in all organisms.⁶ More recently, C. elegans has become increasingly important in large-scale drug screening and as a model for conditions as diverse as cancer, Alzheimer's and Parkinson's diseases, diabetes, Duchenne muscular dystrophy and, perhaps surprisingly for an organism that lacks even an exoskeleton, osteogenesis imperfecta.^{6, 7}

Several features make C. elegans an attractive organism for laboratory studies. For example, the worm's procreative capacity puts rabbits to shame. C. elegans produces about 300 offspring during a 3.5-day reproductive cycle.³ Maintaining a colony is cheap and easy. Naturally C. elegans lives in environments rich in microbes. In the laboratory, C. elegans feeds on a mutant strain of Escherichia coli.¹ C. elegans lacks an exoskeleton. So, it's transparent, which makes viewing specific cells and structures relatively easy.⁴ A male C. elegans has 1048 somatic cells, a hermaphrodite 959. These cells are always in the same place and have the same role.³ “In addition, we can use C. elegans without ethical concerns and there are numerous mutant and transgenic strains,” adds Dr Saul.

C. elegans has five autosomes (non-sex chromosomes). Males have a single X chromosome. Hermaphrodites have two X chromosomes.^{1, 3} “The fact that C. elegans can reproduce by self-fertilisation of hermaphrodites (see Figure 1) or by crosses with males was a great advantage for genetic studies that until the worm's introduction into the laboratory could only be observed in some plants,” says Professor Diego Rayes also from the Universidad Nacional del Sur, Argentina and an independent researcher at INIBIBB-CONICET.

Figure 1

Hermaphroditic C. elegans can reproduce by self-fertilisation, facilitating genetic studies

“This feature of C. elegans makes it possible to maintain genetically identical strains and, at the same time, to carry out crosses. In addition, the fact that C. elegans exists as hermaphrodites allowed researchers to study many mutations of the nervous system, for example those that severely affect locomotion, which generally is not possible in organisms that need to move to copulate and reproduce,” Professor Rayes comments. “In total, C. elegans is the perfect organism to produce fast results and to perform large-scale screenings that wouldn't be possible in rodents, for instance,” Dr Saul adds.

Our genetic footprint

C. elegans is such a good model because our genomes carry the evolutionary footprints left since life emerged on Earth. Four billion years later, evolution still contributes to disease. DNA replication, transcription and translation emerged early in the history of life on Earth and form the basis for genetic diseases. Multicellularity laid cancer's foundation.⁸ Metazoans (multicellular animals) and some plants have components of the innate immune response. In turn, immune aberrations lead to autoimmune and inflammatory diseases.⁸ Indeed, mismatches between our genetic legacy and the environment, such as lifestyle, contribute to many common diseases including obesity, diabetes and heart disease.⁸

Life doesn't evolve by suddenly shedding a vestigial organ here or gaining a new tissue there. New anatomical structures seem to evolve by, in general, co-opting existing tissues, cells or pathways. So, many genes are pleiotropic. In other words, they influence several traits that seem on the surface unrelated.⁸ Genome-wide association studies (GWAS) linked numerous genetic risk factors shared between cancers and, for example, cardiometabolic, inflammatory, immune and hormonal conditions, and obesity.⁹

Some 83% of the C. elegans proteome (the proteins produced) is coded for by genes with a human homolog. In other words, the genes in humans and C. elegans share a common ancestor. About half of C. elegans’ protein-coding genes have functional counterparts (orthologs) in humans.⁴ Dr Saul notes that the exact proportion depends on the algorithm used to analyse the relationships. “Nevertheless, the risk of finding false negatives and false positives is quite high. In fact, C. elegans can have several distinct orthologs for one human gene,” Dr Saul adds. “So, after determining a gene of interest, the function needs to be verified in mammals, human cell cultures or both. We can also check if expression of the putative human ortholog in the worm rescues a certain phenotype in a C. elegans mutant with a defective or missing gene,” Dr Saul comments.

Using such approaches, researchers found that C. elegans shares several pathways (such as RTK/Ras, Wnt and Notch) that are involved in certain cancers and some other human diseases. C. elegans also uses some of the same signalling molecules as humans, including calcium, dopamine, transforming growth factor beta, steroids and insulin.^{1, 3, 10} Pathways controlling protein homeostasis are also highly conserved.⁴

Some genes, however, show antagonistic pleiotropy: they benefit one trait at the expense of another.⁸ The cardiometabolic gene HNF1B increases the risk of prostate cancer and reduces the risk of type 2 diabetes.⁹ The ‘trade-off theory of ageing’ holds that a gene variant that is beneficial for reproduction can cause age-specific decline in performance. As the detrimental trait does not reduce reproduction, the gene accumulates.

For instance, one single nucleotide polymorphism (SNP; rs2157719) in CDKN2A protects against glioma in early life. But in later life, the same SNP increases the risk of several diseases including type 2 diabetes, coronary heart disease, glaucoma and nasopharyngeal cancer.¹¹ C. elegans allows biogerontologists – scientists that study of the biology of ageing – to explore antagonistic pleiotropy and other factors that drive senescence.

The biology of ageing

“Biogerontologists particularly appreciate C. elegans because of its relatively high genetic concordance with humans in general and in terms of ageing-related human diseases specifically. C. elegans also shows numerous human-like ageing phenotypes, such as reduced locomotive abilities and muscle function, a decline in sensory and cognitive capacities, as well as weakened pathogenic and stress defences,” Dr Saul says. In the three days or so after the egg hatches, C. elegans undergoes four larval stages.³ In the laboratory, adult C. elegans lives for between 14 and 21 days. Some mutant worms can, however, live twice as long as the wild-type.³ Dr Saul and colleagues previously identified nine genes, grouped into two clusters, implicated in the ‘health span’ of C. elegans, which have homologues in humans.¹²

In a more recent study, the Humboldt University team modified expression of the genes using RNA interference and measured health-relevant phenotypes in C. elegans 3, 7 and 12 days after emerging into the adult stage – young, middle-aged and elderly worms respectively.¹⁰ “The most important features of health that decline during ageing belong to physiological, physical, cognitive or reproductive functions. Therefore, we tested for changes in stress [heat] resistance, to represent physiological function, and locomotion as marker of physical function. We found that downregulation of five of these genes in worms with advanced age negatively affected locomotion. Downregulating seven of the selected genes in moderately or advanced aged nematodes decreased stress resistance,” Dr Saul reports.

Four genes influenced both stress resistance and locomotor fitness. All seven genes have homologues in human pathways that are important for health including lipid metabolism, body fat deposition and dopamine synthesis.¹⁰ “This provides a further piece of the complex puzzle that is the molecular basis of ageing,” Dr Saul remarks.

GWAS provided another piece of the puzzle. Using GWAS, Dr Saul and colleagues detected several additional genes that are potentially important in maintaining health as we age. “We used RNA interference⁵ to downregulate the orthologs in C. elegans. We then measured several ageing-relevant phenotypes and changes in the transcriptome and metabolome. Some genes were of special interest. These data will be published in near future,” Dr Saul remarks.

Dr Saul suggests that it is too soon to consider whether drugs could influence senescence in humans based on these findings. “In our experiments, downregulation of the target genes resulted in accelerated ageing,” she says. “Thus, we need to prove first that activation or upregulation of the respective genes really results in the opposite, namely decelerated ageing. Furthermore, health is characterised by many more phenotypes than those two tested in our study. To exclude potential trade-off effects, we also need to test the impact of modifying gene expression on, at least, pathogenic defence, cognition, reproduction and growth. The final step should be verification in a mammalian model, before targeting the genes in humans.”

In the meantime, the Humboldt University team is assessing whether natural substances, such as polyphenols in olive oil⁷ and extracts from traditional Chinese medicine,¹³ slow down the ageing process in worms and, hopefully, in humans. The actions of polyphenols in olive oil could contribute to the benefits of the Mediterranean diet, for example. “The uncovering of the genetics of ageing is a complicated and long process,” Dr Saul says. “A lot of work remains to be done.”

For example, life is dynamic. “We frequently observe that treatments, such as drugs, natural extracts or RNA interference, can have beneficial health effects in young individuals but no or even negative effects in old ones, and vice versa,” Dr Saul adds.¹⁴ “On the molecular level, for instance, gene expression following a pathogenic infection can be completely opposing when comparing young and old individuals.”

In addition, the environment influences gene expression and phenotype. “A small change in the experimental protocol can have profound effects on the gene's expression and can lead to completely different responses and, consequently, different results and interpretations,” Dr Saul says. “Therefore, considering only one time point to measure gene expression or phenotypic changes gives very limited insights into the mode of action of a treatment. Unfortunately, due to the cost and time, most studies, especially those investigating gene expression, do not take dynamic changes into account. There is definitely a need for improvement.”

Drug screening

Since 2006, researchers have used C. elegans in large-scale drug screening.⁶ These endeavours gained pace in the last decade, says Professor Rayes. “In the last 10 years, we have made great progress in automating phenotypic studies in C. elegans that allow us to analyse a given characteristic, such as movement, lethality or development. These features, together with the availability of compound libraries, make C. elegans an ideal organism in which to perform the first preclinical tests in drug development,” he says.

“Pharmacological assays with accessible and automated equipment can be performed using the worm. So, we can study an extremely high number of drugs in a short time,” Professor Rayes adds. “These assays allow us to identify different effects, modes of action as well as toxicity of a panel of drugs in a quick, easy and economical way before using mammals.”

C. elegans is, for example, related closely to several parasitic roundworms that are responsible for a heavy burden of human and animal disease worldwide. Studies using C. elegans helped elucidate the mode of action of several anti-helminth medicines and can screen for new agents that overcome resistance to existing drugs, which is increasingly common.⁴

Given their close evolutionary relationship, using C. elegans to screen for anti-helminth drugs is relatively obvious. But some diseases modelled by C. elegans seem, at first sight, counterinitiative, such as osteogenesis imperfecta, liver disease and dementia. C. elegans is, for example, an invertebrate. So, using a nematode to study osteogenesis imperfecta is surprising. But C. elegans produces collagen, an important bone component. Some mutations to the collagen gene in C. elegans create a short stubby worm, aptly called dumpy. Reversing the dumpy phenotype could yield new drugs for skeletal disease.⁶

C. elegans lacks a liver. But researchers can genetically engineer C. elegans to express misfolded alpha1-antitrypsin in the worm's intestine, the same protein responsible for hepatic alpha-1 antitrypsin deficiency. This allows scientists to study drugs that may reduce accumulation of the abnormal protein.⁶

“C. elegans also lacks the genes coding for beta-amyloid or alpha-synuclein, which accumulate and cause neurodegeneration in Alzheimer's and Parkinson's disease, respectively,” adds Professor De Rosa. “Expression of these proteins in C. elegans causes progressive cellular lesions similar to those observed in mammalian models of these pathologies, resulting in locomotion defects, which are easily-scorable phenotypes.” In addition, genetically modifying C. elegans can help uncover drugs’ modes of action. For example, research using C. elegans helped clarify the mechanisms of action of clozapine, a pharmacologically and toxicologically enigmatic atypical antipsychotic.⁶

“These examples do not imply, by any means, that all diseases can be modelled in C. elegans. But they highlight the worm's potential in drug screening,” Professor Rayes adds. “C. elegans will not totally replace the use of vertebrate animals. Any hit found in C. elegans has to be validated in mammals before it can be considered a drug with the potential to enter clinical trials. Nevertheless, C. elegans can shorten drug development and save money.”

C. elegans isn't the only invertebrate with potential for high-throughput drug screening. The fruit fly Drosophila melanogaster is another mainstay of genetic research. Wnt and notch are important human signalling pathways involved in, for instance, cancer. Researchers named both signalling pathways after D. melanogaster mutants. One mutant was wingless (wnt), the other had a notch in the wing. “Genetic manipulation of D. melanogaster is straightforward, studies of phenotypes and behavioural assays are easy to perform,” Professor Rayes says. “In addition, D. melanogaster allows the study of social interactions.

“Drug discovery should include C. elegans and D. melanogaster, which could give a different perspective or provide a more accessible or reliable assay to answer a particular question. Promising results in a specific model need replication to confirm the universality of the finding,” Professor Rayes concludes. “Nevertheless, invertebrate models help to cut research costs and reduce the number of mammals used in research. In my opinion, incorporating invertebrates in the initial steps of drug discovery should be mandatory.” So, this humble worm writhing in your compost is set to make many more discoveries and, possibly, help researchers win more Nobel prizes.

Declaration of interests

Mark Greener is a full-time medical writer and journalist and, as such, regularly provides editorial and consultancy services to numerous pharmaceutical, biotechnology and device companies and their agencies. He has no shares or financial interests relevant to this article.