Fernando Calahorro, Anna Crisford, James Dillon, Lindy Holden-Dye, Vincent O’Connor & Robert J Walker
University of Southampton, UK

https://doi.org/10.36866/pn.99.16

Forward genetics, an approach which maps and characterises genetic mutations that confer specific phenotypes, is one of the most powerful approaches in biology providing new, often unexpected, insights into physiological processes. It has been deployed to great effect in the nematode worm Caenorhabditis elegans. Here we introduce the ‘worm’ to the uninitiated and, focussing on forward genetics, describe a few of our favourite studies that have supported advances in physiology.

The ‘worm’ is a nematode

For those working with C. elegans it is often referred to endearingly as the ‘worm’. This can cause some confusion as to many people ‘worm’ means an earthworm – as exemplified by our marketing department which supplied a photo of an earthworm dangling from a fisherman’s hook to illustrate our research in Caenorhabditis elegans! To be clear, C. elegans is a nematode; it is much simpler than an earthworm: An earthworm has thousands of cells and ~10, 000 neurones but C. elegans (adult hermaphrodite) has precisely 959 somatic cells of which 302 are neurones. The connectivity between neuronal cells was beautifully described and mapped by John White and colleagues in their paper with the running title ‘Mind of a Worm’. This was the first ever, and still is, the only complete connectome for any animal.

C. elegans belongs to the phylum Nematoda- all of the organisms within this phylum are round worms like C. elegans and have the same overall body plan but they have a variety of lifestyles, some are free-living bacteria-eating worms like C. elegans, others are parasitic and infect humans, livestock, pets and even crops. Their sizes span an amazing range; C. elegans is just 0.08 mm wide and 1 mm long but the largest known nematode, a parasite of the sperm whale, Placentonema gigantissima, is the width of a garden hose and the length of a bus. The dimensions of the latter raise an interesting physiological dilemma in terms of long-distance neural signalling given that nematodes are not known to be able to generate fast Na⁺-dependent action potentials!

A 1960s debut for the worm

C. elegans was the inspired model organism of choice of Sydney Brenner in the 1960s. He pioneered its use as a genetic model for biology and his early emphasis on developmental and neurobiological processes quickly drew others into the fold. Over the next couple of decades C. elegans established itself as an exceptionally powerful animal in which to address fundamental questions in biology. A world-wide community of C. elegans researchers has continued to grow over the years, characterised by a willingness to share ideas and resources exemplified by the informal publication The Worm Breeder’s Gazette and co-ordinated cataloguing of a wealth of resources available for the investigation of this animal (Box 1). The worm has had an added significance by inspiring ground-breaking technical and conceptual advances most notably the discovery of RNAi and the development of green fluorescent protein as experimental tools (Figure 1A).

Worm watching catches on

Watching worms down a binocular microscope might not seem like a very sophisticated scientific pursuit but this has been at the forefront of some major discoveries in biology. Worms develop and behave in a very stereotyped manner which means that a skilled worm watcher can readily detect aberrance. Not only that, for an animal with just 302 neurones their behaviour might surprise some in terms of its refinement. In addition to basic behaviours, eating, moving and mating they can make simple associations between environmental cues and show classic conditioning and aversive learning. For example, they will migrate on a thermal gradient to a specific temperature they have learnt to associate with food. They can also learn to avoid eating bacteria that made them feel ‘sick’. Recent papers have even investigated ‘decision-making’ in C. elegans. All these endeavours were set in train by Sydney Brenner who first demonstrated in 1974 that exposure of worms to a chemical mutagen enables one to generate and propagate multiple behavioural mutants within the space of just a few weeks. These mutants were named through a three letter code according to the aberrant phenotype, hence ‘unc’ for uncoordinated, ‘egl’ for egg-laying, eat for feeding etc. Combining this with techniques for genetic mapping, transgenesis which permits cell specific expression of genes of interest and importantly the provision of the complete genome sequence in 1998 (Box 1), the first for any animal, ultimately led the way to decoding the function of specific genes with key biological roles. This is what is meant by forward genetics; it has proven to be an exceptionally important and unbiased approach to liberate meaningful information from the genome. There have been many successful applications of this for physiology (Figure 1B). Below are a few examples of work using the worms where this has been used to best effect.

What makes a worm wriggle?

The physiological depiction of vesicle-mediated neurotransmitter release onto cognate postsynaptic receptors made Sir Bernard Katz a Nobel ‘vordenker’ (1970) and established a field that focussed on a molecular explanation of synaptic transmission. The studies that allowed identification of synaptic proteins were largely biochemical in nature. Forward genetics in C. elegans supplemented this revolution in ‘molecular physiology’ by providing a comprehensive supply of individual genes including many uncs that were suspected of an essential role in neurotransmission because the mutant worms had an abnormal wriggle. An important part of the C. elegans efforts was the identification of molecular components defining the transmitter release site or active zone. Insight into this process was provided by the C. elegans mutant unc-13 which was first characterised to encode a novel class of phorbol ester receptor by Ahmed et al. in 1992. This was picked up by Nils Brose and colleagues who recognised that unc-13’s domain structure, harbouring Ca²⁺ -sensing and phorbol ester binding that predicted lipid-dependent membrane association, was highly suggestive of a role in regulated neurotransmitter release. They cloned and functionally characterised the corresponding mammalian gene family, for which the term munc-13 was coined, and showed it has a fundamental role in vesicle priming and defining the substructure of the active zone (Brose et al. 1995); it was among the first of the truly active zone proteins for the mammalian nervous system . Thus, an awkwardly moving worm prompted mammalian studies that led to the identification of an evolutionary conserved gene family pivotal in synaptic physiology.

Social worms

Mario de Bono noted that different isolates of C. elegans would either feed in clumps of worms together, so-called ‘social’ feeding whilst other isolates, including the N2 strain used as the wild-type by most C. elegans research groups, is a ‘solitary’ feeder. (Some comment has been passed on the fact that one of the social strains derives from Australia, compared to the less social N2 which hails from a Bristol compost heap.) This natural variation in behaviour is due to a single residue difference in a neuropeptide-Y like receptor, npr-1 (de Bono et al. 1998). How does this determine whether a worm is social or solitary? Chasing this question is providing a systems level interpretation of how the worm’s nervous system can integrate multiple environmental cues to bring about flexible behaviour. Important here has been an understanding of the worm’s natural environment, rotting fruit, and the cues it has evolved to respond to especially the relative concentrations of O₂ and CO₂. The excitement is that these sensors which detect molecular gases are embedded in a simple nervous system. The behavioural consequence of this sensing can be quantified in the whole worm by tracking changes in locomotion and scoring aggregation. Dovetailed with cell specific rescue approaches, this is mapping out the individual cells and microcircuits that underpin the behavioural responses to these environmentally salient gaseous cues (Busch et al. 2012). The ability to visualize the activity of these networks using genetically encoded Ca2+ sensors in the intact behaving worm opens a window to the dynamics of these networks. In addition to defining novel sensors of key physiological cues these paradigms, which began with the identification of npr-1 through a forward genetic approach, provide a whole organism view of how a changing environment determines behaviour. It is driving our conceptual understanding regarding the true nature of physiological control as the interface between molecules and behaviour.

Lurching worms

Villu Maricq’s group were led into previously uncharted territory by a specific application of forward genetics, suppressor screens. When a worm is on a bacterial lawn it exhibits a behaviour, called dwelling, in which it frequently changes between backwards and forwards movement so that it stays in pretty much the same location on the food patch to eat. This behaviour is regulated by a C. elegans glutamate receptor, GLR-1 which is an orthologue of mammalian AMPA/Kainate receptors. In a clever piece of model hopping, Maricq’s lab introduced a gain-of-function mutation into C. elegans glr-1 that was known to confer constitutive channel activity in the mouse receptor, originally identified in a mouse locomotor mutant called ‘lurcher’. The worms expressing the gain-of-function channel showed an extremely high frequency of reversals. By subjecting the ‘lurcher’ worms to chemical mutagenesis it was possible to identify mutations in other genes that suppressed this behaviour, so-called suppressor-of-lurcher, or sol genes. sol-1 and sol-2 were found to encode auxiliary AMPA receptor subunits and are important in regulating receptor mediated currents. These studies are helping to elucidate the constituents and dynamics of evolutionary conserved multi-molecular complexes that co-assemble with AMPA receptors to regulate their function. The beauty of this approach is that it has allowed identification of proteins in the GLR-1 signal transduction cascade with no a priori assumption about their identity (Wang et al. 2012).

C. elegans lives for just two to three weeks so finding mutants that can live longer and healthier can be achieved in a much shorter period of time than for other genetic animal models of ageing. As it grows older the worm becomes progressively slower and more ‘wrinkly’. Intriguingly, worms that are about to die show a wave of intense blue autofluorescence propagating from the anterior to the posterior end of the gut. This is a biomarker of calcium-dependent necrosis and is a precise predictor of their forthcoming death (Coburn et al. 2013). The first investigations of the genetics of C. elegans ageing, by Klass and Johnson in the late 1980s, were apparently greeted with some surprise when it was shown that it is possible to extend lifespan through mutation in a single gene, age-1. It was counterintuitive to the view of the day that such a complex phenomenon could be manipulated in this way. The gene age-1 was subsequently shown to lie in a signalling pathway involving the insulin-like growth factor receptor daf-2. Mutations of genes in this pathway have shown that it is possible to extend C. elegans life span more than five-fold. Using epistasis analysis the worm has also provided insight into the organisation of the signalling cascade. Importantly, mechanisms for life-span extension are conserved in mouse and human with key roles for insulin-like growth factor signalling, dietary restriction and even exercise (yes, you can make a worm do exercise!). Not only is the lifespan increased but many of these long-lived worms often appear to remain healthy for longer. Nonetheless, the longest-lived worm at Southampton which notched up 107 days under the careful husbandry of Neil Hopper (Hopper, 2006), equivalent to 400 human years, earned the name ‘Steptoe’ as in latter-life it was rather decrepit.

The attraction of the worm is that it provides the chance to answer core questions in physiology even when the related biology is complex and poorly understood. The world-wide community of worm researchers, first instigated by Sydney Brenner, have a wealth of resources at hand which are carefully catalogued and readily available (you can order your mutant of interest and receive it in the post for $7!). Clever use of forward genetics and model hopping between the worm and mammalian systems has greatly facilitated answering important fundamental physiological questions. We have reported on but a few of the >25,000 papers and the brilliant minds that have been hooked by C. elegans. The quality and resonance of the overall outputs indicates fishing in biology with these worms is actually very smart – the Mad Men in our advertising department were clearly onto something after all!

References

Brose N, Hofmann K, Hata Y, Südhof TC (1995). Mammalian homologues of Caenorhabditis elegans unc-13 gene define novel family of C2-domain proteins. J Biol Chem 270, 25273-25280

Busch KE, Laurent P, Soltesz Z, Murphy RJ, Faivre O, Hedwig B, et al. (2012). Tonic signaling from O₂ sensors sets neural circuit activity and behavioral state. Nat Neurosci 15, 581-591

Coburn C, Allman E, Mahanti P, Benedetto A, Cabreiro F, Pincus Z, et al. (2013). Anthranilate fluorescence marks a calcium-propagated necrotic wave that promotes organismal death in C. elegans. PLoS Biol 11, e1001613

de Bono M, Bargmann CI (1998). Natural variation in a neuropeptide Y receptor homolog modifies social behavior and food response in C. elegans.
Cell 94, 679-689

Hopper NA (2006) The adaptor protein soc-1/Gab1 modifies growth factor receptor output in Caenorhabditis elegans. Genetics 173, 163-175

Wang R, Mellem Jerry E, Jensen M, Brockie Penelope J, Walker Craig S, Hoerndli Frédéric J, et al. (2012). The SOL-2/Neto auxiliary protein modulates the function of AMPA-subtype ionotropic glutamate receptors. Neuron 75, 838-850

Worm facts & resources

The evolutionary distance between C. elegans and humans is 500-600 million years, Pevzner and Tesler; between mouse and human is ~65 million years, O’Leary et al.

Sydney Brenner, (1974) ‘The genetics of Caenorhabditis elegans’. Sydney Brenner established the roundworm Caenorhabditis elegans as a model organism for the investigation of developmental biology.

John Sulston, (1977-1983)  ‘The embryonic cell lineage of the nematode Caenorhabditis elegans’; John Sulston et al. 1983.

John G. White, (1986)  ‘The structure of the nervous system of the nematode Caenorhabditis elegans’. Complete wiring diagram (connectome) for a nervous system. www.wormatlas.org is a clickable map.

Martin Chalfie, (1992)  ‘Green fluorescent protein as a marker for gene expression’. Reporter constructs and cell specific expression.

Expression Pattern data base; Ian Hope. The Hope Laboratory; Expression Patterns for C. elegans promoter::GFP fusions. www.gfpweb.aecom.yu.edu

Caenorhabditis elegans knockout Consortiums. Caenorhabditis Genetics Center (CGC) (University of Minesota); National Bioresource Project (Tokyo Women’s Medical University School of Medicine). www.cgc.cbs.umn.edu ; www.shigen.nig.ac.jp/c.elegans

Andrew Fire and Craig C. Mello, (1998)  ‘Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans’. RNAi technique.

1998  Completion of the C. elegans genome sequence, the first for any animal. Although the C. elegans genome is about 1/30 the size of the human genome it encodes only slightly fewer proteins, approximately 22,000.  www.wormbase.org

2000  ‘A role for Caenorhabditis elegans in understanding the function and interactions of human disease genes’.  ~42% of human disease genes have an orthologue in C. elegans; David B. Sattelle.

2004  ‘A map of the interactome network of the metazoan C. elegans’; Marc Vidal.

2005  ‘Light activation of channelrhodopsin-2 in excitable cells of Caenorhabditis elegans triggers rapid behavioural responses’. Use of channel rhodopsin; Alex Gottschalk.

2013  ‘Heritable genome editing in C. elegans via a CRISPR-Cas9 system’. CRISPR technique.

2013  ‘The million mutation project: a new approach to genetics in Caenorhabditis’ ; Robert H Waterston.