A Transparent Window into Biology: A Primer on Caenorhabditis elegans

Abstract

A little over 50 years ago, Sydney Brenner had the foresight to develop the nematode (round worm) Caenorhabditis elegans as a genetic model for understanding questions of developmental biology and neurobiology. Over time, research on C. elegans has expanded to explore a wealth of diverse areas in modern biology including studies of the basic functions and interactions of eukaryotic cells, host-parasite interactions, and evolution. C. elegans has also become an important organism in which to study processes that go awry in human diseases. This primer introduces the organism and the many features that make it an outstanding experimental system, including its small size, rapid life cycle, transparency, and well-annotated genome. We survey the basic anatomical features, common technical approaches, and important discoveries in C. elegans research. Key to studying C. elegans has been the ability to address biological problems genetically, using both forward and reverse genetics, both at the level of the entire organism and at the level of the single, identified cell. These possibilities make C. elegans useful not only in research laboratories, but also in the classroom where it can be used to excite students who actually can see what is happening inside live cells and tissues.

Keywords: C. elegans; Primer; nematodes; single-cell analysis; transparent genetic system.

Figure 1

Observing C. elegans. (A) Petri dishes sitting on the base of a dissecting stereomicroscope. Bacterial lawns are visible on the surface of the agar inside the dishes but the C. elegans are too small to be seen in this view. (B) C. elegans viewed through the dissecting microscope. The two adults are moving in this view. Tracks in the plate indicate where animals have traveled on the bacterial lawn. (C) An adult hermaphrodite is viewed in a compound microscope. In all pictures, anterior is to the left and ventral is on the bottom. C. elegans moves on either its left or right side; in this image the surface facing the viewer is the left side. Because the animals are transparent, one can see, from left to right on the ventral side, developing oocytes in the gonad (rectangular cells with a clear, circular nucleus inside) followed by the spermatheca (where oocytes are fertilized), and multiple embryos in the uterus. (D) Fluorescent image showing the nervous system labeled with a GFP reporter (sto-6::gfp). Photo credits: (C) Original (modified here): B. Goldstein; (D) J. Kratz.

Figure 2

Life cycle of C. elegans. Animals increase in size throughout the four larval stages, but individual sexes are not easily distinguished until the L4 stage. At the L4 stage, hermaphrodites have a tapered tail and the developing vulva (white arrowhead) can be seen as a clear half circle in the center of the ventral side. The males have a wider tail (black arrowhead) but no discernible fan at this stage. In adults, the two sexes can be distinguished by the wider girth and tapered tail of the hermaphrodite and slimmer girth and fan-shaped tail (black arrowhead) of the male. Oocytes can be fertilized by sperm from the hermaphrodite or sperm obtained from males through mating. The dauer larvae are skinnier than all of the other larval stages. Photographs were taken on Petri dishes (note the bacterial lawns in all but the dauer image). Bar, 0.1 mm.

Figure 3

C. elegans anatomy. Major anatomical features of a hermaphrodite (A) and male (B) viewed laterally. (A) The dorsal nerve cord (DNC) and ventral nerve cord (VNC) run along the entire length of the animal from the nerve ring. Two of the four quadrants of body wall muscles are shown. (B) The nervous system and muscles are omitted in this view, more clearly revealing the pharynx and intestine. (C) Cross-section through the anterior region of the C. elegans hermaphrodite (location marked with a black line in A) showing the four muscle quadrants surrounded by the epidermis and cuticle with the intestine and gonad housed within the pseudocoelomic cavity. Images are modified from those found at www.wormatlas.org (Altun et al. 2002–2015).

Figure 4

Anatomy and study of the C. elegans nervous system. (A) Diagram of the C. elegans nervous system identifying some major nerve bundles and ganglia. Major nerve tracts include the ventral nerve cord (VNC), dorsal nerve cord (DNC), and nerve ring. Major ganglia include the ring ganglia, retrovesicular ganglion (RVG), preanal ganglion (PAG), and dorsal-root ganglion (DRG). Image was produced using the OpenWorm browser utility (openworm.org). (B) Visualization of anterior sensory neurons and their neurite projections by expression of a GFP reporter transgene. The Y105E8A.5::GFP fusion transgene is expressed in amphid, OL, and IL sensory neurons of the head (R. Newbury and D. Moerman, Wormatlas; wormatlas.org). (C) Use of cameleon reporter transgene to detect calcium transients in the C. elegans pharynx. The animal carries a transgene with myo-2, a pharynx-specific myosin gene, fused to YC2.1, a calcium-sensitive fluorescent detector. False-color red in the pharyngeal bulb reflects real-time calcium releases in the cell of the living animal. Image was adapted from Kerr et al. (2000). (D) Electron microscopic section showing synapses. Collections of densely staining vesicles can be seen in neuron 1 at the point of synaptic connection to neurons 2 and 3 (arrows). Synaptic varicosities (V) that contain vesicles can be seen clustered around the active zone. DCV identifies a dense-core vesicle. Image is from D. Hall (Wormatlas; wormatlas.org). (E) Worm behavior on a bacterial plate. Left image shows the standard laboratory N2 strain foraging as individuals evenly dispersed across the bacterial food. Right image shows an npr-1 mutant strain foraging in grouped masses (sometimes called a “social” feeding phenotype). Image is from M. de Bono, taken from Schafer (2005).

Figure 5

C. elegans tissue morphology. (A) Cross-section of the outer layers of the animal showing muscle cells below the epidermis and cuticle viewed by transmission electron microscopy. (B) Single gonad arm dissected out of a hermaphrodite showing germ cell DNA (stained white). Meiosis begins in the region labeled “pachytene” (top right) and continues around the loop of the gonad until oocytes are formed. The stored sperm are located in the spermatheca of the gonad (bottom right). This image is a composite of three gonad arms and dashed lines represent regions not captured in the individual micrographs. (C) The anterior of the animal showing the mouth where food enters, the pharynx with its two bulbs, and the beginning of the intestine viewed with differential interference contrast (DIC). (D) A single body wall muscle cell with six muscle arms (marked with asterisks) extending to the ventral nerve cord (lateral view). The micrograph shows fluorescence from both muscle and neuronal GFP reporters [him-4p::MB::YFP (muscle), hmr-1b::DsRed2 (neuron), and unc-129nsp::DsRed2 (neuron)]. All images are modified from WormAtlas (www.wormatlas.org). Photo credits: (A) D. Hall, (B) J. Maciejowski and E. J. Hubbard, and (C and D) WormAtlas.

Figure 6

C. elegans mutant phenotypes. Wild-type animals (WT) are approximately 1 mm long with a smooth exterior, and they move in a sinusoidal pattern. Rolling (Rol) animals twist their body like a corkscrew and as a result often remain in the same region moving in a circular pattern. Dumpy (Dpy) animals are shorter than wild type. Multivulvae (Muv) hermaphrodites have protrusions along the ventral side (white arrowheads) where vulvae form but are not able to attach to the uterus. Strain sources: D. Eisenmann and A. Golden.

Figure 7

Caenorhabditis species in the animal kingdom. (A) Phylogenetic tree placing Caenorhabditis species (boxed in red) among metazoans based on sequence data from two ribosomal subunits, eight protein coding genes, and mitochondrial genomes. Image was modified from Bourlat et al. (2008). (B) Phylogenetic tree placing C. elegans (boxed in red) among named Caenorhabditis species grown in the laboratory. Species in red have hermaphrodites and males; species in blue have females and males. An ‘o’ denotes branches with low support. Image modified from Félix et al. (2014).