Caenorhabditis

 Caenorhabditis is a genus of primarily free-living soil nematodes. The free-living species Caenorhabditis elegans has become well known after being chosen by Sydney Brenner in the 1960s for a concerted genetic, ultrastructural, and molecular analysis of animal development (1). A few other Caenorhabditis species have been characterized in less detail, largely for comparison, but the findings summarized in this article are for C. elegans unless otherwise indicated.

C. elegans was chosen for a variety of features, including its short life cycle and hermaphroditic mode of development (facilitating genetic analysis), its anatomical and cellular simplicity (<1000 somatic cells in the adult hermaphrodite), and its transparency and small size (facilitating light microscopy of developing animals and ultrastructural analysis, respectively). Its genome size has turned out to be relatively small as well (100 Mb), making molecular biology convenient. An entire field of C. elegans biology has grown out of Brenner's original investigations. As a result of this effort and the relative simplicity of “the worm,” as it is sometimes referred to, C. elegans is now the most completely understood metazoan in terms of genetics, ultrastructure, development, and physiology, and it has taken its place with the fruit fly Drosophila and the laboratory mouse as one of the most important model organisms for research biology (2, 3). Its entire cell lineage from zygote to adult has been determined; its ultrastructure including the complete connectivity of its nervous system has been described in detail; an extensive genetic map has been assembled; and DNA sequencing of its entire genome will be completed in 1998. It is beautifully suited for the genetic approach: identifying genes required for any biological process of interest by mutations that affect it, molecularly characterizing these genes after isolation by positional cloning, and eventually studying the biochemical functions of the corresponding gene products. As in all organisms, the genetic control of development and physiology is still only beginning to be understood in C. elegans. The rapidly accumulating detailed knowledge of the worm's biology, as well as the recent realization that many developmental and physiological mechanisms are remarkably conserved throughout the animal

kingdom, make C. elegans a powerful experimental system for investigating a variety of the most important questions in current biological research.

1. General Description

C. elegans inhabits soil in most parts of the world and feeds on soil bacteria. The adults are about 1 mm in length, and the two sexes, hermaphrodites and males, are morphologically distinguishable (Fig. 1). The hermaphrodites produce both eggs and sperm and can self-fertilize. Males can mate with hermaphrodites to give cross progeny; hermaphrodites cannot fertilize each other. A hermaphrodite can produce about 300 self progeny, in addition to cross progeny if mated. Embryos begin development in the hermaphrodite uterus and are laid at the gastrulation stage. They hatch as first-stage juveniles (L1 larvae), which as in all nematodes grow through three subsequent larval stages punctuated by molts, before a final molt to the sexually mature adult. The adult reproductive period lasts for about 4 days, after which the animals live for an additional 2 weeks. 

Figure 1. Anatomy of hermaphrodite and male C. elegans adults. Major structural features mentioned in the text are label Hermaphrodite. (b) Male. (c) Hermaphrodite, bright-field photomicrograph. Scale bar 0.1 mm. P, pharynx; I, intestine; O vulva; DG, distal gonad. (d) Hermaphrodite, cross section through the anterior, viewed toward the anterior. d, v, l, r, Drosophila  muscle; h, hypodermis; I, intestine; g, gonad; pc, pseudocoelom; nc, nerve cord. Note the treads (alae) on the left and right .

C. elegans is one of the simplest metazoans and has fixed numbers of somatic cells: 959 in the adult hermaphrodite and 1031 in the adult male. In the laboratory it can be cultured conveniently on agar plates spread with Escherichia coli bacteria, or in liquid culture when larger quantities are desired for biochemical work. Individual animals can be observed and transferred from plate to plate with a platinum wire “worm pick” under a dissecting microscope, which is sufficient for scoring many

mutant phenotypes. Taking advantage of the animal's transparency throughout the life cycle, it is possible to follow individual cells during development at higher magnification using a compound microscope, preferably equipped with Nomarski differential interference-contrast optics.

2. Genetics and the Genome

 C. elegans is diploid, with five autosomes (I–V) and a sex chromosome (X). Hermaphrodites are XX and males XO; sex is determined by the X chromosome to autosome ratio. Males arise spontaneously in self-fertilizing hermaphrodite populations at a frequency of about 0.2%, as the result of X chromosome nondisjunction.

The haploid genome size of C. elegans is 100 Mb, about 10 times that of yeast, one-half that of Drosophila, and one-thirtieth that of mammals. Based on frequency of predicted coding units in the genomic DNA sequence, which is now nearly complete, the estimated total number of functional genes is about 13,000. Over 1000 genetic loci have been identified by mutation following chemical mutagenesis and mapped to the six linkage groups. Positional cloning of mutationally identified genes is facilitated by an extensive physical map of overlapping clones, which is anchored to the genetic map at many locations. The DNA sequence of the entire genome will soon be completely known.

3. Anatomy

C. elegans has the typical nematode body plan: an outer tube of hypodermis with attached musculature and neurons, surrounding an interior space (the pseudocoelom), which contains the gut and, in adults, the gonad (see Fig. 1). The hypodermis secretes a three-layered, collagenous cuticle that is shed and replaced at each larval molt. Four bands of body-wall muscles run the length of the animal. The neuromuscular system bends the body only in the dorsal–ventral direction, so that the animal always lies on one side or the other on a solid surface. Specialized hypodermal cells on each side secrete “treads,” known as alae, on which the worm moves. The digestive system consists of a muscular pharynx in the head, which crushes bacteria and pumps them into the intestine, which empties at the anus near the tail. An excretory system controls the hydrostatic pressure on which the worm depends to maintain its body shape.

 The entire nervous system of the hermaphrodite consists of only 302 neurons and 56 supporting and glial cells. The major ganglion is the nerve ring at the base of the pharynx, which sends processes down ventral and dorsal nerve cords to a secondary ganglion in the tail. Motor neurons from the cords enervate the body-wall muscles. Sensory neurons extend into the nerve ring from chemoreceptors and touch receptors in the head. Hermaphrodites have specialized neurons that control egg-laying; males lack these but have additional neurons that provide input from sensory structures in the tail and control male mating behavior.

 The hermaphrodite reproductive system is comprised of a bilobed gonad, with one lobe extending anteriorly and one posteriorly from the uterus near the middle of the animal. The gonad consists of a somatic sheath surrounding the germ cells and has several specialized regions. The distal arm of each lobe contains mitotically dividing germ-cell nuclei in a common cytoplasm. The nuclei enter meiosis as they move away from the distal tip. The earliest nuclei to mature differentiate into sperm during the fourth larval stage; at the molt to adulthood the germ line switches sex, and subsequent meiotic nuclei are recruited to form oocytes as they round the bend into the proximal arm, which contains the oviduct. At the proximal end of the oviduct is the spermatheca, containing stored sperm,

through which the oocytes pass and become fertilized on their way to the uterus. A muscular vulva connects the uterus to the outside and serves as the egg-laying apparatus. In the male, the gonad is single lobed and produces only sperm, which are stored in the vas deferens and released through the cloaca in the tail. The fan-shaped male tail is specialized for mating, which is accomplished by deposition of sperm through the hermaphrodite vulva into the uterus, where they move to the           spermatheca and compete for oocytes with the resident hermaphrodite sperm.

 4. Fertilization and Embryonic Development

Following fertilization, a tough chitinous egg shell forms around the zygote, and embryonic cleavage begins. The spheroidal embryos, about 70 µm in length, are viable if dissected out of the hermaphrodite at this time, but normally they remain in the uterus for about 2 h until the onset of gastrulation, when they are laid through the vulva. Gastrulation is simple; only 53 cells move from the exterior to the interior, but the result is a triploblastic embryo with outer ectodermal precursor cells that will form the hypodermis and nervous system, inner endodermal precursors that will form the gut, and in between a layer of mesodermal precursors that will give rise to muscles and the somatic gonad. By 6 h after fertilization, midway through embryogenesis, the embryo consists of about 600 cells. At this point, cell proliferation essentially ceases and a process of morphogenesis begins which literally squeezes the spheroidal embryo into the shape of a worm as organogenesis proceeds internally and cuticle is formed externally. During this time about 40 cells undergo programmed cell death and are engulfed by neighboring cells. At the end of embryogenesis the worm, about 3.5 times the length of the original embryo, digests the shell from the inside and hatches out of the egg.

 The process of embryogenesis is essentially invariant at the cellular level. It proceeds by a stereotyped series of cell divisions (Fig. 2), whose timing and relative spatial orientations are the same in every embryo. This feature and the transparency of the embryo made it possible for John Sulston and his colleagues to trace out the entire embryonic cell lineage from fertilization to hatching, so that the ancestry of each of the 558 cells in the L1 is known (4). Most cells are born close to their final locations; only about 12 cells undergo long-range migrations during embryogenesis.

Figure 2. Early divisions in the C. elegans embryonic cell lineage, showing cells and tissues derived from each of the major branches. The vertical axis shows time after fertilization and total number of cells in the embryo. Horizontal lines indicate times of cell divisions. P0 through P4 are germ line cells; the cells named AB, MS, E, C, and D are somatic founder cells for the various branches of the lineage. The number of cells of each different type produced in each branch is indicated. Note that many cells are programmed to die during embryonic development, especially in the AB and MS branches.

Genetic and molecular analysis of the cleavage stage has revealed that cell fates in the early embryo  (Fig. 2) are determined by a combination of maternally derived factors, which segregate asymmetrically to specific cells during cleavage, and cell-signaling mechanisms by which certain cells induce new cell fates in their neighbors. This patterning process appears to proceed by evolutionarily conserved mechanisms that are common to all embryos; for example, the signaling pathways so far known to be employed include several that are also important for insect and vertebrate development, such as the wingless (Wnt), and Notch pathways (5).

5.  Larval Development

5.1.  Larval Stages 

After hatching as an L1, the worm grows through three more larval stages, L2–L4 (Fig. 3), separated by molts at which a new cuticle is secreted by the underlying hypodermis and the old one shed. The newly hatched L1, 250 µm long with 558 cells in the hermaphrodite and 560 in the male, includes functional digestive, neuromuscular, and neurosensory systems and a gonad primordium consisting of two somatic and two germ-line cells. Most of the cells in the L1 divide no further during larval development. However, 55 are blast cells that undergo additional divisions during larval development to produce, primarily, the adult reproductive structures and the neurons that control them: the hermaphrodite and male gonads, the vulva and egg-laying muscles in the hermaphrodite, and the sex muscles and specialized tail mating structures in the male. Additional motor neurons are also produced in the nerve cords for finer control of the body-wall muscles. As in the embryo, the invariance of these processes, the transparency of all stages, and the small number of cells involved made it possible to map the larval cell lineages in both sexes from L1 to adulthood (6). Consequently, the entire cell ancestry of C. elegans is known, from fertilized egg to adult.

 Figure 3. The C. elegans life cycle. Clock circle indicates hours of development after fertilization at 25°C. Eggs containing developing embryos are laid at about 2 h. The L1 larvae, hatching at about 14 h, undergo four more molts at the times indicated as they grow to adulthood. See text for further explanation.

5.2.   Cell Interactions during Larval Development 

Elaboration of larval structures involves cell interactions, again occurring by evolutionarily conserved signaling pathways such as those involving homologues of Notch, Wnt, epidermal growth factor (EGF), and transforming growth factor (TGF)-b-superfamily ligands as the signaling molecules. Application of the genetic approach to the process of vulval development, for example, has revealed details of a ras-based pathway by which the developing gonad signals underlying hypodermal cells to differentiate using an EGF-like ligand. The molecular components are very similar to those of ras pathways found in mammalian cells, in which conversion of ras to an oncogene causes a variety of cancers, and the C. elegans pathway has become an important model system for cancer research.

 Cell interactions during larval development also include guidance cues by which several cells, including the growing axonal processes of motorneurons and the distal tip cells of the enlarging hermaphrodite gonad, find their way along the basement membrane of the pseudocoelom en route to their final destinations. Again, application of the genetic approach in C. elegans has shown that this process is accomplished by a conserved mechanism: The so-called netrin ligands and their receptors that guide these cells in the worm are remarkably similar to the molecules that guide axonal growth from the neural floor plate to the spinal cord in developing avian and mammalian embryos, making C. elegans a potentially useful model system for study of nerve regeneration.

The cell divisions and molts in larval development occur in a precisely timed sequence. Again, application of the genetic approach has identified many of the genes in the timing mechanism and is providing new information on how developmental clocks function.

5.3. The Dauer Larva 

Like most free-living nematodes, C. elegans can molt to an alternative form of the L3, called a dauer larva (German for “enduring larva”) when conditions are unsuitable for reproduction. Dauer larvae, often simply called dauers, do not feed, are relatively resistant to drying, and can live for up to a year if desiccation is prevented. If conditions improve, they can molt to the L4 stage and resume the normal developmental pathway, with no change in subsequent lifespan. Dauer development is triggered by lack of food and overcrowding, signaled by a pheromone of unknown nature that stimulates chemosensory structures in the head. This process is of interest for at least two reasons. First, the signaling mechanism involves homologues of ligands, receptors, and downstream components of the evolutionarily conserved TGF-b pathway, which is of widespread importance but not fully understood in mammalian development. Second, entry into the pathway of dauer development appears to turn off genes that normally limit the C. elegans lifespan to less than 3 weeks, allowing the dauer to live much longer. This finding, and the experimentally convenient short normal lifespan, make C. elegans an exciting model system for studying genetic control of aging, which is under active investigation (7).

6. Behavior and the Nervous System

 C. elegans can move forward or backward and change direction, by coordinated flexing of its body-wall muscles. It is touch-sensitive, moving forward in response to a touch on the tail and backward in response to a touch on the head. Chemosensors in the head, connected by sensory neurons to the ring ganglion, can detect a variety of ions as well as volatile odorants and elicit either an attractive or repulsive response. For example, C. elegans is attracted to Na⁺ , K⁺ , Cl^– , and several alcohols and ketones; it is repelled by Cu²⁺ , acid pH, D-tryptophan, and benzaldehyde. Males are attracted by a pheromone of unknown nature that is produced by hermaphrodites. Although some of its relatives have photoreceptors, C. elegans does not appear to respond to light or use it as a sensory cue. It does, however, sense and respond to temperature and will move to a preferred point in a temperature gradient.

 Is an animal with only 302 neurons capable of learning? Simple conditioning experiments suggest that C. elegans not only can habituate to several stimuli, but also is capable of associative learning, that is, learning to use a normally neutral stimulus to predict the arrival of a second more significant stimulus. For example, if presence of food is paired with one of two equally attractive ions and absence of food with the other during conditioning, the conditioned animals will preferentially move toward a source of the paired ion when tested subsequently in the absence of food, and this preference lasts up to 7 h after training. In addition, C. elegans not only can sense a temperature gradient, but also can “remember” the temperature at which it has previously fed and move to the same temperature when placed in a new gradient without food. The genetic approach should allow the genes involved in learning and memory in C. elegans to be identified and the mechanisms of the proteins they encode to be elucidated.

C. elegans does not show obvious circadian rhythms, but it exhibits much higher frequency ) ultradian) rhythms, such as a regular defecation cycle that is repeated about once every 45 s when the animal is feeding, regardless of the temperature. As in other organisms, the mechanisms of the temperature-compensated molecular clocks that control such cycles (like the human heartbeat) are just beginning to be understood. Application of the genetic approach to cyclical behaviors in C. elegans is identifying the genes and proteins that control ultradian rhythms.

 7.  Current Investigation and Online Information about C. elegans

Knowledge about C. elegans genetics, development, and behavior is accumulating rapidly in several areas of currently exciting biological research, some of which are mentioned above. These include mechanisms of pattern formation in development, origins of left–right asymmetry in animal body plans, cell fate determination, programmed cell death, cell migration and guidance, developmental timing mechanisms, physiological sensory mechanisms, organization of animal genomes, and animal evolution.

References

1.  S. Brenner (1974) Genetics 77, 71–94. 

2. W. B. Wood et al. (eds.) (1988) The Nematode Caenorhabditis elegans, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 

3. D. L. Riddle, T. Blumenthal, B. J. Meyer, and J. R. Priess (eds.) (1997) C. elegans II, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 

4.J. Sulston, E. Schierenberg, J. White, and J. Thomson (1983) Dev. Biol. 100, 64–119. 

5. R. Schnabel and J. R. Priess (1997) In C. elegans II (D. L. Riddle, T. Blumenthal, B. J. Meyer, and J. R. Priess, eds.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 361–382.

6.  J. Sulston and H. Horvitz (1977) Dev. Biol. 56, 110–156. 

7. C. Kenyon (1997) In C. elegans II (D. L. Riddle, T. Blumenthal, B. J. Meyer, and J. R. Priess, eds.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 791–813.