Gastrulation in the nematode Caenorhabditis elegans

Gastrulation in Caenorhabditis elegans has been described by following the movements of individual nuclei in living embryos by Nomarski microscopy. Gastrulation starts in the 26-cell stage when the two gut precursors, Ea and Ep, move into the blastocoele. The migration of Ea and Ep does not depend on interactions with specific neighboring cells and appears to rely on the earlier fate specification of the E lineage. In particular, the long cell cycle length of Ea and Ep appears important for gastrulation. Later in embryogenesis, the precursors to the germline, muscle and pharynx join the E descendants in the interior. As in other organisms, the movement of gastrulation permit novel cell contacts that are important for the specification of certain cell fates.     

Anaerobiosis in the nematode Caenorhabditis elegans

In Caenorhabditis elegans, mortality rates and changes in concentrations of carbohydrate stores and anaerobic end products were determined in anoxic (test) and normoxic (control) animals at two different temperatures (10 and 20 degrees C). The anoxic tolerance of the free-living nematode proved to be well-developed: at 10 degrees C, about 50% of animals had survived a period of 50 h of anoxia. The carbohydrate stores (approximately 30 mmol glycosyl units kg-1 freshweight (FW)) were reduced by two-thirds within 24 h of anoxia at both temperatures. L-lactate, acetate, succinate, and propionate were identified as the main anaerobic end products. The amounts and proportions of the end products were dependent on temperature. They did not accumulate very much in the tissues, but were mainly excreted. During anoxia, the metabolism of C. elegans was depressed to 3-4% of the aerobic value. The food-source Escherichia coli was found to be at least partly alive in the gut of the animals. To separate between anaerobiosis in animals and bacteria, cleaning procedures were applied, and additional control measurements were made: anaerobic end products produced either by E. coli alone or by bacteria-free (axenic) bred nematodes were quantified at identical incubation conditions.     

Polyploid tissues in the nematode Caenorhabditis elegans

During larval development, the number of somatic nuclei in C. elegans hermaphrodites increases from 558 to 959 (J. E. Sulston and H. R. Horvitz, Dev. Biol. 56, 110-156, 1977; J. E. Sulston et al., Dev. Biol. 100, 64-119, 1983). At the same time, the animals increase about 60-fold in volume. We have measured the DNA contents of several classes of nuclei by quantitating the fluorescence of Hoescht 33258 stained DNA (D. G. Albertson et al., Dev. Biol. 63, 165-178, 1978). Probably all embryonic nuclei, including those of neurons, muscles, hypodermis, and intestine, are diploid at hatching. Neurons, muscles, and nondividing hypodermal nuclei remain diploid throughout larval development. The DNA content of the intestinal nuclei doubles at the end of each larval stage, reaching 32C by the adult stage. New hypodermal cells, generated by division of seam cells in the larval stages, undergo an additional round of DNA replication before fusing with the major syncytium (hyp7, Sulston et al., 1983). Thus the larval hyp7 syncytium comprises a fixed number of diploid embryonic nuclei plus an increasing number of tetraploid postembryonic nuclei. Some of the endoreduplications that occur in the intestinal and hypodermal lineages of C. elegans may correspond to nuclear or cellular divisions in another nematode Panagrellus redivivus (P. W. Sternberg and H. R. Horvitz, Dev. Biol. 93, 181-205, 1982).     

Xenobiotic detoxification in the nematode Caenorhabditis elegans

The nematode Caenorhabditis elegans is an important model organism for the study of such diverse aspects of animal physiology and behavior as embryonic development, chemoreception, and the genetic control of lifespan. Yet, even though the entire genome sequence of this organism was deposited into public databases several years ago, little is known about xenobiotic metabolism in C. elegans. In part, the paucity of detoxification information may be due to the plush life enjoyed by nematodes raised in the laboratory. In the wild, however, these animals experience a much greater array of chemical assaults. Living in the interstitial water of the soil, populations of C. elegans exhibit a boom and bust lifestyle characterized by prodigious predation of soil microbes punctuated by periods of dispersal as a non-developing alternative larval stage. During the booming periods of population expansion, these animals almost indiscriminately consume everything in their environment including any number of compounds from other animals, microorganisms, plants, and xenobiotics. Several recent studies have identified many genes encoding sensors and enzymes these nematodes may use in their xeno-coping strategies. Here, we will discuss these recent advances, as well as the efforts by our lab and others to utilize the genomic resources of the C. elegans system to elucidate this nematode's molecular defenses against toxins.     

Egg-laying defective mutants of the nematode Caenorhabditis elegans

We have isolated 145 fertile mutants of C. elegans that are defective in egg laying and have characterized 59 of them genetically, behaviorally and pharmacologically. These 59 mutants define 40 new genes called egl. for egg-laying abnormal. Most of the other mutants are defective in previously identified genes. The egl mutants differ with respect to the severity of their egg-laying defects and the presence of behavioral or morphological pleiotropies. We have defined four distinct categories of mutants based on their responses to the pharmacological agents serotonin and imipramine, which stimulate egg laying by wild-type hermaphrodites. These drugs test the functioning of the vulva, the vulval and uterine muscles and the hermaphrodite-specific neurons (HSNs), which innervate the vulval muscles. Mutants representing 14 egl genes fail to respond to serotonin and to imipramine and are likely to be defective in the functioning of the vulva or the vulval and uterine muscles. Four mutants (representing four different genes) lay eggs in response to serotonin but not to imipramine and appear to be egg-laying defective because of defects in the HSNs; three of these four were selected specifically for these drug responses. Mutants representing seven egl genes lay eggs in response to serotonin and to imipramine. One egl mutant responds to imipramine but not to serotonin. The remaining egl mutants show variable or intermediate responses to the drugs. Two of the HSN-defective mutants, egl-1 and her-1(n695), lack HSN cell bodies and are likely to be expressing the normally male-specific program of HSN cell death. Whereas egl-1 animals appear to be defective specifically in HSN development, her-1(n695) animals exhibit multiple morphological pleiotropies, displaying partial transformation of the sexual phenotype of many cells and tissues. At least two of the egl mutants appear to be defective in the processing of environmental signals that modulate egg laying and may define new components of the neural circuitry that control egg laying.     

Mutant sensory cilia in the nematode Caenorhabditis elegans

Eight classes of chemosensory neurons in C. elegans fill with fluorescein when living animals are placed in a dye solution. Fluorescein enters the neurons through their exposed sensory cilia. Mutations in 14 genes prevent dye uptake and disrupt chemosensory behaviors. Each of these genes affects the ultrastructure of the chemosensory cilia or their accessory cells. In each case, the cilia are shorter or less exposed than normal, suggesting that dye contact is the principal factor under selection. Ten genes affect many or all of the sensory cilia in the head. The daf-19 (m86) mutation eliminates all cilia, leaving only occasional centrioles in the dendrites. The cilia in che-13 (e1805), osm-1 (p808), osm-5 (p813), and osm-6 (p811) mutants have normal transition zones and severely shortened axonemes. Doublet-microtubules, attached to the membrane by Y links, assemble ectopically proximal to the cilia in these mutants. The amphid cilia in che-11 (e1810) are irregular in diameter and contain dark ground material in the middle of the axonemes. Certain mechanocilia are also affected. The amphid cilia in che-10 (e1809) apparently degenerate, leaving dendrites with bulb-shaped endings filled with dark ground material. The mechanocilia lack striated rootlets. Cilia defects have also been found in che-2, che-3, and daf-10 mutants. The osm-3 (p802) mutation specifically eliminates the distal segment of the amphid cilia. Mutations in three genes affect sensillar support cells. The che-12 (e1812) mutation eliminates matrix material normally secreted by the amphid sheath cell. The che-14 (e1960) mutation disrupts the joining of the amphid sheath and socket cells to form the receptor channel. A similar defect has been observed in daf-6 mutants. Four additional genes affect specific classes of ciliated sensory neurons. The mec-1 and mec-8 (e398) mutations disrupt the fasciculation of the amphid cilia. The cat-6 (e1861) mutation disrupts the tubular bodies of the CEP mechanocilia. A cryophilic thermotaxis mutant, ttx-1 (p767), lacks fingers on the AFD dendrite, suggesting this neuron is thermosensory.     

Colchicine binding in the free-living nematode Caenorhabditis elegans

The [3H]colchicine-binding activity of a crude supernatant of the free-living nematode Caenorhabditis elegans was resolved into a non-saturable component and a tubulin-specific component after partial purification of tubulin by polylysine affinity chromatography. The two fractions displayed opposing thermal dependencies of [3H]colchicine binding, with non-saturable binding increasing, and tubulin binding decreasing, at 4 degrees C. Binding of [3H]colchicine to C.elegans tubulin at 37 degrees C is a pseudo-first-order rate process with a long equilibration time. The affinity of C. elegans tubulin for [3H]colchicine is relatively low (Ka = 1.7 x 10(5) M(-1)) and is characteristic of the colchicine binding affinities observed for tubulins derived from parasitic nematodes. [3H]Colchicine binding to C. elegans tubulin was inhibited by unlabelled colchicine, podophyllotoxin and mebendazole, and was enhanced by vinblastine. The inhibition of [3H]colchicine binding by mebendazole was 10-fold greater for C. elegans tubulin than for ovine brain tubulin. The inhibition of [3H]colchicine binding to C. elegans tubulin by mebendazole is consistent with the recognised anthelmintic action of the benzimidazole carbamates. These data indicate that C. elegans is a useful model for examining the interactions between microtubule inhibitors and the colchicine binding site of nematode tubulin.     

Trehalose extends longevity in the nematode Caenorhabditis elegans

Trehalose is a disaccharide of glucose found in diverse organisms and is suggested to act as a stress protectant against heat, cold, desiccation, anoxia, and oxidation. Here, we demonstrate that treatment of Caenorhabditis elegans with trehalose starting from the young‐adult stage extended the mean life span by over 30% without any side effects. Surprisingly, trehalose treatment starting even from the old‐adult stage shortly thereafter retarded the age‐associated decline in survivorship and extended the remaining life span by 60%. Demographic analyses of age‐specific mortality rates revealed that trehalose extended the life span by lowering age‐independent vulnerability. Moreover, trehalose increased the reproductive span and retarded the age‐associated decrease in pharyngeal‐pumping rate and the accumulation of lipofuscin autofluorescence. Trehalose also enhanced thermotolerance and reduced polyglutamine aggregation. These results suggest that trehalose suppressed aging by counteracting internal or external stresses that disrupt protein homeostasis. On the other hand, the life span‐extending effect of trehalose was abolished in long‐lived insulin/IGF‐1‐like receptor (daf‐2) mutants. RNA interference‐mediated inactivation of the trehalose‐biosynthesis genes trehalose‐6‐phosphate synthase‐1 (tps‐1) and tps‐2, which are known to be up‐regulated in daf‐2 mutants, decreased the daf‐2 life span. These findings indicate that a reduction in insulin/IGF‐1‐like signaling extends life span, at least in part, through the aging‐suppressor function of trehalose. Trehalose may be a lead compound for potential nutraceutical intervention of the aging process.     

Metabolism and aging in the nematode Caenorhabditis elegans

Research into the causes of aging has greatly increased in recent years. Much of this interest is due to the discovery of genes in a variety of model organisms that appear to modulate aging. Studies of long-lived mutants can potentially provide valuable insights into the fundamental mechanisms of aging. While there are many advantages to the use of model organisms to study aging it is also important to consider the limitations of these systems, particularly because ectothermic (poikilothermic) organisms can survive a far greater metabolic depression than humans. As such, the consideration of only chronological longevity when assaying for long-lived mutants provides a limited perspective on the mechanisms by which longevity is increased. Additional physiological processes, such as metabolic rate, must also be assayed to provide true insight into the aging process. This is especially true in the nematode Caenorhabditis elegans, which has the natural ability to enter into a metabolically reduced state in which it can survive many times longer than its normal lifetime. The extended longevity of at least some long-lived C. elegans mutants may be due to a reduction in metabolic rate, rather than an alteration of a metabolically independent genetic mechanism specific for aging.     

Protein oxidation during aging of the nematode Caenorhabditis elegans

The nematode Caenorhabditis elegans has proven a robust genetic model for studies of aging, including the roles of oxidative stress and protein damage. In this review, we focus on the genetics of select long-lived (e.g., age-1, daf-2, daf-16) and short-lived (e.g., mev-1) mutants that have proven useful in revealing the relationships that exist among oxidative stress, life span, and protein oxidation. The former are known to control an insulin/IGF-1-like pathway in C. elegans, while the latter affect mitochondrial function.     

A methyl viologen-sensitive mutant of the nematode Caenorhabditis elegans

A methyl viologen (paraquat)-sensitive mutant, mev-1 (LG III), in Caenorhabditis elegans was about 4 times more sensitive to methyl viologen than the wild type. This mutant was also hypersensitive to oxygen. The brood size was about 1/4 that of the wild type. The average life span was determined to be 9.3 days as compared to 14.3 days for the wild type. The activity of superoxide dismutase (SOD), a scavenging enzyme for superoxide anion, was about half the wild-type level. We suggest that oxygen radicals may be involved in the normal aging mechanism in C. elegans.     

Organization of neuronal microtubules in the nematode Caenorhabditis elegans

We have studied the organization of microtubules in neurons of the nematode Caenorhabditis elegans. Six neurons, which we call the microtubule cells, contain bundles of darkly staining microtubules which can be followed easily in serial-section electron micrographs. Reconstruction of individual microtubules in these cells indicate that most, if not all, microtubules are short compared with the length of the cell process. Average microtubule length varies characteristically with cell type. The arrangement of microtubules gives an overall polarity to each bundle: the distal ends of the microtubles are on the outside of the bundle, whereas the proximal ends are preferentially inside. The distal and proximal ends each have a characteristic appearance indicating that these microtubules may have a polarity of their own. Short microtubules in processes of other neurons in C. elegans have also been observed.     

Post-embryonic cell lineages of the nematode,Caenorhabditis elegans

The number of nongonadal nuclei in the free-living soil nematode Caenorhabditis elegans increases from about 550 in the newly hatched larva to about 810 in the mature hermaphrodite and to about 970 in the mature male. The pattern of cell divisions which leads to this increase is essentially invariant among individuals; rigidly determined cell lineages generate a fixed number of progeny cells of strictly specified fates. These lineages range in length from one to eight sequential divisions and lead to significant developmental changes in the neuronal, muscular, hypodermal, and digestive systems. Frequently, several blast cells follow the same asymmetric program of divisions; lineally equivalent progeny of such cells generally differentiate into functionally equivalent cells. We have determined these cell lineages by direct observation of the divisions, migrations, and deaths of individual cells in living nematodes. Many of the cell lineages are involved in sexual maturation. At hatching, the hermaphrodite and male are almost identical morphologically; by the adult stage, gross anatomical differences are obvious. Some of these sexual differences arise from blast cells whose division patterns are initially identical in the male and in the hermaphrodite but later diverge. In the hermaphrodite, these cells produce structures used in egg-laying and mating, whereas, in the male, they produce morphologically different structures which function before and during copulation. In addition, development of the male involves a number of lineages derived from cells which do not divide in the hermaphrodite. Similar postembryonic developmental events occur in other nematode species.     

The initiation of spermiogenesis in the nematode Caenorhabditis elegans

Spermiogenesis in nematodes involves the activation of sessile spherical spermatids to motile bipolar amoeboid spermatozoa. In Caenorhabditis elegans males spermiogenesis is normally induced by copulation. Spermatids transferred to hermaphrodites as well as some of those left behind in the male become spermatozoa a few minutes after mating. Spermiogenesis can also be induced in vitro by the ionophore monensin (G.A. Nelson and S. Ward, 1980, Cell 19, 457-464) and by weak bases such as triethanolamine. Both triethanolamine and monensin cause a rapid increase in intracellular pH from 7.1 to 7.5 or 8.0. This pH increase precedes the subsequent morphological events of spermiogenesis. Triethanolamine or monensin must be present throughout spermiogenesis for all cells to form pseudopods, but once pseudopods are formed the inducers are unnecessary for subsequent motility. The pH induced spermiogenesis is inhibited by drugs that block mitochondria or glycolysis. Protease treatment can also induce spermiogenesis without increasing intracellular pH, apparently bypassing the pH-dependent steps in activation and the requirement for glycolysis. These results show that the initiation of spermiogenesis in C. elegans, like some steps in egg activation and the initiation of sea urchin sperm motility, can be induced by an increase in intracellular pH, but this pH change can be bypassed by proteolysis.     

The embryonic cell lineage of the nematode Caenorhabditis elegans

The embryonic cell lineage of Caenorhabditis elegans has been traced from zygote to newly hatched larva, with the result that the entire cell lineage of this organism is now known. During embryogenesis 671 cells are generated; in the hermaphrodite 113 of these (in the male 111) undergo programmed death and the remainder either differentiate terminally or become postembryonic blast cells. The embryonic lineage is highly invariant, as are the fates of the cells to which it gives rise. In spite of the fixed relationship between cell ancestry and cell fate, the correlation between them lacks much obvious pattern. Thus, although most neurons arise from the embryonic ectoderm, some are produced by the mesoderm and a few are sisters to muscles; again, lineal boundaries do not necessarily coincide with functional boundaries. Nevertheless, cell ablation experiments (as well as previous cell isolation experiments) demonstrate substantial cell autonomy in at least some sections of embryogenesis. We conclude that the cell lineage itself, complex as it is, plays an important role in determining cell fate. We discuss the origin of the repeat units (partial segments) in the body wall, the generation of the various orders of symmetry, the analysis of the lineage in terms of sublineages, and evolutionary implications.