Biomedical subjects
Publications and source records attributed to J N Thomson.
Mutations do not always arise as single events. Many new mutations actually occur in the cell lineage before germ cell formation or meiosis and are therefore replicated pre-meiotically. The increased likelihood of substitutions caused by these clusters of new mutant alleles can change the fundamental theorem of neutral evolution.
The meiotic segregation of the holocentric chromosomes of Caenorhabditis elegans in both spermatogenesis and oogenesis is described. The extended kinetochore typical of the mitotic chromosome could not be differentiated on meiotic bivalents; instead microtubules appeared to project into the chromatin. The meiotic spindles formed during spermatogenesis contain centrioles and asters, while in oogenesis the spindles are acentriolar and barrel shaped. The formation of the acentriolar spindle was studied in fixed specimens by anti-tubulin immunofluorescence. Microtubule arrays were seen first to accumulate in the vicinity of the meiotic chromosomes prior to congression. At later stages, elongated spindle structures up to 13 mu in length were observed parallel to the surface of the embryo. Further development of the spindle appeared to involve its shortening into a barrel shape and rotation so that one spindle pole was opposed to the membrane. By anaphase the pole-to-pole spindle length reached a minimum of 3-4 mu. One end of each chromatid in the meiotic bivalent was labelled by in situ hybridization of a probe DNA to show that in oogenesis the chromatids were associated end-to-end in the bivalent. Furthermore, either the right or the left ends of the homologues could be held in association. At metaphase I the bivalents were oriented axially, such that kinetic activity was restricted to one end of each pair of sister chromatids. At metaphase II the chromosomes were also aligned axially.
Identification of the genes orchestrating neurogenesis would greatly enhance our understanding of this process. Genes have been identified that specify neuron type (for example cut and numb in Drosophila and mec-3 in Caenorhabditis elegans) and process guidance (for example, unc-5, unc-6 and unc-40 in C. elegans and the fas-1 gene of Drosophila). We sought genes defining synaptic specificity by identifying mutations that alter synaptic connectivity in the motor circuitry in the nematode C. elegans. We used electron microscopy of serial sections to reconstruct the ventral nerve-cords of uncoordinated (unc) mutants that have distinctive locomotory choreographies. Here we describe the phenotype of mutations in the unc-4 gene in which a locomotory defect is correlated with specific changes in synaptic input to a subset of the excitatory VA motor neurons, normally used in reverse locomotion. The circuitry alterations do not arise because of the inaccessibility of the appropriate synaptic partners, but are a consequence of changes in synaptic specificity. The VA motor neurons with altered synaptic inputs are all lineal sisters of VB motor neurons; the VA motor neurons without VB sisters have essentially the same synaptic inputs as in wild-type animals. The normal function of the wild-type allele of unc-4 may thus be to invoke the appropriate synaptic specificities to VA motor neurons produced in particular developmental contexts.
In normal development both the anterior and posterior blastomeres in a 2-cell C. elegans embryo produce some descendants that become muscles. We show that cellular interactions appear to be necessary in order for the anterior blastomere to produce these muscles. The anterior blastomere does not produce any muscle descendants after either the posterior blastomere or one of the daughters of the posterior blastomere is removed from the egg. Moreover, we demonstrate that a daughter of the anterior blastomere that normally does not produce muscles appears capable of generating muscles when interchanged with its sister, a cell that normally does produce muscles. Embryos develop normally after these blastomeres are interchanged, suggesting that cellular interactions play a major role in determining the fates of some cells in early embryogenesis.
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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.
Three types of microtubules are seen in the neuronal processes of the nematode Caenorhabditis elegans. Single cytoplasmic microtubules of most neurones have 11 protofilaments whereas those of six touch receptor cells have 15 protofilaments. The axonemes of sensory cilia have nine outer doublets with a variable number (up to seven) of singlet microtubules. Mutations in 11 genes affect the appearance of these microtubules.
Eight pairs of chemosensory neurons in Caenorhabditis elegans take up fluorescein dyes entering through the chemosensory organs. These are amphid neurons ADF, ASH, ASI, ASJ, ASK, and ADL and phasmid neurons PHA and PHB. When filled with dye, the processes and cell bodies of these neurons can be examined in live animals by fluorescence microscopy. Using this technique, we have identified five genes, unc-33, unc-44, unc-51, unc-76, and unc-106, that affect the growth of the amphid and phasmid axons. These genes were found to affect the axons of the mechanosensory PDE neurons as well. The unc-33 mutation specifically affects neuronal microtubules. Sensory dendrites in this mutant have a superabundance of microtubules. Moreover, many of these microtubules are abnormal in diameter, and some form hooks or multiple tubules.
Monoclonal antibodies were generated using mice immunized with total homogenates of Caenorhabditis elegans adults or early larvae. Two of them were shown to distinguish a certain class of neuronal or supporting cells in the nervous tissue of this animal. Their histological specificities were studied in detail by indirect immunofluorescence on a whole mount preparation of animal head (or tail); for one of the antibodies further analysis was done by immunoelectron microscopy with the aid of a colloidal gold probe. An application of this antibody to a mutant of C. elegans is also described.
The endosomal compartment of giant HeLa cells was labelled with a transferrin-horse radish peroxidase (HRP) conjugate. Serial thin sections from the leading lamella of a cell are presented; they show that the endosomal compartment contains a tubular system connected to vesicular structures. In addition, small (approximately 50 nm) coated vesicles are seen in the leading lamella.
The neural pathways for touch-induced movement in Caenorhabditis elegans contain six touch receptors, five pairs of interneurons, and 69 motor neurons. The synaptic relationships among these cells have been deduced from reconstructions from serial section electron micrographs, and the roles of the cells were assessed by examining the behavior of animals after selective killing of precursors of the cells by laser microsurgery. This analysis revealed that there are two pathways for touch-mediated movement for anterior touch (through the AVD and AVB interneurons) and a single pathway for posterior touch (via the PVC interneurons). The anterior touch circuitry changes in two ways as the animal matures. First, there is the formation of a neural network of touch cells as the three anterior touch cells become coupled by gap junctions. Second, there is the addition of the AVB pathway to the pre-existing AVD pathway. The touch cells also synapse onto many cells that are probably not involved in the generation of movement. Such synapses suggest that stimulation of these receptors may modify a number of behaviors.
The two postembryonic touch receptor neurons in the nematode Caenorhabditis elegans arise from essentially identical cell lineages and have the same ultrastructural features. The cells are found in different positions in the animal, however, and differ in neuronal branching, connectivity, and function. These structural and functional differences are not seen when cells are placed in similar positions by mutation or laser-induced damage. Thus, some, but probably not all, of the differentiated properties of these cells are a consequence of their cellular environment.
Mutations in two nonessential genes specifically block the phagocytosis of cells programmed to die during development. With few exceptions, these cells still die, suggesting that, in nematodes, engulfment is not necessary for most programmed deaths. Instead, these deaths appear to occur by cell suicide.
HeLa cells bind horse spleen ferritin when the two are incubated at 0 degrees C. Since the majority of this bound ferritin is located in coated pits, we conclude that the ferritin binds to a specific receptor which takes part in an endocytic cycle. When substrate-attached and well-spread giant HeLa cells are briefly labelled at 0 degrees C with ferritin, ferritin particles are found to be concentrated towards the cell periphery, where they exist largely outside coated pits. This peripheral concentration is a property of circulating (and not just newly synthesized) receptors because it is not affected by prior incubation of giant cells in cycloheximide. However, coated pits are themselves roughly uniformly distributed over the surface of these cells. These results provide evidence that the membrane internalised by coated pits on these cells is returned to the cell surface at the leading edge of the cell. Because of this separation of the sites of endocytosis and exocytosis, a flow of membrane must occur across the cell surface. This flow is composed of lipid plus receptors. The implications of this for capping and for cell spreading are discussed.
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.
The nervous system of C. elegans is arranged as a collection of process bundles. Processes within bundles are generally unbranched and occupy defined positions relative to their neighbors. Small groups of processes are often closely associated together and run adjacent to one another for relatively long distances. We have defined the set of neurons that have processes adjacent to the processes of a given neuron as the neighborhood of that neuron. Synapses in C. elegans are made en passant between adjacent processes. Of the 1165 pairs of adjacent processes that were analyzed, 520 (45%) had synaptic contacts. The set of neurons that make synaptic contact with a given neuron is therefore, on average, 45% of that neuron's neighborhood. Neurons make synaptic contacts with fewer classes of partners than they have the potential for, as they are limited in their choice of partner to those that inhabit their neighborhood. Some classes of neurons have processes that make abrupt transitions from one neighborhood to another. There is usually some identifiable cue at the transition point, such as the termination of a closely associated process or a discontinuity at the junction of one process bundle with another. Neurons that inhabit more than one neighborhood have a more extended set of synaptic partners than those that are confined to a single neighborhood.
Light microscopy of the mitotic chromosomes of Caenorhabditis elegans suggests that non-localized kinetochores are present, since the chromosomes appear as stiff rods 1 to 2 micrometers in length and lack any visible constriction. The holokinetic structure was confirmed by reconstructions of electron micrographs of dividing nuclei in serially sectioned embryos. In prophase the kinetochore appears as an amorphous projection approximately 0.18-0.2 micrometer in diameter in cross section and in longitudinal section it appears to be continuous along the chromatin. At prometaphase and metaphase the kinetochore is a convex plaque covering the poleward face of the chromosome and extending the length of the chromosome. In longitudinal section the kinetochore is a trilaminar structure with electron dense inner and outer layers of 0.02 micrometer, and an electron lucent middle layer of 0.03 micrometer. The inner layer is adjacent to a more electron dense region of chromatin. The kinetochore was also seen as a band extending the length of the chromosome in whole mount preparations of chromosomes stained with ethanolic phosphotungstic acid. Most gamma ray induced chromosome fragments segregate normally in embryonic mitoses, but some fragments display aberrant behavior. Similar behavior was seen in embryos carrying a genetically characterized free duplication. It is suggested that mitotic segregation of small fragments may be inefficient because the probability of attachment of microtubules to the kinetochore is proportional to kinetochore length.
Nuclei occupy characteristic positions in most cells. In Caenorhabditis elegans, nuclei can be observed in living animals. Ordinary movements can distort the cells and displace their nuclei, but the extent of displacement is limited and nuclei return to their resting positions when the muscles relax. We have isolated five mutants in which the nuclei of certain epithelial cells are not elastically anchored but float freely within the cytoplasm. These mutations define a single gene, anc1, on linkage group 1. Mitochondrial positioning, observed by staining live animals with rhodamine 6G, is also disturbed in these cells. Additional defects, including abnormal tonofilaments and inappropriately positioned desmosomes, have been found by electron microscopy. The anc1 product may be a cytoskeletal component of nematode epithelial cells. Although the Anc1 phenotype is fully expressed in the newly hatched larvae, mutants develop and reproduce normally. Despite mispositioning of organelles, cuticle deposition and moulting are essentially normal. These mutations represent the null phenotype of the gene. At least three independent isolates revert spontaneously at high frequency (10(-5) to 10(-4) ). We suggest that anc1 is a member of a family of cytoskeletal genes.