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(A) Nematode Strains
Caenorhabditis elegans var. Bristol (strain N2) was obtained from Sydney Brenner and grown on Escherichia coli OP50 on petri dishes at 20°C, as described previously (Brenner, 1974). Hermaphrodite stocks were propagated by self-fertilization; male stocks were propagated by crossing with hermaphrodites.
Aphelencoides blastophthorus, Longido-rus macrosoma, Panagrellus redivivus (Panagrellus silusiae), and Turbatrix aceti were obtained from David J. Hooper, Rothamsted Experimental Station, Har penden, Hertfordshire, England. Ascaris lumbricoides var. suis was obtained from the local slaughterhouse; eggs were squeezed out of adult uteri and development was initiated by incubation in 0.1 M sulfuric acid at 25°C (Rogers, 1960); at appropriate intervals, samples were washed with insect tissue culture medium (Shields et al., 1975) and larvae were released from the eggs by gently rolling the tip of a Pasteur pipet over them.
(1) Study of Living Specimens
(a) Mounting. An agar slab about 0.5 mm thick was prepared by flattening a drop of 5% agar on a microscope slide with a second, siliconized slide placed across it; the siliconized slide was supported by "spacer" slides raised by one or two thicknesses of adhesive tape. After the agar had hardened, the siliconized slide was removed and a small drop (about 2 microlitre) of 10% polyvinylpyrrolidone (molecular weight 44,000; BDH Chemicals, Ltd., Poole, England) in S medium (Sulston and Brenner, 1974) was placed on the agar, care being taken not to break its surface. A selected nematode was transferred to this drop, using either a sharpened wooden stick or a human eyelash attached with Plasticine to a stick. An animal successfully transferred would thrash wildly. The center of a 12 x 12-mm coverslip was very thinly coated with E. coli OP50 scraped from a petri dish with a wire loop. The coverslip was lowered gently onto the agar, care again being taken not to break the agar surface. The volume of liquid was such that the pressure of the coverslip prevented the worm from thrashing without excessively inhibiting its movement. All of these operations were performed at a room temperature of about 20°C.
Excess agar protruding beyond the coverslip was removed with a razor blade. The
edges of the remaining agar block were sealed with silicone grease or Vaseline
to prevent dehydration. Air bubbles which formed when the coverslip was
positioned were gradually absorbed by the agar. After a period of quiescence
(less than 1 hr), the nematode generally moved into the bacterial lawn and
started to feed.
Nematodes mounted for observation in the light microscope could be safely removed afterward for other types of study (e.g., electron microscopy).
(b) Observation. Nematodes were observed in either a Zeiss
Universal or a Zeiss Standard RA microscope equipped with a Plan 100 objective
and Nomarski differential interference contrast optics. To reduce heating, illumination
was kept as low as possible; a heat filter was routinely used and, occasionally,
a broadband green interference filter was also employed. Air temperature was
maintained between 19 and 22°C.
Because C. elegans is small and transparent, internal structures in intact living specimens could be readily examined.
Nuclei and large nucleoli were clearly resolved by Nomarski optics,
which permits visualization of changes in refractive index and has a very shallow
depth of field (e.g., Fig 2). Cell
boundaries, however, were not always visible. The nuclei of different types
of cells were generally distinguishable and are described under Results.
This technique is nondestructive and allows the nematode to live under reasonably natural conditions. While mounted for observation, the nematode moved freely between the coverslip and the surface of the agar block. Attracted by the bacterial lawn, it generally stayed near the middle of the coverslip. If the nematode strayed out from under the coverslip, it could be transferred to a petri dish and remounted.
C. elegans can only flex in a dorso-ventral direction. Hence, because it was restricted to the plane between the agar and the coverslip, the nematode was observed lying on its side. A properly mounted animal usually remained lying on the same side. However, for a period just before molting, nematodes actively and repeatedly invert their positions with a rapid twisting movement. On occasion, such "flips" proved very inconvenient, as they made it difficult to continue observing cells which had moved from the side nearest the coverslip to the side nearest the agar slab (because of interference introduced by the body of the nematode).
(c) Cell lineages. Lineages were determined by continually observing given nuclei as they migrated, divided, and died during the course of postembryonic development. Because cell boundaries were often not visible, these lineages depict the behavior of nuclei and not necessarily the behavior of cells; it is possible that some nuclear divisions (or movements) are not accompanied by concomitant cellular divisions (or movements). During periods of nuclear activity, sketches of those nuclei of interest and of appropriate adjacent landmarks (usually other nuclei) were made as frequently as possible. During periods of nuclear quiescence, observations were recorded at intervals of 0.5-2 hr. When lineages in a given nematode were to be followed for more than 1 day, the animal was refrigerated overnight at 6-8°C during a suitable quiescent period. Nematodes were generally chilled during the early period of a given larval stage. (Individuals refrigerated just prior to or during lethargus —a 2-hr period preceding each of the four larval molts during which there is little pharyngeal pumping or body movement — often did not recover.) When returned to 20°C, most individuals recommenced development after a 0.5- to 2-hr lag. Generally, nematodes were refrigerated for no more than 15 hr.
When appropriately mounted, healthy nematodes developed as rapidly as on petri dishes. Some individuals, particularly after refrigeration, developed more slowly. Occasionally, a younger worm was damaged during the mounting process and barely developed at all; such individuals were discarded. To obtain a standard time scale for the lineage charts presented below, observed time intervals were normalized according to the observed length of the appropriate intermolt period.
On the lineage charts, the time of a molt is defined as the moment when the head of the nematode breaks through the old cuticle after lethargus. Lethargus is indicated by dotted regions along the time scales. The time of a cell division reflects the time a metaphase plate is visible (see Results, Cell Division). The time of a cell death indicates the time of maximal nuclear refractility (see Results, Programmed Cell Death).
(d) Photography. Photomicrographs were taken with a Zeiss microflash illuminator. All photomicrographs are of living specimens.
(2) Cell Assignments
Nuclei observed with Nomarski optics were assigned to specific cell types by tracing them through to the adult stage, in which they have been identified by reconstruction from serial section electron micrographs available in this laboratory and prepared as described by Ward et al. (1975).
(3) Camera Lucida Drawings
Drawings were made using a Zeiss camera lucida. Specimens were mounted on slabs of 5% agar as described above, except (1) no bacteria were added, and (2) the molten agar contained l-phenoxy-2-propanol (Bird, 1971) as an anesthetic. The concentrations of phenoxypropanol used were 0.2% v:v for L1's, 0.3% for L2's and L3's, and 0.5% for L4's and adults. (Ln denotes animals of the nth larval stage.)
The line drawings of nematodes presented below are based upon camera lucida drawings. They show nuclei, nucleoli (when visible), and other appropriate morphological landmarks. These drawings depict typical individuals, but, as there are minor variations from animal to animal, they cannot always be used to identify nuclei in animals in which lineages have not been traced directly. Also, minor distortions in nuclear position sometimes are evident in anesthetized nematodes.
(4) Feulgen Staining
A small drop of 10% ovalbumin was placed on a glass slide coated with a thin layer of collagen prepared according to Bornstein (1958). A large number of nematodes were spread evenly over the surface of the slide with a paper strip; alternatively, single worms were transferred with a wooden stick or platinum wire. When necessary to prevent evaporation, the slide was kept cool over ice. The slide was placed in Carnoy's fixative (Pearse, 1968) for at least 1 hr; overnight fixation was preferable. After rehydration through 50 and 30% ethanol (10 min each), the slide was incubated in 1 N HCl at room temperature for 10-20 min and then in 1N HCl at 60°C for 10-12 min. It was transferred to Schiff's reagent for 1.5-2 hr. Schiff's reagent was prepared by bubbling sulfur dioxide gas through 1% basic fuchsin, essentially as described by Rafalko (1946). After staining, the slide was rinsed twice in distilled water and dehydrated through a graded series of alcohols (30, 50, 70, and 90%, 10 min each). After two 30-min baths in absolute ethanol and two 30-min baths in xylene, the slide was mounted in Depex mounting medium (Gurrs, London, England). Stained specimens were examined using bright field illumination through a blue filter.
A coumarin dye laser microbeam system developed by J. G. White was used to kill specific cells in individual nematodes. This system produces a focused spot about 1.5 micrometer in diameter and can destroy a given cell with no apparent damage to its neighbors. It is based upon a system developed by Berns (1972) and will be described in a future publication by Dr. White.
In describing the anatomy and development of C. elegans, we have adopted the following system to name specific cells (or nuclei) and their daughters. Upper-case letters indicate blast cells of unknown embryonic lineage; for example, M is a mesoblast present in the newly hatched L1. In some cases, a set of blast cells undergoes similar patterns of cleavage and produces morphologically similar groups of daughters; each member of such a set is denoted with a common upper-case letter followed by a specific numeric designation, e.g., six blast cells on each side of the lateral hypodermis are V1-V6. When a blast cell divides, each daughter is named by adding to the name of its mother cell a single lower-case letter representing its position immediately after division relative to its sister cell. A cell which divides along a dorso-ventral axis has its daughters indicated by a "d" and a "v"; e.g., M divides to produce M.d and M.v. Left-right divisions are indicated by "l" and "r"; e.g., M.d divides to produce M.dl and M.dr. Anterior-posterior divisions are indicated by an "a" and a "p"; e.g., M.dl divides to produce M.dla and M.dlp. As indicated in these examples, a period separates the name of the original blast cell from the labels which define specific progeny of subsequent divisions.
Within the lineage trees, "d," "l," and "a" daughters are represented by left branches, and "v," "r," and "p" daughters are represented by right branches; these branches are labeled accordingly (except in lineages with most divisions anterior-posterior, in which the labels a and p are omitted from the branches). An oblique division is indicated on the branches by labels with two (or three) characters; the first character from these labels defines the branch assignments. For example, E.r divides along an anterior/ventral-posterior/dorsal axis; hence, the left branch is labeled "av," and the right branch is labeled "pd" (Fig. 22). The daughters of such a division are named by using only the first of the characters which indicate the division axis, i.e., E.ra and E.rp. Thus, the number of characters which follow a blast cell name directly implies the number of divisions which generated the progeny cell; e.g., E.rap was produced by the third division of E.
In the B lineage, the precise fates of certain progeny cells are not predetermined. For example, B.alaa and B.araa, generated on the left and right sides respectively, recentralize. Their relative anterior-posterior position after recentralization is indeterminate. The anterior cell ("Balpha") follows one lineage program, whereas the posterior cell ("Bbeta") follows another. In other words, either B.alaa or B.araa can become Balpha. Thus a greek letter following a blast cell name indicates a cell derived from that blast cell via one of a number of alternative lineage routes.
Entirely lower-case names are used as abbreviations for cell types in lineage charts, camera lucida drawings, and photographs. For example, "bm" identifies a body muscle. In the lineage charts, this name follows the systematic name based on the lineage history of the cell, e.g., M.dlpp; bm. The abbreviations used are: bm, body muscle; cc, coelomocyte; dep, anal depressor muscle; exc, excretory cell; exc gl, excretory gland; g, neuron or glial cell; hsn, hermaphrodite-specific neuron; i, intestinal nucleus; im, intestinal muscle; pig, posterior lateral ganglion; rect gl, rectal gland; se, seam cell; set, tail seam cell; sph, anal sphincter muscle; sy, syncytial nucleus; um1, type 1 uterine muscle; um2, type 2 uterine muscle; vcn, ventral cord neuron; vh, ventral hypodermal nucleus; vm1, type 1 vulval muscle; vm2, type 2 vulval muscle.
The nomenclature used in this paper is somewhat different from that used previously (Sulston, 1976) to describe the cell lineages of the ventral nervous system of C. elegans. We believe that the revised nomenclature is easier to use in referring to the relatively large number of cells described in this paper.
Adapted by Yusuf KARABEY for WORMATLAS, 2003