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Culture Caenorhabditis elegans (Bristol), strain N2, was maintained according to Brenner (1974). Turbatrix aceti and Aphelencoides blastophthorus were kindly given to us by David Hooper, Rothamsted Experimental Station, Harpenden, Herts, England; Panagrellus redivivus was obtained from Rothamsted in 1976, and is the strain studied by Sternberg and Horvitz (1981, 1982). T. aceti and P. redivivus were maintained in the same way as C. elegans; A. blastophthorus was grown on NGM plates infected with mixed fungi.
General Biology of C. elegans C. elegans is a free-living nematode which has two sexual forms: a self-fertilising hermaphrodite and a male. Development begins in the egg, and continues through four larval stages (L1-L4) to the adult. A newly hatched L1 is depicted in Fig. 1. The general anatomy of the newly hatched L1 larva has been described by Sulston and Horvitz (1977). As far as it goes this account is accurate except for the precise cell count in the head and the omission of the intestino-rectal valve (virL and virR, see Figs. 1 and 8c). Postembryonic cellular development has been described for the gonad by Kimble and Hirsh (1979), for the male tail by Sulston et al. (1980), and for other systems by Sulston and Horvitz (1977). Other references will be found in the tissue-by-tissue description below.
FIG. 1. General anatomy of newly hatched L1 larva, left lateral aspect, body wall cut away to midline. Part of the intestine is also cut away to show two of the four longitudinal bands of body muscle embedded in the hypodermis. Neuronal cell bodies are represented schematically. Most of the sensilla (amphids, cephalics, inner labials, outer labials, together with a number of accessory neurons) are grouped around the mouth (Ward et al, 1975; also shown schematically in Fig. 18); elsewhere are the anterior deirids (paired, lateral, over rear bulb of pharynx), the phasmids (paired, lateral, posterior to lumbar ganglia), and the microtubule neurons (longitudinal lateral mechanoreceptors, one pair anterior and one pair posterior; Chalfie and Sulston, 1981). Body cavity is termed a pseudocoelom, since intestine is in direct contact with body wall. Principal changes during postembryonic development are: overall increase in size; growth and maturation of the gonad; formation of vulva (midventral) in hermaphrodite, and copulatory bursa (posterior) in male, both with associated musculature; cell division in hypodermis and ventral nervous system. Sensilla generated postembryonically are: postdeirids (paired, lateral, between gonad and anus) and ventral microtubule neurons in both sexes; numerous sensilla in the male copulatory bursa (Sulston et al, 1980).
Light Microscopy C. elegans eggs were transferred from plates which contained ample bacteria to water in a watch glass; alternatively, young eggs were obtained by cutting gravid hermaphrodites in water. With the help of a 50X dissecting microscope about 50 eggs of approximately the required age were selected; they were transferred to a layer of 5% agar, any that were in contact were moved apart with a fine hair, and they were viewed by Nomarski optics (Sulston and Horvitz, 1977; Sulston et al, 1980). A single egg which happened to be at the required stage and in an appropriate orientation was chosen for observation. Representative specimens are shown in Fig. 2.
FIG. 2. Photomicrographs of embryos, (a-h) Nomarski optics, Microflash, anterior to left. Bar = 10 micrometer. A few landmark features are marked on the photographs, but no attempt has been made to label all the cells; for reliable identifications the line drawings should be used, preferably in conjunction with at least some tracing of the lineage. (a) Just before first cleavage. Midplane. Male and female pronuclei are apposed; first polar body visible beneath eggshell at anterior end (where it typically but not invariably resides). (b) Beginning of gastrulation. Left lateral aspect, midplane; cf. Fig 5. Ea and Ep are moving dorsally, into the interior. P4 is recognisable by its small size, by the germinal plasm or nuage (arrowed; Strome and Wood, 1982; Krieg et al, 1978) around its nucleus, and by the distinctness of its nuclear membrane. (c) Late gastrulation (ca. 210 min). Ventral aspect, superficial plane; cf. Fig. 6. The cleft through which the MS cells have just entered is starred. (d) ca. 280 min. Dorsal aspect, superficial plane; cf. Fig. 7a. Small neuroblasts anteriorly; larger hypodermal cells, loaded with granules, posteriorly. Furrows can be seen between hypodermal cells (cf. Fig. 10). White arrow, dying ABarpaaapp; black arrows, ADEshL and ADEshR. (e) ca. 260 min. Ventral aspect, superficial plane; cf. Fig. 7b. Small neuroblasts over entire surface. Dying cells (arrowheads) are engulfed by their sisters (arrowed): ABplpappaa, ABplpppapa, ABprpppapa. (f) ca. 280 min. Dorsal aspect, midplane. int, cylinder of intestinal cells, nine nuclei in this focal plane, cytoplasm heavily loaded with granules; ph, cylinder of pharyngeal cells, less distinctive, contain few granules, (g) First movement (ca. 430 min). Left lateral aspect, midplane; cf. Fig. 8c. int, intestine; ph, pharynx; white arrowhead, anterior sensory depression; black arrowhead, rectum; arrows, dorsal hypodermal ridge, heavily loaded with granules, (h) Threefold, rolling. Only the anterior two-thirds of the embryo is in focus. White arrowhead points to mouth, linked by pharynx (ph) to intestine (int). Arrows point to germ cells. (i) ca. 430 min; electron micrograph of transverse section to show phagocytosed cells. Recent death (starred) lies within a ventral hypodermal cell; older death (arrowed) lies within a pharyngeal muscle. Pharyngeal lumen seen at upper right, surrounded by desmosomes between muscles and marginal cells. Outer surface of embryo, seen at lower left, is covered by a tenuous membrane in addition to the hypodermal basement membrane. Bar = 1 micrometer. (j) ca. 470 min; electron micrograph of transverse section, to show protrusion of lobes from germ cell (Z2) into two intestinal cells (int). Germ cells are united via lobes, but Z3 is not visible in this section. Germinal plasm or nuage (arrowheads; cf. Pig. 2b) visible around Z2 nucleus. Lumen (lu) of intestine is sealed by desmosomes; its wall carries microvilli. Bar = 1 micrometer.
T. aceti eggs normally develop within the parent; they are both mechanically and osmotically fragile, and were therefore mounted on 1% agar in isotonic medium. Agar was dissolved in boiling water to a concentration of 2%, cooled to about 50°C, and mixed with an equal volume of a solution containing 180 mM NaCl, 8 mM KC1, 36 mM CaCl2, 36 mM MgS04, 10 mM Hepes, pH 7.2 (cf Laufer et al, 1980). A single gravid adult was cut in half in water, and the extruded eggs were immediately pipetted onto a prepared agar layer. Even with these precautions, development continued for only about 7 hr, representing one-quarter of embryogenesis in T. aceti. However, the successive cell patterns were reproducible (and also similar to those of C. elegans), so the lineage could be traced unambiguously as described below (Strategy of Observation).
P. redivivus eggs were mounted like those of T. aceti, except that the gravid adults were placed directly on the agar layer and cut in half with scissors; the eggs were gently moved away from their parents with a fine hair.
A. blastophthorus eggs were teased out from agar/ fungal blocks into water, and mounted like those of C. elegans.
The egg shell excludes the usual fixatives and embedding media, and must be made permeable in some way before conventional methods can be applied.
The eggs examined in the course of the present work were first treated with NaOCl (2% available chlorine, 5-10 min); they were then fixed with Os04 (1%, 1 hr) and embedded and sectioned in the usual way (Ward et al, 1975). In an improved version of this method the egg is treated with chitinase or l-phenoxy-2-propanol after the NaOCl, and then prefixed with glutaraldehyde (Albertson and Thomson, 1982).
Permeabilisation with hypochlorite is always rather erratic; a better method for dealing with an individual egg is to puncture the shell with a laser microbeam (Schierenberg and Cole, in von Ehrenstein et al, 1981).
For reconstruction of the anterior sensilla, L4 larvae and adults were prepared as follows. The nematode was transferred to 3% glutaraldehyde in 0.1 M Hepes, pH 7.4, and immediately cut in the posterior half. After about 2 min a second cut was made in the anterior half; after 1 hr the head was washed three times with 0.1 M Hepes, pH 7.4, and then postfixed with 1% Os04 in the same buffer for 1 hr. The specimen was embedded as usual (Ward et al, 1975), and about 200 serial sections were cut from the anterior tip.
Strategy of Observation
All of the embryonic lineage was followed by direct observation. This method currently gives the best resolution in space and time, but has the disadvantage that the number of cells which can be followed in a single individual is limited by the short-term memory of the observer. Events were recorded by sketching the nuclei, using a colour code to indicate depth. A camera lucida was used at first, but the effective resolution was reduced by this accessory, and the additional illumination required tended to damage the specimen. The best aid proved to be a pair of gossamer cross hairs in one eyepiece, under which the nucleus of interest could be located with the help of a gliding stage. The light was blocked whenever the specimen was not being viewed.
The earlier part of the lineage was also analysed using videotape recordings,
and much of this work has already been described (Deppe et al, 1978). The
advantages of this technique are considerable: a permanent record is created, in
which the cell lineage can be followed at leisure and in which long-range
comparisons of cell movements and the timing of events can be made.
However, it was not possible to trace the later cell divisions, particularly those taking place in the interior of the embryo, in this way. In order to resolve a small dividing nucleus it is necessary to be able to focus through it without jitter or excessive electronic noise, and at frequent intervals; this is not easily achieved with current equipment. Nevertheless, videotaping remains the technique of choice for studying early em-bryogenesis both in the wild type and in mutants and experimental animals.
Fortunately for the direct observer, it is unnecessary to follow the lineage from the beginning for each terminal cell. The developing egg displays a succession of reproducible patterns, in which previously identified cells can be recognised. Some of the more useful ones are shown in Figs. 5-8. Although these diagrams were prepared with the aid of a camera lucida they are not intended to show the absolute positions of cells, which in any case vary appreciably from one individual to another; what is reproducible is the neighbourhood of each cell at a given time. The patterns change rapidly, but the behaviour of each cell is characteristic and provides an additional check on its identity. An inexperienced observer should be able to identify nuclei in the diagrams unambiguously by starting one division earlier and checking the arrangement of sister cells.
When placed on an agar layer under a coverslip, the embryo adopts a predictable, though age-dependent, orientation. At the four-cell stage it turns to display either the left or the right side; at gastrulation (100-150 min) it turns from left to dorsal or from right to ventral (these turns are only about 45°, because of the arrangement of blast cells); finally, at 350-400 min, the growing tail forces a return to a lateral aspect. No means of constraint compatible with good resolution was found to prevent these turns, but they were controlled by selection of obliquely oriented embryos at appropriate times.
Cell deaths were recognised by a characteristic increase in refractility, followed by shrinkage and disappearance (Sulston and Horvitz, 1977; Robertson and Thomson, 1982).
The final step in our analysis was to identify the surviving cells in terms of the known larval and adult anatomy. As many cells as possible were identified at 430 min (by comparison with serial section reconstructions of animals at this stage), because thereafter observation is much more difficult on account of movement of the embryo. The most reliable assignments at this time are for cells which have already formed desmosomes or other structural connections (i.e., hypodermis, body muscles, sensory nervous system, alimentary tract) but some useful clues to the identity of other neurons can be gleaned from the initial outgrowth of their processes.
The cells which were not identifiable at this stage (mainly interneurons and motorneurons) were followed in small groups until the animal hatched. These observations are tedious because from 450 min onwards the embryo rotates continually about its longitudinal axis, and it is necessary to train the eye to rapid pattern recognition for each cell group in turn. After hatching, some cells were identified from previously known L1 anatomy (J. G. White et al, unpublished) and the remainder were traced into the adult. This is a relatively easy task because the larvae do not rotate and the patterns change only slowly, so that an entire ganglion can be followed in one individual.
The lineage is based on a minimum of two direct observations for unique events, and one direct observation on each side for bilaterally symmetrical events. However, the great majority of events received considerably more confirmation than this. In addition, the earlier part of the lineage, extending to the terminal divisions in the intestine and the lateral hypodermis, was followed independently by videorecording.
Perhaps it is too much to hope that we have entirely avoided errors. It is worth emphasising, however, that any which have arisen are clerical in nature; there are no guesses, and the validity and reproducibility of a particular event can readily be checked by further observation.
The system of lineage nomenclature is essentially that described by Sulston and Horvitz (1977). Certain key blast cells are given arbitrary names comprising uppercase letters and numbers; their progeny are named by adding lowercase letters indicating the approximate division axis according to an orthogonal coordinate system (a, anterior; p, posterior; l, left; r, right; d, dorsal; v, ventral); the next generation of cells is named by appending further letters in the same way; and so on. Existing blast cell names have been retained as far as possible, but certain changes are desirable to avoid confusion whilst conforming to our system: MS was formerly MSt, U was E, Y was C, W was "P0.a," QL was Q1, and QR was Q2. It should be noted that AB and B are entirely separate names, as are P0-P4 and P1-P12. A pair of cells may be designated by the use of internal parentheses, e.g., MS(a/p)pa means MSapa and MSppa. Pairs of identical postembryonic cells, lying on the left and right sides of the animal, have previously been given identical names; they are now distinguished by the addition of symmetry operators, as defined in the next paragraph. Sometimes developmental stages are named after the number of progeny generated by a particular founder cell; e.g., MS8 means that MS has divided into eight cells.
Functional names are listed alphabetically in the Appendix, and follow a variety of systems:
Neurons and supporting cells other than those in the pharynx: White et al, (in preparation). This system is largely self-explanatory (see Appendix), but note that it uses symmetry operators as suffixes to distinguish cells which differ only in position (A, anterior; P, posterior; D, dorsal; V, ventral; L, left; R, right).
Pharynx: Albertson and Thomson (1976), with the addition of symmetry operators.
Hypodermis: See Figs. 12-14 ; symmetry operators are used for the arcades and hypodermal rings 1 and 2.
Muscle: see Fig. 15.
Gonad: Kimble and Hirsh (1979).
Intestine: see Fig. 8c.
In descriptions of cell division the terms "equal" and "unequal" refer to the relative sizes of the daughters; "symmetrical" means that the daughters not only are equal in size but also are (or subsequently become) disposed symmetrically across the midline; "equational" means that the daughters are supposedly of equal developmental potential.
Adapted by Yusuf KARABEY for WORMATLAS, 2003