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Results

(1) Gross Morphology
Like other nematodes (e.g., Chitwood and Chitwood, 1974), C. elegans has an elongated cylindrical body with tapered ends (figure 1 and figure 2). An external cuticle covers the hypodermal body wall. Beneath the hypodermis are four longitudinal rows of body muscles located subventrally and subdorsally. The mouth leads into the bilobed muscular pharynx, which pumps food into the tubular intestine to the rectum and anus. The nervous system consists of a circumpharyngeal nerve ring, dorsal and ventral nerve cords, and a variety of sensory receptors and ganglia.
The reproductive system of the adult hermaphrodite produces both sperm and oocytes. Two reflexed gonadal arms, each of which contains an ovary, oviduct, spermatheca, and uterus, terminate at the vulva, located midway along the ventral side. The reproductive system of the adult male consists of a single testis which empties into the vas deferens; the vas deferens unites posteriorly with the rectum, forming a cloaca. Accessory sexual structures are located in the male tail.

(2) Newly Hatched Hermaphrodite
(a) Ectoderm. The ectoderm, which comprises the majority of cells in the L1, can be broadly divided into the nervous system with its supporting cells and the hypodermis. Embedded in the hypodermis are various blast cells and a number of cells of specialized functions.
The hypodermis essentially consists of four longitudinal ridges (dorsal, ventral, lateral right, and lateral left) joined cir-cumferentially by thin sheets of cytoplasm which separate the muscle cells from the cuticle. Hypodermal nuclei, generally large and flat with large nucleoli (figure 3), are located in these four ridges. The dorsal ridge, which is filled with refractile granules in the late embryo and the young L1, normally contains nuclei only in the head. The ventral ridge contains hypodermal nuclei in the head and tail but not along the body in the young L1; as described below (see Cell Lineages, Ectoderm), a number of hypodermal nuclei are produced in the ventral ridge during later development. The lateral hypodermal ridges are almost bilaterally symmetrical and contain nuclei throughout their lengths. Twenty-four of these nuclei are arranged in six similar ventro-lateral pairs located on each side along the body; each pair includes one hypodermal precursor (V) and one precursor (P) of the ventral nervous system (figure 4 and figure 9). Anterior to the first of these pairs on each side there are two hypodermal precursors (H1 and H2). Each side of the tail has a single precursor (T). There are 11 other hypodermal nuclei on each side of the nematode. These nuclei are not bilaterally symmetrical in location; the set on the left appears to be displaced posteriorly relative to that on the right. Except for the precursor cells, almost all of the lateral hypodermal nuclei lie in a single syncytium (White, 1974). Some syncytial hypodermal nuclei do not occupy rigidly defined positions and seem to be moved passively during development.

Fig. 3 Left lateral hypodermis, posterior region of young L1 hermaphrodite, lateral view; Nomarski optics, hsn, hermaphrodite-specific neuron; sy, syncytial hypodermal nuclei. Bar = 20 micrometer.

Three gland cells (rect gl) (Chitwood and Chitwood, 1974) ring the junction between the intestine and the rectum (figure 4). Two other cells (B and F) are located dorsal to the rectum; these cells divide only in the male. Another pair of cells is located laterally; the cell on the left (K) divides in both hermaphrodites and males. Two additional cells (C and E) are located ventral to the rectum and posterior to the preanal ganglion. C lies more ventrally and further right than E. Both C and E divide only in the male.
The cells of the so-called excretory system (Bird, 1971) lie in close association with the hypodermis. The nucleus of the 'H"-shaped excretory cell (exc) lies just left of the midline, ventral to the rear bulb of :he pharynx. Immediately anterior to it is the nucleus of the duct cell. Two gland cells (exc gl) which feed into the excretory duct are located just posterior to the posterior-most region of the ventral ganglion. Although normally quiescent, the duct pulsates in dauer larvae. [The dauer larva is a developmental variant specialized for dispersal and/or survival in harsh conditions (Cassada and Russell, 1975)]. The rate of excretory duct pulsation in dauer larvae of other nematodes depends upon the osmolarity of the medium (e.g., Weinstein, 1952), indicating that one function of the "excretory" system is osmoregulation. In the L1, the nuclei of the nervous system are generally distinct morphologically from those of the hypodermis. Neurons and glial cell nuclei are small and granular with no visible nucleoli (figure 2, figure 7, and figure 8); in older larvae and adults, these nuclei often acquire visible nucleoli. The adult anterior sensory nervous system has been described in detail (Ward et al., 1975; Ware et al., 1975); we have observed no postembryonic cell divisions in this system. Its cell bodies lie in groups anterior and posterior to the ring of nerve fibers which runs circumferentially around the isthmus of the pharynx. The posterior groups, known as the lateral ganglia, also include some interneuron and motoneuron cell bodies. In the young L1, the amphid sheath cell bodies (Ward et al., 1975) lie behind the pharynx and are embedded in the hypodermis; during larval development, they gradually move anteriorly and medially (figure 4 and figure 9). The ventral ganglion contains two blast cells (G1 and G2) and lies ventral and posterior to the nerve ring, extending to the excretory glands. The sublateral ganglia lie dorsolateral to the excretory glands. The retrovesicular ganglion, with 12 mature nuclei and 1 neu-roblast (PO.a), lies ventral and posterior to the excretory cell and merges with the ventral nerve cord. The ventral nerve cord is the major longitudinal fiber bundle in the nematode and runs along the right side of the ventral hypodermal ridge; the dorsal nerve cord contains fewer fibers and runs along the left side of the dorsal hypodermal ridge. In the L1, 15 motoneuron cell bodies are present in the ventral cord. At the posterior end of the ventral cord is the pre-anal ganglion, which contains six nuclei. Two lumbar ganglia are present in the post-anal region; the ganglion on the left contains eight nuclei, and that on the right contains nine nuclei. A small dorso-rectal ganglion with two nuclei is also present in the tail.

FIG. 4. Young L1 hermaphrodite, lateral views, showing nuclei as seen in central, left, and right optical sections. Not all nuclei in the head ganglia are shown. These figures are somewhat schematic, as nuclei are not arranged precisely into three planes. Left-right pairs of P cells (e.g., Pl/2) are not finally named until after their subsequent migration into the ventral cord (see Cell Lineages, Development). bm, body muscle; cc, coelomocyte; dep, anal depressor muscle; exc, excretory cell; exc gl, excretory gland; hsn, hermaphrodite-specific neuron; im, intestinal muscle; rect gl, rectal gland; sph, anal sphincter muscle; sy, syncytial hypodermal nuclei.

On each lateral side of the nematode, four isolated cell bodies with neuronal-like nuclei are embedded in the hypodermis immediately beneath the cuticle (figure 4 and figure 9). The small subventral neuron (hsn) near the gonad primordiurn in the hermaphrodite is absent in the male (figure 5). Two other lateral neuronal-like cells are located (a) just anterior to V1 and (b) subdorsally, near V3. A fourth nucleus with similar morphology is present between V3 and V4, anterior to the hermaphrodite-specific neuron; although this nucelus becomes more hypodermal-like during development, the cell nonetheless appears to be a neuron or supporting cell in the adult.
Also lying subdorsally in the lateral hypodermis between V4 and V5 is a pair of neuroblasts: one on the left side (Q1) and one on the right side (Q2).

(b) Mesoderm. At hatching, the L1 contains 81 body muscle cells (bm) which control its locomotion. The nuclei of these cells are ovoid with a granular nucleo-plasm surrounding a spherical nucleolus (figure 6). During the L2 stage, the nucleo-plasm becomes smooth and remains so throughout the rest of development. The somatic muscle nuclei are distributed almost symmetrically in four longitudinal rows located subventrally and subdorsally (figure 4 and figure 23). Each dorsal quadrant contains 21 somatic muscle cells, the ventral right quadrant contains 20, and the ventral left quadrant contains 19. The ventral asymmetry appears to result from a gap on the left side somewhat posterior to the gonad primordiurn.
Four specialized muscle cells are present in the body of the newly hatched larva Two are intestinal muscles (im). These muscles run subventrally along the posterior-most region of the intestine and attach to it and to the thin layer of hypodermis just ventral to the lateral cords; their filaments are oriented longitudinally. The intestinal muscle nuclei are located slightly dorso-laterally to the ventral body muscle rows and are morphologically similar to but somewhat smaller than body muscle nuclei. Slightly posterior to these cells is a single saddle-shaped muscle (sph), which almost surrounds the posterior end of the intestine and probably functions as a sphincter muscle to control defecation. The nucleus of this sphincter muscle is located somewhat left of the central midline. An H-shaped "anal depressor" muscle cell (dep), with its nucleus located centrally in the crossbar of the H, is located posterior to the anus and opens the anus during defecation.
A single mesodermal blast cell (M) is present on the right side of the young L1 somewhat posterior to the gonad primordium (figure 6). This cell has a large, relatively flat nucleus and a large nucleolus.

FIG. 5. Anatomical differences between the young hermaphrodite and the young male. The top two figures show lateral views of the posterior halves of a young L1 male and hermaphrodite. The sex-specific characteristics shown are (a) position of a left coelomocyte (cc), (b) presence of hermaphrodite-specific neurons (hsn), and (c) size of the B and C nuclei. Four male-specific cephalic companion neurons, also present at hatching, are best seen during the L4 stage, as shown in the bottom figure (left lateral view). The position of H2.aa in the lateral ganglion is indicated.

Four coelomocytes (cc) (Chitwood and Chitwood, 1974) are also present in the L1. These glandular cells are located in the pseudocoelom adjacent to the somatic musculature. In the young L1, their nuclei are granulated and do not contain visible nucleoli (figure 6). During larval development, the cytoplasm of the coelomocytes acquires both granules (of high refractive index) and vacuoles (of low refractive index), giving these cells a very characteristic appearance (figure 6). In the L1 hermaphrodite, all four coelomocytes are located sub-ventrally between the pharynx and the gonad primordium; the two on the right are anterior to the two on the left (figure 4 and figure 23). In the male, one of the left coelomocytes is located posterior to the gonad primordium (figure 5). We consider these coelomocytes to be mesodermal in origin because, as described below (see Cell Lineages, Development), other coelomocytes are generated by blast cells which produce muscles during larval development.

FIG. 6. Mesoderm, left lateral views; Nomarski optics. Bar = 20 micrometer. (a) Body muscle (bm) and mesoblast (M) nuclei in young L1 hermaphrodite; go, gonad. (b) Ventral left coelomocyte (cc) in young L1 hermaphrodite. i, intestinal nucleus; nuc, nucleus; vac, vacuole. (c) Dorsal right coelomocyte (cc, M.drpa) in L4 hermaphrodite. Note opacity of the well-fed intestine.

Located centrally, dorsal to the rear bulb of the pharynx, is a single cell with a muscle-like nucleus and a nucleolus slightly smaller than that of a body muscle. This cell is often squashed in appearance. Electron micrographs reveal that this head mesodermal cell forms gap junctions (White et al., 1976) with adjacent body muscles.

(c) Intestine. The intestine of the L1 is a hollow tube which normally contains 20 large, round nuclei with large nucleoli. (figure 2); its cytoplasm is usually filled with refractile granules. Along most of the length of the intestine, nuclei are located dorsal and ventral to the lumen; in the anterior-most region, however, a ring of four nuclei surrounds the lumen. This anterior-most region has a thinner internal boundary than the rest of the intestine; electron micrographs reveal that the microvilli in this region are approximately half the length of those found elsewhere. The number of intestinal nuclei is not rigidly determined, as individuals with 19, 21, and 22 nuclei have been observed. The intestine has a left-handed twist of 180° about the longitudinal axis of the nematode in the region of the primordial gonad. As such a twist can introduce a left-handed superhelical twist, we suspect that the bilateral asymmetry of the position of the intestine -its anterior part is displaced slightly left of the midline, and its posterior part, slightly right -is caused by this rotational twist.
Pale at hatching, the cytoplasm of the intestine becomes filled with refractile droplets as the nematode feeds. Lethargus (or starvation), causes the intestine to again become relatively pale. Subsequent feeding restores the refractile droplets, which are therefore probably involved in energy storage. Because of the opacity of the well-fed intestine, hypodermal nuclei on the lower side of the nematode are difficult to observe between molts.

(d) Gonad. The gonad primordium consists of four cells in the young L1 (figure 4). The two larger cells are adjacent; the two smaller cells are slightly anterior and posterior to them, respectively. All four nuclei have relatively large nucleoli (figure 2). The gonad primordium lies obliquely, with its anterior cells located slightly right and its posterior ones located slightly left. This bilaterally asymmetric positioning is probably related to the twist in the intestine (see above, Intestine). The gonad primordium of the male is morphologically identical to that of the hermaphrodite.

(e) Pharynx. The morphology of the pharynx of the adult hermaphrodite has recently been described in detail (Albertson and Thomson, 1976). No postembryonic cell divisions or deaths have been seen in the pharynx.

(3) Newly Hatched Male
At hatching, the L1 male is very similar in structure to the L1 hermaphrodite. We have found three criteria by which the sex of a young nematode can be determined (figure 5): (1) In the male, a left coelomocyte lies posterior to the gonad primordium. (2) The male is missing a neuronal-like nucleus embedded in each side of the lateral hypodermis near the gonad primordium. (3) Ectodermal nuclei B and C near the anus enlarge soon after hatching in the male; these nuclei divide only in the male.
In addition, the male head contains four neuronal-like nuclei which are absent in the hermaphrodite. These nuclei are located close to the cell bodies of the cephalic neurons and presumably are the cell bodies of the four male-specific sensory processes in the cephalic sensilla discovered by Ward et al. (1975). Although present in the newly hatched larva, these "cephalic companion" cell nuclei are more easily distinguished in the L4, in which they have relatively large distinct nucleoli (figure 5), possibly reflecting increased activity at this stage of development.

(1) Cell Division
Although the size and shape of different blast cells vary, the overall process of cell division as observed with Nomarski optics during the postembryonic development of C. elegans is quite uniform (figure 7). For several hours before division, the nucleus of a blast cell generally has a prominent nucleolus and a smooth nucleoplasm. About 10 min before division, the nucleoplasm becomes coarsely granular, often producing a "rosette" around the nucleolus. In rapid succession during the next few minutes, the nucleolus disappears, the nuclear membrane breaks down, and the metaphase plate forms. During early anaphase, the plate splits and the two halves begin to move toward the poles; individual chromosomes are usually not visible. For a short time, nuclear components cannot be seen; the daughter nuclei then gradually appear. Finally, in some cells, daughter nucleoli are formed.

(2) Programmed Cell Death.
In a number of the ectodermal cell lineages, certain cells undergo a series of morphological changes which we interpret as programmed cell death (Saunders, 1966) (figure 8). Blobs appear at the perimeter of the nucleus. The nucleoplasm becomes less granular and somewhat more refrac-tile. There is then a marked increase in the refractility of the nucleus, giving it a characteristic flattened appearance. The internal refractility diminishes slightly, leaving a refractile shell at the surface of the nucleus. The nucleus shrinks and it gradually disappears. Generally, other nuclei completely obliterate the site, although in some cases a permanent gap remains. Either way, no subsequent trace of the dead cell can be detected either by Feulgen staining or by electron microscopy.
Most cell deaths are of posterior daughters from anterio-posterior divisions. As described below (see Cell Lineages), the pattern of cell deaths in the male differs from that in the hermaphrodite.

(3) Development
To the best of our knowledge, the results described in this section include all non-gonadal cell divisions and deaths that occur during the postembryonic development of C. elegans. However, very late developmental events in the male tail could have been missed. Because the detailed anatomy of the adult male tail is not known, many of its nuclei seen with the light microscope could not be assigned to specific cell types.
Cell lineages not specifically described for the male are identical to those which occur in the hermaphrodite.

FIG. 7 Cell division. Sequential photographs of an L1 hermaphrodite, lateral view; Nomarski optics vcn, ventral cord neurons. 0 min, interphase; 16 and 21 min, P10 prophase; 24 min, P10 metaphase; 26 min 10 anaphase; 27 mm, P10 telophase; 29 min, P9 prophase; 33 and 34 min, P9 metaphase. Bar = 20 micrometer

FIG. 8. Cell death. Sequential photographs of an L1 hermaphrodite, lateral view; Nomarski optics. The arrow points to the dying cell, P11.aap. Bar = 20 micrometer.

(a) Ectoderm.
V cells, hermaphrodite (figure 9 and figure 10): All 12 of the ventrolateral hypodermal precursor cells (V) divide about midway through the L1 stage. The anterior daughters enter the hypodermal syncytium. The posterior daughters remain separate from the syncytium; together with one head hypodermal cell (on each side) present at hatching, the posterior daughters form a distinct row of cells embedded in each lateral hypodermal ridge. Such cells have been named "seam" cells. Seam cells display a characteristic periodic activity. Toward the end of each intermolt period, the cytoplasm of the seam cells fills with refractile blobs (figure 11). This activity continues until midway through lethargus. That this activity is seen prior to the L4 molt (when the seam cells do not divide; see below) as well as prior to the other three molts (when the seam cells do divide) suggests that it is related to molting rather than to cell division.

FIG. 9. H, V, and T.a; hermaphrodite, lateral views; development of the left lateral hypodermis. Seam cells are indicated by dotted lines. hsn, hermaphrodite-specific neuron; plg, posterior lateral ganglion.

FIG. 10. H, V, and T lineages, hermaphrodite and male; development of the lateral hypodermis. All divisions are anterior-posterior. g, neuron or glial cell; se, seam; set, tail seam (a separate group of cells which shows seam-like cytoplasmic activity in the late L4 male); sy, syncytial.

FIG. 11. Seam cell. Cytoplasmic activity in H1.aa of late L4 hermaphrodite, lateral view; Nomarski optics. The seam is outlined with a dotted line. Bar = 20 micrometer.

The Vn.p seam cells undergo two rounds of division at about the time of the L1 molt; each produces a longitudinal row of four small nuclei. With the exception of V5.pap, the posterior daughters of the second round of divisions are themselves seam cells that divide at the L2 and L3 molts. The posterior daughter of each of these divisions is itself a seam cell. All of the other hypodermal progeny nuclei become part of the syncytium. In the L4 and adult, the seam cells form a continuous lateral band.
During the L2 stage, the lineage of V5 becomes distinct from that of the other V cells. On each side of the nematode, V5.paa and V5.pap undergo a series of divisions to produce four daughter nuclei. Together with two other nuclei on the left side (Q1.paa and Q1.pap; see below, Q Cells), these nuclei compose the posterior lateral ganglia, which consist of six cells on the left side and four cells on the right side. Ultrastructural studies of the posterior lateral ganglia show that they are positioned beneath the "postdeirids" (Chitwood and Chitwood, 1974) and contain the cell bodies associated with these sensilla. The proposed homology (Chitwood and Chitwood, 1974) between the postdeirids and the deirids in the head is supported by the observation that each of these sensilla has a single neuron containing the neuro-transmitter dopamine (Sulston et al., 1975). We have now identified the dopaminergic cell in each posterior lateral ganglion as being V5.paaa. This assignment was suggested by correlating the formaldehyde-induced fluorescence of the dopaminergic cell with the position of the V5.paaa cell body as visualized by Feulgen staining, using methods described previously (Sul ston et al., 1975); it was confirmed by killing selected cells of known lineage with a laser microbeam and showing that the death of V5.paaa or of any of its ancestors (but not of any other cell) leads to loss of the formaldehyde - induced fluorescence. These experiments failed to reveal any regulative capacity of the cells involved in the development of the posterior lateral ganglia; no extra divisions occurred to replace those cells destroyed with the laser. H cells, hermaphrodite (figure 9 and figure 10): The lineages of the hypodermal precursors of the head (H1 and H2) appear to be related to those of the V cells. Both generate seam cells as well as hypodermal nuclei in the syncytium, and both utilize similar stem cell patterns of cell division. However, there is an inversion of the pattern of the divisions of H1 compared to that of the V cells; the anterior daughter H1.a is the blast cell of the H1 lineage, whereas the posterior daughter Vn.p is the blast cell of the V lineage. It should be noted that H1.a and H1.p slowly reverse their anterior-posterior positions; however, this positional reversal is probably not the cause of the inversion of the pattern of divisions, because H1.a has the morphological appearance of a blast cell before any movement of its nucleus occurs. H1.aa has the appearance of a seam cell; unlike the "equivalent" Vn.pa cells, however, H1.aa does not divide. The lineage of H2, which is related to those of H1 and the V cells, produces seam cells, syncytial hypodermal nuclei, and one neuron-like cell (H2.aa), which is positioned in the mass of cell bodies posterior to the nerve ring (figure 5 and figure 9). That H1 and H2 together produce only half the number of syncytial hypodermal nuclei expected from two V cells may relate to the fact that postembryonic growth of the head is much less than that of the body (Hodgkin, 1974).

T cell, hermaphrodite (figure 9, figure 10, and figure 12): The tail precursor-cell T generates both hypodermal and nerve cells. Its anterior daughter T.a produces a lineage initially like that of a V cell; however, this similarity in the pattern and timing of cell divisions is not reflected in the morphology of the progeny. In the neuronal lineage of T.p (figure 10 and figure 12), there are two anterior-posterior positional inversions. First, about 1 hr after it is formed, T.pp begins to move slowly past T.pa. This migration appears to be an active process; neighboring cells seem to hinder rather than assist the movement. Later, T.pppap moves anterior to its sister; its nucleus becomes considerably deformed as it squeezes past other cells to reach its final position.
The fates of the progeny of the T cells in the adult have not yet all been established; at least some of these progeny appear to be associated with the phasmids, post-anal sensilla located on each side of the nematode (Chitwood and Chitwood, 1974).

FIG. 12. T, K, P11, and P12; hermaphrodite, left lateral views; development of the hermaphrodite tail. Only left and central nuclei are drawn.

Q cells (figure 13 and figure 14): The left and right lateral neuroblasts, Q1 and Q2, display bilaterally symmetrical patterns of cell division. However, their progeny are not symmetrical in their movements: Q2.a, Q2.p, and Q2.ap all migrate anteriorly, while Q1.ap migrates posteriorly.
The fate of Q2.pp is variable. In hermaphrodites, it becomes less and less distinct and usually disappears, although in one individual it was observed to survive into the adult. In two males, Q2.pp underwent typical programmed cell death. Perhaps its fate is weakly sex specific.

FIG. 13. Q, left lateral views; development of the lateral neuroblasts. Sometimes, Q2.paa and Q2.pap move directly to their final positions, without circling around one another.

FIG. 14. Q, G, K, and I lineages. Q1 and Q2 are lateral neuroblasts; G1 and G2 are in the ventral ganglion; K is in the tail; In are intestinal nuclei. Divisions are anterior-posterior unless otherwise indicated. g, neuron or glial cell; i, intestine.

P cells, hermaphrodite (figure 12, figure 15, figure 16, figure 17, and figure 18): The P precursor cells are responsible for the postembryonic development of the ventral nerve cord and its associated ganglia. One of us has described the development of these systems in detail elsewhere (Sulston, 1976). For completeness, we summarize these observations below.
About the middle of the L1 stage, the ventro-lateral P cells appear to be "loosened" from their hypodermal neighbors. Cytoplasmic extensions of the P cells grow into the ventral cord. The nuclei of P1 and P2 migrate into these extensions; the residual cytoplasm soon follows (figure 15). After these movements of P1 and P2, similar migrations of successively more posterior pairs occur roughly in order along the cord; P11 and P12 migrate into the pre-anal ganglion.
There is some variability in the anterior-posterior order of a given left-right pair of P cells after they have entered the ventral cord; in at least some cases, the subsequent development of a cell depends not upon its side of origin, but rather upon its anterior-posterior position. For this reason, we number the P cells after their migration into the cord (figure 16). We have determined the extent of this variability for the following pairs. P1 generally arrives from the right side (in 14 out of 16 individuals examined), P11 arrives from the left (7 out of 7), and the entries of P3 and P5 are approximately random.
Soon after its migration, each P cell divides. All 12 P cells follow identical initial programs of division, each producing five neurons and one hypodermal cell (figure 17). In addition, a single neuroblast in the retrovesicular ganglion follows a program identical to that of the anterior daughters of the P cells (Pn.a); by analogy, we have named this neuroblast PO.a.
As cell divisions continue, the neuroblasts and their progeny move freely past the juvenile cells and the new hypodermal cells (Pn.p); the extent of this movement is variable, leading to different patterns of intercalation of new neurons with the juvenile and hypodermal cells in different individuals. Gradually, the ventral cord becomes evenly filled with nuclei. Toward the end of the period of division, the pattern of nerve cells is modified by a fixed program of cell deaths in the anterior and posterior regions of the cord. There is also an extra division of the most posterior hy-podermal cell (P12.p), followed by the death of its posterior daughter (P12.pp).
Late during the L3 stage, the six ventral hypodermal nuclei (P3.p, P4.p, P5.p, P6.p, P7.p, P8.p) in close proximity to the developing gonad (figure 18) divide. The daughters of P5.p, P6.p, and P7.p move closer together and continue to divide to produce the 22 cells which form the vulva. The daughters of P3.p, P4.p, and P8.p become ventral hypodermal nuclei. In the adult, the ventral hypodermal nuclei formed during the development of the ventral cord lie in the hypodermal syncytium (White et al., 1976).

FIG. 15. Ventral cord and muscle development. L1 hermaphrodite, left lateral view, central plane; Nomarski optics. The nucleus of P10 is just entering the cord. The intestine is pinched between M.d and M.v. Bar = 20 micrometer.

FIG. 16. P and I; hermaphrodite, lateral views; development of the ventral nervous system and the intestine.

FIG. 17. P lineages, hermaphrodite and male; development of the ventral nervous system. Dotted lines indicate the times nuclei migrate into the ventral cord. P0.a is so named for convenience; its actual ancestry is unknown. Divisions are anterior-posterior unless otherwise indicated. g, neuron or glial cell; vh, ventral hypodermal cell.

FIG. 18. P3.p-P8.p; hermaphrodite, left lateral views midway along ventral side; development of the vulva.

The fine-structure neuroanatomy of the ventral cord of the adult C. elegans hermaphrodite has been elucidated by White et al. (1976). The neurons in the ventral cord can be assigned to morphologically distinct classes which recur periodically along the length of the cord. The number of cells of each class is constant in different individuals, but slight variations in the relative positions of different classes occur. These anatomical observations can be explained by the developmental processes described above according to the following model. Each morphological class consists of those cells with an equivalent lineage history (Table 1); the positional variations result from the differential migrations which occur during development. The correlation of cell type with lineage history has been directly confirmed by using serial-section electron micrographs to reconstruct the retrovesicular ganglion and part of the ven­tral cord of individuals of known lineage (Table 1). However, three daughters lo­cated in the retrovesicular ganglion are not allocated as expected from their li­neage history. As discussed below (see Discussion, Mechanisms of Determination), they may reflect end effects which seem to affect a number of aspects of the postembryonic development of C. elegans. G cells (figure 4 and figure 14): G1, located near the anterior end of the ventral ganglion, divides transversely late during the L1 stage to produce daughters with neuronal-like nuclei. Also in the ventral ganglion, G2 divides during the L2 stage. Like G1, G2.a then divides transversely and pro­duces two daughters with neuronal-like nuclei.

*Column 1 indicates morphological classes as defined by White et al. (1976). Column 2 shows the predicted lineage history of cells of each morphological class according to the model. Column 3 indicates neurons of each morphological class which, by their positions and neighbors, are consistent with having the lineage listed; to date, morphological assignments have been completed only for the anterior ventral cord (containing the progeny of PO.a-P6). Assignments for neurons in boldface in Column 3 have been established in individuals of known lineage. Three neurons located in the retrovesicular ganglion are not allocated as expected from this model: P0.aaaa and P1.aaaa are nonmotor neurons, and P0.apa is of morphological class VA. Three progeny (P0.aap, P1.aap, P2.aap) undergo programmed cell death and thus are not found in the adult. All electron microscopy was done by White et al. (1976).

K cell (figure 12 and figure 14): The solitary K precursor cell in the tail divides once about 3.5 hr before the L1 molt. K.p joins the two other neurons in the dorso-rectal ganglion. K.a is a hypodermal cell which completes a bilaterally symmetrical pair with a cell located right of the midline and present in the young L1.

Other cell deaths: On two occasions, other cell deaths were observed early during the L1 stage. One was immediately anterior to the excretory cell and the other was in the dorsal region of the lateral ganglion. These two cell deaths may normally be embryonic events which, in some individuals, are delayed until after hatching.

Male: Below, we first describe those ectodermal lineages which occur in the hermaphrodite and are modified in the male (V, T, P); we then describe male-specific lineages derived from cells present in the newly hatched L1 which do not divide in the hermaphrodite (B, F, C, E).

V and T cells, male (figure 10 and figure 19): The V and T lineages of the male are iden­tical to those of the hermaphrodite until L2 lethargus; thus, the male carries all tail and posterior lateral cell bodies found in the hermaphrodite. Further divisions in the V5, V6, and T lineages then generate nine similar sets of neuronal-like nuclei on each side of the nematode as well as a number of larger hypodermal-like nuclei. Numerically, these nine sets correspond to the nine "rays" found on each side of the male tail (figure 1 and figure 20). These rays lie in a noncellular cuticular webbing, which forms a leaf-shaped fan. The entire struc­ture, consisting of fan and rays, probably functions as a mechanosensory organ during copulation.
Preliminary experiments with the laser microbeam suggest how the V and T cell lineages lead to the development of the rays. Destruction of the precursor (e.g., V6.ppppp) of one of the nine sets of progeny cells with the laser results in the loss of a particular ray. In this way, the ancestry of the nine rays has been assigned (figure 10). Destruction of the precursor of ray 5, 7, or 9 also resulted in the loss of one of the three dopaminergic cells on that side of the male tail (Sulston etal., 1975). That only 6 of 18 morphologically similar rays have dopaminergic neurons suggests why mutant males lacking dopamine are still capable of mating (Sulston et al., 1975): at least some of the 12 other rays may act as alternative sensory elements.

FIG. 19. V5, V6, and T; male, left lateral views of the left side; development of the rays of the male tail. There is extensive rearrangement of the cells later, during L4 lethargus.

FIG. 20. Male tail, ventral view. One spicule is extended and one is retracted. As indicated, the sensory tips of the rays lie either on the ventral (inside) surface (indicated by a solid circle) or on the dorsal (outside) surface (dotted circle).

The precise lineages of the six dopaminergic cells were then determined by destroying some of the progeny of the precursors of rays 5, 7, and 9. Usually, destruction of "aaa" (e.g., V6.pppppaaa) eliminated the dopaminergic cell, but did not eliminate the ray; destruction of "apa" (e.g., V6.pppppapa) eliminated neither the dopaminergic cell nor the ray; and destruction of "app" (e.g., V6.pppppapp) eliminated the ray, but did not eliminate the dopaminergic cell. These results suggest that, in each set, aaa is the neuron and app forms the structural element. Destruction of any one of the hypodermal nuclei "p" (e.g., V6.pppppp) associated with these sets produced no lesion detectable in the light microscope. As in the development of the posterior lateral ganglia (see above, V cells, hermaphrodite), laser experiments failed to reveal any potential for regulation.
Although 18 similar lineages generate 18 morphologically similar rays, lineally equivalent daughters do not all differentiate identically: Only six of the aaa progeny are dopaminergic. It will be interesting to determine if the other aaa progeny are indeed neurons and, if so, to identify what transmitter(s) they employ.

P cells, male (figure 17 and figure 21): In the development of the male ventral nerve cord, there are fewer programmed cell deaths during the L1 stage and extra cell divisions during the L3 stage; both aspects of this "reprogramming" involve only the Pn.aap daughters. In these ways, the male produces 14 extra cells in the ventral cord.
In the male, ventral hypodermal nuclei P3.p-P8.p (which generate the vulva in the hermaphrodite) do not divide; instead, P10.p and P11.p divide to produce 16 male-specific nuclei in the region of the pre-anal ganglion. We do not know the fate of these nuclei in the adult male; preliminary observations suggest that some may be neurons. In 4 out of 17 individuals observed, P9.p divided once, yielding two ventral hypodermal nuclei.

B and F cells, male (figure 21 and figure 22): Some of the descendants of the B and F cells form the cloaca; others produce the copulatory spicules, elongated cuticular structures used to open the vulva of the hermaphrodite during mating (figure 1 and figure 20). The specific fates of the daughters of these lineages have not been established. Most of these progeny cells are probably structural, as they have clear nucleo-plasms with prominent nucleoli; a few, designated "g" in figure 22, are more compact with granular nucleoplasms and seem to be more like neurons or glial cells. However, unlike the neurons of the ventral cord and hermaphrodite tail, these cells often contain visible nucleoli soon after they are formed (possibly reflecting relatively rapid growth). Because of these ambiguities, any distinctions made between neurons and structural cells for these lineages are rather subjective.
B divides obliquely late during the L1 stage. During the L2 stage, its anterior-dorsal daughter (B.a) divides transversely; B.al and B.ar divide twice each, producing a row of four nuclei on the right and left sides of the tail. Both the anterior-most (B.alaa and B.araa) and posterior-most (B.alpp and B.arpp) of these nuclei recen-tralize. One member of each of these pairs becomes located anterior to the other, but there is no fixed pattern as to the order these cells assume; all four possible final configurations have been observed. The subsequent lineage of each of these four cells is determined not by its ancestry (whether it was derived from B.al or B.ar), but rather by its newly assumed position (anterior or posterior). In other words, positional influences play a determining role in the developmental fates of these cells.

E cell, male (WA editor's note; new name= U) (figure 21 and figure 22): E.lp and E.rp are associated with the vas deferens where it joins the cloaca. E.rap and E.raa are in the pre-anal ganglion. Usually, E.la remains posterior to the preanal ganglion, but, in two individuals, it behaved like E.ra and migrated anteriorly and divided.

FIG. 21. B, C, E, F, P10.p, and P11.p; male, lateral views; ectodermal development in the male tail. There is extensive rearrangement of the cells later, during L4 lethargus. Only left and central cells are shown.

FIG. 22. B, C, E, and F lineages, male; ectodermal development in the male tail. g, neuron or glial cell.

C cell, male (WA editor's note; new name= Y) (figure 21 and figure 22): The C cell generates compact, neuronal-like nuclei. C.a remains in the pre-anal ganglion, while C.p divides transversely and ultimately produces a pair of five-celled ganglia which flank the cloaca.

(b) Mesoderm.
M cell, hermaphrodite (figure 6, figure 23, figure 24, and figure 25): All of the postembryonic mesodermal cell lineages derive from a single mesoblast, M. M divides dorso-ventrally about midway through the L1 stage (figure 15). Three subsequent divisions generate 16 daughter cells, 4 in each of the muscle quadrants. Two of the ventral cells (M.vlpa and M.vrpa) divide longitudinally once more, so that by the L1 molt 18 progeny cells are present. During the L2 stage, two of the dorsal cells (M.dlpa and M.drpa) differentiate into coelomocytes. It is interesting that all four of these "specialized" cells are lineally equivalent daughters from the four muscle quadrants. Two of the daughters of the "extra" ventral divisions (M.vlpaa and M.vrpaa) migrate anteriorly. The other 14 progeny cells (6 dorsal, 8 ventral) become normal body muscles and are used for locomotion. They intercalate with juvenile body muscles posterior to the gonad primordium. The precise pattern of this intercalation varies among different individuals. A typical arrangement is shown in figure 24. The mature adult thus contains 95 body muscle cells; the ventral right quadrant and each dorsal quadrant contains 24 of these cells, and the ventral left quadrant contains 23.

FIG. 23. M; hermaphrodite, lateral views; mesodermal development during the L1 stage. bm, body muscle; cc, coelomocyte; dep, anal depressor muscle; im, intestinal muscle; sph, anal sphincter muscle.

M.vlpaa and M.vrpaa continue their movement until late during the L2 stage, stopping when they reach a position midway along the developing gonad. Late during the L3 stage, these sex myoblasts begin a series of three longitudinal divisions. Each cell produces 2 sets of 4 daughters by the L3 molt, generating a total of 16 new cells located in 4 sectors around the dorso-ventral axis of the developing vulva. In each set, the cell nearest the vulva (e.g.,M.vlpaaapp) becomes a type 1 vulval muscle (vml); its sister.
(M.vlpaaapa) becomes a type 2 vulval muscle (vm2). vml attaches to the vulva more ventrally than does vm2 and attaches to the body wall more dorsally than vm2; vm1 is located more superficially than vm2. The cell farthest from the vulva in each set (e.g., M.vlpaaaaa) becomes a type 2 uterine muscle (um2); its sister (m.vlpaaaap) becomes a type 1 uterine muscle (um1). Both um1 and um2 form thin sheets with most of their filaments in a circumferential orientation. um1 attaches to the ventral side of the uterus close to the vulva and to the lateral ridge of the body wall. um2 attaches solely to the uterus in a region further from the vulva; in contrast to um1, um2 extends to the dorsal side of the uterus.
During the L4 stage, the nuclei of the sex muscles assume their final positions around the developing gonad. During the L4 molt, all of the 16 sex muscle cells cytes twitch in characteristic directions. In the adult, these muscles are used in laying eggs.

FIG. 24. M; hermaphrodite, left lateral views of the left side; mesodermal development. cc, coelomocyte.

M cell, male (figure 25 and figure 26): The mesodermal cell lineage in the male begins exactly as in the hermaphrodite. However, cells homologous with those which in the hermaphrodite differentiate into coelomocytes (M.dlpa and M.drpa) are blast cells in the male. The six male sex mesoblasts grow in size and, during the L3 stage, migrate posteriorly. Late during the L3 stage, these 6 cells begin a series of divisions, producing a total of 42 daughters. Most of the progeny of the ventral cells move to positions along the ventral side of (M.dlpa and M.drpa) are blast cells in the male. The six male sex mesoblasts grow in size and, during the L3 stage, migrate posteriorly. Late during the L3 stage, these 6 cells begin a series of divisions, producing a total of 42 daughters. Most of the progeny of the ventral cells move to positions along the ventral side of the nematode. Some of the daughters of the posterior dorsal cells migrate posteriorly into the post-anal region of the tail; some of the others move ventrally. Most of the daughters of the anterior dorsal cells migrate to the ventral side; most then continue their movements, some in an anterior direction and some in a posterior direction. These migrations, which have not yet been followed in detail, often occur concomitantly with the divisions which generate these cells; there is substantial variation among individuals regarding the precise order of events as well as the orientation of the division planes of the migrating cells. For example, sometimes a given cell will migrate from the dorsal to the ventral side and then divide, whereas sometimes this same cell will divide on the dorsal side and then both of its daughters will migrate to the ventral side.
We have not yet identified these 42 daughter cells in the adult male, but we believe they must include a variety of male-specific muscles located in the tail and, usually, one coelomocyte (located near the dorsal midline somewhat anterior to the anus). Approximately 16 of the muscles are located anterior to the anus and run diagonally in a ventral-posterior to lateral-anterior direction. Other male-specific muscles control the movements of the spicules.

FIG. 25. M lineage, hermaphrodite and male; mesodermal development. Divisions are anterior-posterior unless otherwise indicated. bm, body muscle; cc, coelomocyte; um1, type 1 uterine muscle; um2, type 2 uterine muscle; vml, type 1 vulval muscle; vm2, type 2 vulval muscle.

FIG. 26. M; male tail, left lateral views of the left side; mesodermal development. cc, coelomocyte. The muscles continue to migrate in the L4.

(c) Intestine.
I cells (figure 14 and figure 16): Fifteen minutes after the beginning of L1 lethargus, the intestinal divisions take place. Normally, the six anterior-most nuclei do not divide; any of the four posterior-most nuclei may also fail to divide. Thus, division of 10-14 of the 20 juvenile intestinal nuclei produces the 30-34 nuclei normally found in the adult.
Other patterns of division occasionally occur. Once, for example, an intestinal nucleus located immediately behind the anterior six appeared to die during division; after anaphase, no daughter nuclei were formed. Concomitantly, the adjacent anterior nucleus divided, the only time this nucleus has been observed to do so.

(d) Gonad. The cell lineages of the gonad have not been determined. As suggested by Hirsh et al. (1976), the morphology of the gonad offers a convenient means of staging the postembryonic development of C. elegans. For comparison with the timing of other postembryonic events, various stages of gonad development in both the hermaphrodite and the male can be seen in figure 9, figure 24, and figure 26.

(4) Other Nematodes
Postembryonic developmental events similar to those we have observed in C. elegans also occur in other nematodes. We have studied the postembryonic development of a variety of nematode species in two ways: (1) observation of Feulgen-stained specimens to determine whether or not a postembryonic increase occurs in the number of cell bodies in the ventral cord, and (2) examination of living specimens using Nomarski optics to determine whether or not cell divisions could be observed directly. Based upon these criteria, postembryonic nongonadal cell divisions occur in all nematodes we have studied.

Panagrellus redivivus (the "sour-paste nematode") and Turbatrix aceti (the "vinegar eelworm") are free-living nematodes in a different family from that of C. elegans (de Coninck, 1965). Aphelencoides blastophthorus is plant parasitic and in a different order (de Coninck, 1965). All three of these species showed development of their ventral nerve cords and cell divisions in other tissues that appeared to be very similar to these events in C. elegans. A. blastophthorus may contain slightly more ventral cord neurons than C. elegans.

 Ascaris lumbricoides var. suis, an intestinal parasite of pigs, is in an order different from both C. elegans and A. blastophthorus (de Coninck, 1965). It also showed development in its ventral cord similar to that seen in C. elegans. For microscopy, embryos and L1 larvae were artificially released from eggs at different developmental stages as described under Materials and Methods. (Normally, A. lumbricoides larvae do not hatch until the L2 stage.). The first cleavage of the egg occurred on Day 4; by Day 14, the embryos had reached the "comma" or "tadpole" stage (Chitwood and Chitwood, 1974). At Day 17, the eggs contained moving larvae with nuclei sparsely distributed in the ventral cord. By Day 21, the number of nuclei in the ventral cord had increased severalfold to a number comparable to that in C. elegans L2 larvae. Some specimens with intermediate cord counts contained cells in the ventral cord in the process of division. Because of difficulties in visibility, cell divisions in A. lumbricoides were not observed using Nomarski optics.

Longidorus macrosoma, a raspberry ectoparasite, is in a different subclass from the other nematode species examined (de Coninck, 1965). Its ventral cord was quite distinctive, containing about 140 neuron-like nuclei in the L1. As in the other nematodes, substantial development occurs after the L1 stage, increasing the number of ventral cord neurons to about 1000 by the beginning of the L3 stage. Cell division was not observed using Nomarski optics.

We have not traced lineages in any of these other nematodes.
Postembryonic increases in the number of cells in the ventral cord, hypodermis, and intestine of the free-living nematode Rhabditis anomala have been described by Wessing (1953). The increase in the number of ventral cord nuclei in P. redivivus has been previously reported by Hechler (1970).

Adapted by Yusuf KARABEY for WORMATLAS, 2003