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The development of the embryo has proved to be essentially invariant (Fig. 3-See complete Fig. 3 here). There seems to be no naturally occurring indeterminacy like that found postembryonically in the gonad (Kimble and Hirsh, 1979), the ventral hypodermis, and the male tail (Sulston and Horvitz, 1977).
FIG. 3. The embryonic cell lineage of C. elegans. All interconnecting lines between the separate panels have been drawn, so that the pages can be copied, trimmed, and pasted together to give a complete chart. Vertical axis represents time at 20°C, from 0 min at first cleavage to 800 min at hatching. Many of the observations were made on eggs which were developing at slightly different rates (due to temperature variation and the effect of prolonged illumination); these primary results were normalised, by means of certain prominent cell divisions, to the course of events in eggs which were kept at 20°C and viewed infrequently. The precise times of individual events were not our primary concern, and should not be taken too seriously; the likely error varies from ±10% at the beginning of the lineage to ±2% at 400 min. Horizontal axis represents the direction of cell division. The majority of divisions have a marked anterior-posterior bias, and are shown with anterior to the left and posterior to the right, without any label. Only when this would lead to ambiguity in naming the daughters is an alternative direction indicated (l, left; r, right; d, dorsal; v, ventral); thus, our system is taxonomic rather than fully descriptive. The natural variation seen suggests that the precise direction of cell divisions is unimportant, at least in later development. Note that the daughters of a left-right division are not necessarily bilaterally symmetrical: for example, all the cells derived from ABalaaapa lie on the left of the animal and the right-hand daughters lie nearer the midline. Each terminal branch of the embryonic lineage is labelled either with X (indicating cell death; the position of the X on the time axis indicates the time of maximum refractility) or with a lineage name followed by a functional name. Large arrowheads denote cells which divide postembryonically, and small arrowheads denote nuclei which divide postembryonically, Symbols O and --- link precursors which give rise to bilaterally symmetrical groups of cells, the symbol ~ being included for cases of imperfect symmetry (see: Sublineages; Symmetry and Asymmetry), cord, ventral cord; gang, ganglion; lumb, lumbar; d-r, dorsorectal; p-a, preanal, r-v, retrovesicular; lat neur, isolated neuron lying laterally (WA editor's note: MSpappal is shown as MSpappaa in this diagram and MSpappar is shown as MSpappap (cf. FIG15)(R. Lee pers. comm., and Richards et al., 2013.)
FIG. 4. Marker events and count of living nuclei during embryogenesis. Fertilisation is normally at -50 min. Note that the comma stage is defined in a precise way, as the moment at which the ventral surface of the tail lies perpendicular to the long axis of the egg.
Cell Divisions and Cell Movement The majority of cell divisions take place during the first half of embryogenesis. During the second half, the embryo changes greatly in external appearance: it elongates more than threefold, moves actively, synthesises cuticle, and initiates pharyngeal pumping before breaking out of the egg. This does not mean, however, that in the first half of embryogenesis there is no differentiation. On the contrary, by 430 min (the stage shown in figure 8), gastrulation and organogenesis are complete and the majority of the desmosomes seen in the L1 larva have already been made; subsequent events involve principally stretching and functional maturation of cells.
The Founder Cells The fertilised egg cleaves into a larger anterior and a smaller posterior daughter. The latter (P1) is a stem- like cell which continues to cleave unequally for a further three rounds of division (figure 9); it is then named P4, and is the ancestor of the germ line. The anterior daughters AB, C, and D are called founder cells, and divide approximately equally with characteristic periods (Chitwood and Chitwood, 1974; Deppe at al, 1978); the anterior daughter EMS divides unequally into the founder cells MS and E, which then proceed to divide equally. At one time the various founder cells were thought to give rise to discrete tissue types and were named accordingly; as will be seen later, it is actually not possible to classify all of them in a precise way and so this terminology will not be used here. We use the term "founder cell" (Schierenberg and Cassada, 1982) in preference to the classical "stem cell," which nowadays has a rather different connotation; an alternative expression is "embryonic blast cell" (Laufer et al, 1980).
In addition to dividing with a characteristic period, the clone of cells derived from each founder cell behaves in a characteristic way, as follows:
AB. Starting from the anterior part of the egg, the cells spread over the entire surface except for the posterior dorsal region. Some of them lie inside the head temporarily at an early stage. Towards the end of gastrulation the AB pharyngeal precursors enter the interior through the ventral side of the head, and, later still, four muscles and the rectal cells sink into the ventral surface of the tail.
MS Divides to eight cells on the ventral surface. In the course of the next two rounds of division the pharyngeal precursors sink inwards and form two rows in the head; meanwhile, the body muscle, coelomocyte, and somatic gonad precursors insinuate themselves between the intestine and the surface layer of AB cells.
E. Generates only intestine. Divides ventrally into two cells, which are the first to enter the interior in the course of gastrulation. Further division leads to a cylinder of cells with distinctively granular cytoplasm lying along the body axis.
C. Starting from the posterior dorsal side of the embryo, the cells spread anteriorly and posteriorly. The most posterior ones are body muscle precursors, which travel round to the ventral side and enter the interior immediately after D. Most of the remaining cells are employed in forming the dorsal hypodermis over the posterior two-thirds of the body; their nuclei migrate contralaterally (see Migrations).
D. Generates only body muscle. Enters the interior after division to four cells, at the same time as MS(a/p)p.
P4. Generates only germ line. Enters the interior after E, and divides into two cells which extend lobes into the intestine (figure 17).
Gastrulation At 100 min after first cleavage, when the egg comprises 28 cells, gastrulation begins (figure 5). The first cells to enter the interior are Ea and Ep, which constitute the endoderm; they sink inwards from the ventral side, near the posterior end of the embryo. Next, at 120-200 min, are P4 and the progeny of MS. The entry zone widens and lengthens, spreading first posteriorly as most of the remaining myoblasts (derived from C and D) enter (180-230 min), and then anteriorly as the AB-derived part of the pharynx enters (210-250 min). The ventral cleft closes from posterior (230 min) to anterior (290 min). As gastrulation proceeds, the clone of E cells and the precursors of the pharynx form a central cylinder, while the body myoblasts insinuate themselves between this cylinder and the outer layer of cells. Although most of the myoblasts enter the body cavity during gastrulation, two do not. These are ABp(l/r)pppppa, which do not sink inwards until the time of their terminal divisions at 290 min.
FlGS. 5-8. Drawings of embryos. Circles and ovals represent nuclei, traced by
means of a camera lucida, the thickness of the lines being inversely related to
depth; outlines of the egg, embryo, and internal structures are traced with thin
lines (regardless of depth). Anterior is towards the top of the page. Dying
nuclei are stippled.
FIG. 5. Embryo, 100 min, left dorsal aspect; all nuclei included; cf. Fig. 2b. This stage has already been well characterised (Krieg et al, 1978), and the observer quickly learns to recognise all the nuclei; it is a useful starting point both for lineages and for ablation experiments. An embryo in the orientation shown will present a dorsal aspect until it turns at 350 min; an embryo with the MS cells uppermost will present a ventral aspect. The intestinal precursors are entering the interior, leaving a characteristic depression on the ventral side.
Later Cell Movements
At 250 min the ventral side is occupied largely by neuroblasts. During the following hour the latter undergo their last major round of division, and their progeny become covered by a sheet of hypodermis which grows circumferentially from a row of lateral cells on each side. Although there is a sufficient latero-ventral movement of cells to fill the gap left by the entry of the pharyngeal precursors, there is not a general flow of neural tissue through the ventral cleft. Dorsal and lateral neuroblasts sink directly inwards and their progeny become covered by adjacent hypodermal cells.
As in postembryonic development, many cells move short distances past their neighbours but only a few embark upon long-range migrations. It seems that the lineage not only generates cells of the correct types but also places them, for the most part, in appropriate positions. The way in which this may have come about is suggested later (see Conclusion).
The cells that migrate furthest are: the postembryonic mesoblast M and its contralateral homologue mu int R (figure 16); the somatic gonad precursors Z1 and Z4 (figure 16); CANL and CANR, which move from the anterior end to a point midway along the body (Figs. 7a, 8a, 14); HSNL and HSNR, which move from the posterior end to a point midway along the body (Figs. 8a, 14); ALML and ALMR, which move posteriorly from the anterior end of the intestine (Figs. 8a, 14). All these migrations are longitudinal. The most extensive circumferential migration is that of the head mesodermal cell (hmc) and its contralateral homologue, but many of the body muscles also move circumferentially as they assemble into rows.
FIG. 6. Embryo, 200 min, ventral aspect, late gastrulation. The orientation of the egg can be recognised by the prominent ventral cleft; cf. Fig. 2c. All progeny of MS, E, D, and P4 are shown, together with AB-derived pharyngeal precursors and C-derived myoblasts; other AB progeny (unlabelled) are included for perspective. MSaa and MSpa have each formed a chain of eight cells, of which all but the posterior two have entered the interior. The derivatives of ABaraap have also entered, and the remainder of the AB pharyngeal precursors will soon follow. Posteriorly, the mesodermal precursors derived from C and D are also entering the cleft. The germ line and the intestine can be seen lying more dorsally. The division of ABplpappa (earlier than its neighbours and very unequal) is a useful time point. Embryos of this age, but viewed from the dorsal side, were used as starting points for lineages of the dorsal pharynx and dorsal body muscle.
FIG. 7a. Embryo, 260 min, dorsal aspect, superficial nuclei; cf. Fig. 2d. Landmarks: nuclei of hyp4-hyp7, cell deaths; time points: division of various neuroblasts. The dorsal hypodermal cells have very granular cytoplasm and form prominent transverse ridges. In figure 7 some licence has been allowed in depicting cell deaths, because of their importance in pattern recognition; in fact, they do not all become refractile simultaneously. WA editors' note: the positions of the descendants of Caaa and Cpaa are inadvertantly reversed in this fig (Loer C. et al, WBG 10(3)120).
FIG. 7b.Embryo, 270 min, ventral aspect, superficial nuclei; cf. Fig2e. Landmarks: excretory cell, cell deaths; time point: division of mother of excretory cell. The gap anterior to the excretory cell contains pharyngeal and buccal precursors which are entering the interior.
The group of cells which forms most of the dorsal hypodermis exhibits nuclear migration as opposed to cellular migration (see figure 10). These cells are born subdorsally, in two longitudinal rows; a cytoplasmic process grows circumferentially from each cell across the dorsal midline, and after a time the nucleus migrates along this process until it lies on the opposite side of the embryo. This type of migration is analogous to that seen postembryonically in the P cells (Sulston and Horvitz, 1977), and it is therefore interesting that mutations at two loci interfere with both processes (Sulston and Horvitz, 1981).
Programmed Cell Death
In the course of the lineage, one in six of all cells produced subsequently dies; their identity and the approximate times of their deaths are predictable. The mode of death is similar to that seen previously in the postembryonic lineages (Sulston and Horvitz, 1977; Robertson and Thomson, 1982). In some cases death occurs several hours after birth, so that it is possible for the cells to function in some manner before being discarded. A good example is the pair of tail spike cells, which fuse together, form a slender bundle of filaments in the tip of the tail, and then die. At the other extreme are the majority of programmed deaths, which occur 20 to 30 min after birth; these cells are born with very little cytoplasm, and die without differentiating in any obvious way. The limited sexual dimorphism seen in the embryo is a consequence of differential cell death (see Nervous System).
All the dying cells are promptly phagocytosed by their neighbours (cf. Robertson and Thomson, 1982). During the first wave of deaths, when the surviving cells are relatively large and rounded, this process can be followed by light microscopy; the engulfing cell is almost always the sister of the dying cell at this time (Fig. 2e). Later on, cell boundaries cannot usually be resolved by light microscopy but electron micrographs show that all dying cells (identified by their high electron density) lie within other cells (Fig. 2i); the principal phagocytes now seem to be the hypodermis (both seams and syn-cytia) and the pharyngeal muscles, though one death (possibly MSpaapp, which tends to be delayed) was found in the anterior intestine.
Other Nematode Species
Previous research showed convincingly that the pattern of early cleavage was uniform in the nematodes examined (all of which belong to the class Secernentea (Chitwood and Chitwood, 1974). However, authors working on different species disagreed about the tissues to which certain founder cells gave rise; these disagreements may have been due either to the difficulties of interpreting observations on fixed specimens or to genuine differences between the species.
The most interesting discrepancy is in the origin of the somatic gonad, which was reported to arise from the founder cell P4 in the nematodes Turbatrix aceti (Pai, 1927), Ascaris megalocephala (Boveri, 1892; but not sustained in later reports), and Bradynema rigidum (zur Strassen, 1959). Given the consistency of the early cleavage pattern of nematode embryos, a switch in the origin of such a vital tissue would be surprising indeed. Other discrepancies are in the fates of C progeny (said to be only five, and exclusively ectodermal, in T. aceti (Pai, 1927)) and D progeny (reported to form the rectum, by several authors; reviewed by Chitwood and Chitwood (1974)). However, the latter assignment seems to be hypothetical, since it rests solely on the observation that the D cells enter the interior of the embryo. In order to go some way towards resolving these uncertainties, we have followed a few lineages in other nematode species.
The results for T. aceti are shown in fig. 11. We find that this nematode is identical with C. elegans in the following respects: the origin of the somatic gonad and the germ line (cell assignments based on morphology at hatching); the origin and behaviour of "M" and its contralateral homologue (followed to the equivalent of 320 min, cf. figure 16); the behaviour of the progeny of C and D (followed explicitly to the equivalent of 230 min, by which time the mesoblasts are distinctive). T. aceti differs from C. elegans within the endoderm lineage, and it will not be surprising if it also differs in details of the MS and AB lineages.
We have also followed the origin of the founder cells and certain later lineages in the embryo of Panagrellus redivivus. This nematode is of interest because Sternberg and Horvitz (1981,1982) have shown that its postembryonic lineage is quite similar to that of C. elegans, and that the newly hatched animal contains the same set of blast cells with one addition. We find that this extra blast cell, known as T3, has the embryonic ancestry Caappa; it is therefore homologous with the neuron PVR of C. elegans. On the basis of Nomarski microscopy, PVR is absent from P. redivivus (Sternberg, personal communication). P. redivivus proved to have the same endodermal lineage as T. aceti, to which it is closely related (Ritter, 1975); in one individual, however, division of Ea(1/r)ap resulted in an intestine having 20 cells instead of the usual 18.
zur Strassen (1959) based his conclusion -that in B. rigidum P4 yields the somatic gonad- upon the observations that P4 divides in the embryo and that the newly hatched larva contains only one germ cell. Although we were not able to obtain B. rigidum, we investigated another member of the order Tylenchida, Aphelencoides blastophthorus, which similarly contains a single germ cell at hatching. We find that P4 does not divide in the embryo of A. blastophthorus, and becomes the solitary larval germ cell. Comparison with the detailed drawings provided by zur Strassen (1892) shows that the early cleavages of the two nematodes are very similar and reveals the reason for the discrepancy: he had inadvertently reversed anterior and posterior, so that the dividing cell which he took to be P4 was in fact one of the AB group.
In conclusion, although differences of detail have been seen, the general pattern of cell fates shown in figure 9 is correct for the two rhabditids C. elegans and T. aceti, and may well be conserved throughout the class Secernentea.
The hypodermis is a sheet of cells which forms the outer surface of the nematode and secretes the cuticle. Its postembryonic development has been described by Sulston and Horvitz (1977), and other aspects have been discussed by White (1974) and by Singh and Sulston (1978), but no comprehensive account has previously been given.
FIG. 8. This figure illustrates the arrangement of all left and
central nuclei at 430 min after first cleavage; the right-hand side is a mirror
image of the left, except where otherwise indicated. The three parts roughly
represent three planes of focus (from superficial to central), but there is
considerable overlap between them. The anterior sensory depression (not a mouth
opening -see text) is at the top and the lengthening tail, terminating in the
spike, curves round to the left. On the ventral side of the tail the rectal
opening has appeared, and on the ventral side of the head the excretory duct
leads to the excretory pore. Since the major hypodermal migrations are complete
at this stage, the ancestry of nuclei in hyp3-hyp11 can be inferred from figure
FIG. 8a. Left lateral ectoderm. No midplane nuclei are included, but the outlines of the pharynx, intestine, and gonad are shown for reference. Nuclei which will soon divide are labelled with the names of both presumptive daughters. The parent of QL and V5L is named QV5L. The pattern on the right is identical, except that: the homologue of hyp11 is PVR; an extra hyp7 nucleus (ABarpaappp) lies dorsal to H2R; there is no hyp2 DR.
FIG. 8b. Left and central mesoderm (excluding pharynx). M, mu int R, hmc, and MSpppaaa lie in the midplane. Unlabelled nuclei are in body muscles. The pattern on the right is identical, except that ABprpppppaa and ABprpppppap become a body muscle and the sphincter muscle, respectively, and lie slightly more anteriorly than their left-hand homologues.
FIG. 8c. Intestine, gonad, left central pharynx, and ectoderm; cf. Fig. 2g. The pattern on the right is identical, except that: the homologue of G2 is W, of 16 is M1, of U is B, and of K is K'; an asymmetric neuron RIS (ABprpappapa) lies anterior to AVKR.
In the newly hatched L1 the anterior part of the hypodermis consists of a series of cylindrical syncytia linked together by desmosomes. These cylinders are numbered hyp1 to hyp7 from the mouth posteriorly (figure 12). The anterior and posterior arcades, which are historical names for the specialised hypodermis which lines the mouth (Chitwood and Chitwood, 1974), follow the same plan. hyp7 extends back on the dorsal side and encircles the body again at the anus; the midventral surface is occupied by the P cells (ventral cord blast cells). The tail is completed by three mononucleate cells (hyp8,9,11) and a binucleate cell (hyp10). All the hypodermal syncytia arise as mononucleate cells which subsequently fuse together.
FIG. 9. Generation of the founder cells, and a summary of cell types derived from them. Areas of circles and sectors are proportional to number of cells. Stippling represents typically ectodermal tissue and striping typically mesodermal tissue.
On each side of the animal there is a longitudinal row of specialised hypodermal cells, called seam cells (H0-H2, V1-V6, T); they remain separate from the rest of the hypodermis, and are responsible for making the lateral cuticular ridges known as alae (Singh and Sulston, 1978). All except HO are blast cells in the wild type, and even HO has been seen to divide in certain mutants (E. Hedgecock, personal communication).
The location of the larger hypodermal nuclei in the L1 is shown in Figs. 13 and 14. During postembryonic development many more nuclei, generated by division of the seam cells and P cells, are added to hyp7, which comes to occupy most of the body surface. For this reason hyp7 has been called the large hypodermal syncytium.
Embryonically, the hypodermis is derived from the founder cells AB and C. The dorsal nuclei undergo a strange contralateral migration in midembryogenesis (see Migrations). The finely tapering spike of the tail is formed by a process which passes posteriorly through hyp10 and contains a bundle of filaments; the process is formed by a binucleate cell (ABp(l/r)ppppppa) which subsequently dies. The dorsal ridge of hyp7 seems to act as a storage organ during late embryogenesis; the concentration of refractile granules in it becomes more and more marked while that in the intestine diminishes.
The pattern of generation of the postembryonic blast cells (nearly all of which are hypodermal at hatching) is more intriguing than informative. Most of the seam cells are made from ABarpp, but V3 and V5 are closely related to the P cells. The special origin of V5 is not unexpected, in view of its unique postembryonic programme (Sulston and Horvitz, 1977); on the other hand, there is no known difference in behaviour between V3 and the other seam cells. By ancestry, the P cells fall neatly into the equivalence groups previously revealed by postembryonic laser ablation (Sulston and White, 1980); embryonic laser ablations are consistent with the hypothesis that the equivalence groups are determined cell autonomously, although these experiments are not conclusive (see Cell Interaction Experiments).
FIG. 10. Contralateral migration of dorsal hypodermal nuclei. Dorsal aspect. Cell boundaries visible by Nomarski microscopy (because of surface depression) are shown as dotted lines. Numbers are to provide continuity and have no other significance. Cpapp(a/p) and Caapp(a/p) (see figure 7b) countermigrate similarly, but are only visible from the ventral side at this stage. WA editors' note: the positions of the descendants of Caaa and Cpaa are inadvertantly reversed in this fig (Loer C. et al, WBG 10(3)120).
It is likely that the hypodermis is primarily responsible for the overall architecture of the animal, but the way in which it achieves this is largely unknown. One hint comes from ablation experiments in the head, which suggest that tension in the head hypodermis is necessary for elongation of the tail (see Cell Interaction Experiments). Certainly the cuticle is not involved in the shaping process, because the first sign of cuticle formation is at 600-650 min. At this time the seam cells acquire large Golgi bodies visible by Nomarski microscopy (cf Singh and Sulston, 1978), and cuticle can be seen at the mouth and in the rectum. The paired lateral alae characteristic of the L1 do not appear until just before hatching, and are apparently generated by circumferential contraction of the seam-specific cuticle (as is the case for dauer larva alae (Singh and Sulston, 1978)).
FIG. 11. Partial embryonic lineage of Turbatrix aceti. Conventions as for Fig. 3. Times (at 22°C) are approximate, for reasons given in text; the time scale is adjusted to facilitate comparison with Fig. 3. Arrows represent continued division, not followed.
The excretory system has been described by Mounier (1981) and Nelson et al. (1983). It is derived from AB. Internally, it comprises four cells: the excretory cell, the duct cell, and two gland cells (which eventually become fused (Nelson et al, 1983)). A fifth cell surrounds the excretory pore, and forms the interface between the excretory duct and the hypodermal syncytia: it is known as the excretory socket or pore cell. In the embryo, the socket cell is G1; after hatching, G1 becomes a neuroblast and the socket function is taken over by G2; finally, G2 divides into a neuroblast (G2.a) and the mature excretory socket (G2.p).
Regulative interaction is seen within the pairs excretory duct/G1 and G2/W (see Cell Interaction Experiments).
The complete adult nervous system has been described by White et al. (in preparation). Regions which were described previously are: the anterior nervous system (Ward et al, 1975; Ware et al, 1975); the ventral cord (White et al, 1976); the pharynx (Albertson and Thomson, 1976); the male tail (Sulston et al, 1980).
FIG. 12. Schematic longitudinal section to illustrate cells and syncytia forming surface coverings of newly hatched L1. Part of the digestive tract is dotted to indicate that boundaries are not shown; for details of this region see Albertson and Thomson (1976) (pharynx) and figure 17 (remainder). Inset (above) indicates three-dimensional arrangement in central region; the four longitudinal grooves between hypodermis and intestine are occupied by body muscle. Commas indicate two cells meeting in plane of drawing. During postembryonic development: hyp7 enlarges (by fusion) into the P-cell region; in the hermaphrodite a vulva is formed and Y becomes a neuron; in the male there are extensive changes in the tail (Sulston and Horvitz, 1977; Sulston et al, 1980).
Of the 222 neurons present in the newly hatched L1 hermaphrodite, 2 arise from founder cell C, 6 from MS, and the rest from AB. All the supporting cells of the sensilla arise from AB.
FIG. 13. Arrangement of larger hypodermal nuclei in newly hatched L1. Schematic cylindrical projection, viewed from within animal. Brackets indicate pairs of nuclei, of bilaterally symmetrical origin, whose anterior-posterior ordering is uncertain. WA editors' note: the positions of the descendants of Caaa and Cpaa and ABarppaapa and ABarpppapa are inadvertantly reversed in this fig (Loer C. et al, WBG 10(3)120).
The lineage patterns are complex, and will be discussed later. Most neurons are born fairly close to their ultimate positions, though a few migrate long distances (see Migrations) and many migrate short distances relative to their neighbours. There are two occasions upon which mass movements of neuroblasts and neurons are noticeable. The first (230-290 min) leads to closure of the ventral cleft at the end of gastrulation. In the second (about 400 min) the anterior neurons move towards the tip of the head, and the rudiments of the sensilla are formed; the neurons then move posteriorly again, the sensory cell bodies laying down their dendritic processes as they go. At the same time, a depression appears in the tip of the head (figure 8c); this does not involve mor-phogenetic cell death, and is presumably a way of providing more surface area for the sensilla. The depression is not a primordial mouth, because it subsequently everts; the buccal cavity arises further inside, between the arcade cells.
FIG. 14. Arrangement of neuronal and larger hypodermal nuclei in newly hatched L1; based on camera lucida drawings, (a) Entire animal: left lateral aspect. Pattern on the right is identical, except that: additional hyp7 nucleus (ABarpaappp) lies dorsal to H2R; homologue of QL is QR, of hyp11 is PVR. (b) Ring, ventral, and retrovesicular ganglia: left lateral aspect. Note that arrangement of ring ganglion cells around posterior bulb of pharynx is very variable at this stage. Anatomy anterior to the ring is not wholly known, since cells in this region were mostly identified by their processes in the embryo. (c) Ventral and retrovesicular ganglia: ventral aspect, (d) Preanal and left lumbar ganglia, rectal cells: left lateral aspect. WA editors' note: the positions of the descendants of Caaa and Cpaa are inadvertantly reversed in this fig (Loer C. et al, WBG 10(3)120).
After 430 min the tip of the head elongates and the pharynx grows forward through the mass of neurons surrounding the developing nerve ring; at the same time the head becomes thinner. The pattern of neurons changes rapidly at first but stabilises after about 2 hr. In late embryogenesis it is possible to recognise all the neurons in the ring ganglion by their positions; at hatching the arrangement of the most posterior ones changes in an unpredictable way, perhaps as a result of pharyngeal movements.
FIG. 15. Arrangement of body muscles at 430 min; schematic cylindrical projection, viewed from within animal. The shapes of the muscles are not intended to be realistic, but each cell is defined uniquely by its position in the pattern. Note that, with the exception of ABprpppppaa, the assignments are bilaterally symmetrical (WA editor's note: MSpappaa is shown as MSpappal in this diagram and MSpappap is shown as MSpappar (cf. FIG3)(R. Lee pers. comm., and Richards et al., 2013.)
At about 470 min sexual dimorphism becomes visible for the first time: in the hermaphrodite the cephalic companions (CEM) die, whilst in the male the hermaphrodite-specific neurons (HSN) die. It appears that these decisions are not made at the time that the cells are born, because all six behave at first in the same way in both sexes. In the hermaphrodite the CEMs have time to grow into the cephalic sensilla, where they form desmosomes with the sheaths and the cephalic neurons; in the male the HSNs migrate anteriorly at the same rate as they do in the hermaphrodite.
In postembryonic development a periodically repeated sublineage generates five classes of motorneurons in the ventral cord (Sulston and Horvitz, 1977; White et al, 1976). In the embryo, however, there is no such repeated sublineage to produce the three classes of juvenile motorneurons (DA, DB, and DD) which are interspersed along the ventral cord (figure 14). All that can be said is that classes DA (together with SAB, the analogue of DA in the retrovesicular ganglion) and DD are each generated semiclonally, whilst DB neurons have a variety of unique origins and are not closely related to one another.
Mesoderm (Excluding the Pharynx)
The anatomy of the larval mesoderm has been described by Sulston and Horvitz (1977).
Of the 81 body muscles present in the L1, 80 are generated in a symmetrical fashion by MS, C, and D; the remaining one is generated by AB. A schematic cylindrical projection of their arrangement is shown in figure 15. The pattern of overlaps between the spindle-shaped cells is already apparent at 430 min, and allows unambiguous assignment at this stage.
The unique AB body muscle is one of a group of four muscles generated preanally by AB. The two mother cells of this group (ABp(l/r)pppppa) remain on the outside of the embryo until their division at 295 min. The other three members of the group become the anal muscle, the sphincter muscle, and one of the two intestinal muscles.
The postembryonic mesoblast (M) is born on the left, next to the pharynx. It migrates posteriorly, following a distinctive path between the two germ line cells (figure 16); it remains on the midline for some time, but then gradually shifts to the right-hand side of the intestine. The contralateral homologue of M migrates in a similar way, preceding M along the midline between the germ cells, but then differentiates into the second intestinal muscle.
The head mesodermal cell (hmc) is one of a pair of homologues (sisters to the somatic gonad cells) which migrate to the dorsal midline. There the two cells align themselves anterior-posteriorly and appear very similar until late embryogenesis, when the anterior one dies.
FIG. 16. Migration of M, its contralateral homologue (mu int R) and the cells of the gonad from 250 to 400 min. Ventral aspect. Nuclei are drawn at 320 min.
The four coelomocytes are generated symmetrically. Their reproducible and sexually specific arrangement arises as a result of later movements, the reasons for which are not understood.
The alimentary tract is a single tube which comprises the following components: (mouth), buccal cavity, pharynx, pharyngo-intestinal valve, intestine, intestino-rectal valve, rectum, (anus). Part of it is shown schematically in figure 17.
Buccal cavity, pharynx, and pharyngo-intestinal valve. The pharynx is a pump which ingests bacteria and crushes them; it is a complex organ, comprising muscles, structural cells, neurons, and glands. Its anatomy in the adult has been described by Albertson and Thomson (1976); their account needs amendment only in that the m2 muscles are binucleate. The buccal cavity, which is formed by the arcade cells and the anterior end of the pharynx, has been described by Wright and Thomson (1981).
FIG. 17. Part of alimentary tract, 430 min, schematic. Except for absence of germ line lobes, arrangement is similar in L1. int, rep, vpi1, and vpi2 bear microvilli on their inner surfaces.
All the mechanical elements of these tissues (arcade, epidermal cells, muscles, marginal cells, valve) can be recognised in the 430-min embryo by the pattern of desmosomes which they form. The neurons and glands of the pharynx were followed until they settled into the mature pattern of nuclei which persists essentially unchanged from late embryogenesis to the adult. A series of cell fusions, which take place either before or soon after hatching, yield the multinucleate cells seen in the adult. In muscle class m1, all six cells fuse together; in each of the muscle classes m2, m3, m4, and m5, the six cells fuse in pairs -DL with DR, L with VL, and R with VR; in gland class g1, AR fuses with P.
The arcade, pharynx, and pharyngo-intestinal valve are generated by two granddaughters of MS and three great-great-granddaughters of AB. These precursors, however, do not yield exclusively these tissues. At the anterior end, there are no obvious lineal boundaries between the future hypodermis, arcade, and pharynx, in spite of the specialisations which become apparent later. Conversely, there is no functional boundary between pharyngeal components derived from MS and those derived from AB. For example, apparently identical cells arise respectively from MS and AB in muscle rings m3, m4, and m5 (see Appendix), and indeed three MS muscle cells go so far as to fuse with seemingly identical AB partners (m4VR with m4R, m5VL with m5L, m5VR with m5R).
Descendants of the precursors enter the body cavity from the ventral side during late gastrulation. First to enter are the MS cells (120-200 min), next are the ABaraap cells (210 min), and last are the remaining AB cells (220-250 min). At first the dividing cells form a cylinder anterior to the intestine; gradually a distinct boundary appears at the surface of the developing pharynx; then, at about 400 min, it is compressed posteriorly and becomes almost spherical, but subsequently it gradually elongates, first anteriorly and then posteriorly. The transient compression coincides with a flux of anterior sensory neurons towards the tip of the head (see Nervous System) but the causal relationship between these events is unknown.
The three g1 gland cells migrate in a reproducible way. Their movements approximately follow the subsequent course of their secretory processes, and may be responsible for laying down the latter. The cell bodies of the anterior muscles and epidermis also move substantially in late embryogenesis (compare figure 8c with Albertson and Thomson (1976)).
The structural elements of the mature pharynx have an exact threefold rotational axis of symmetry, yet there is no trace of a threefold axis in their lineages; rather, the lineages show approximate bilateral symmetry and the third symmetry element arises by piecemeal recruitment of cells.
After 430 min (figure 8c) the pharynx continues to elongate, and within an hour the two bulbs and the isthmus are apparent. A refractile thread gradually appears along the axis of the pharynx and protrudes from the mouth. At 600-650 min the formation of L1 cuticle begins. The straight-sided cylinder of the buccal cavity appears, still plugged by the tip of the thread, and the pharyngeal lumen becomes outlined. One hour before hatching, the g1 glands become active, just as they do before ecdysis (Singh and Sulston, 1978). Half an hour later the pharynx begins to pump spasmodically, the mouth plug falls away, and the refractile thread is broken up and discharged into the intestine.
Intestine. The intestine comprises a chain of paired cells (figure 17). At hatching they are mononucleate, but subsequently most of them become binucleate by nuclear division (Sulston and Horvitz, 1977). Occasionally an extra cell is found in a newly hatched larva, presumably as a result of an extra division in the E lineage (cf. Other Nematode Species: P. redivivus). The anterior ring of four cells (int1) is specialised in having shorter microvilli than the rest of the intestine.
The intestine is derived exclusively from founder cell E, which gives rise to no other tissue. The daughters of E are the first cells to enter the body cavity during gastrulation (90 min). By 300 min they have formed two rows of eight cells, one on the left and one on the right. The anterior pair divide dorso-ventrally to yield int1, which attaches to the pharyngo-intestinal valve. Before attaching to int1, the rest of the anterior intestine undergoes a 90° left-handed twist, so providing half the total twist noted by Sulston and Horvitz (1977); the remainder seems to be due to packing of the posterior nuclei, because no twist is seen in the attachment of the intestine to the intestino-rectal valve (Fig. 17).
Intestino-rectal valve and rectum. The cells which form these structures are shown schematically in figure 17. Some of them have been given new names: rectal epithelium was formerly rectal gland, U was E, and Y was C. K is a blast cell; its contralateral homologue K' is a blast cell in the C. elegans mutant lin-4 (Chalfie et al, 1981) and also in Panagrellus redivivus (Sternberg and Horvitz, 1982). F, U, B, and Y are blast cells in the male. All these cells underlie cuticle in the L1. During late larval development of the hermaphrodite Y withdraws from the hypodermis, without division, and becomes a neuron.
There is some similarity between the intestino-rectal valve and the pharyngo-intestinal valve: in both, the intestine attaches to a ring of two cells which do not bear microvilli, which attaches in turn to a ring of three cells which do bear microvilli. Only the intestino-rectal valve is a true valve, in that it can be closed actively by means of a sphincter muscle which surrounds it.
At hatching, the gonad comprises two germ line cells (Z2 and Z3) and two somatic cells (Z1 and Z4). Its subsequent development has been described by Kimble and Hirsh (1979), and its structure in the adult by Hirsh et al. (1976) and by Klass et al (1976).
The germ cells and somatic cells have separate embryonic origins; the former are the daughters of founder cell P4, and the latter arise by identical lineages from MSa and MSp. After their birth, the somatic cells migrate posteriorly and attach to the germ cells. At this stage the gonad is oriented transversely across the animal (figure 16), but, probably as an inevitable consequence of the elongation and narrowing of the embryo, it gradually adopts the oblique position shown in figure 8c. The homologous origin of the somatic cells is concordant with their equivalent behaviour in Postembryonic laser ablation studies (Kimble, 1981).
Electron micrographs of a 470-min embryo show that the germ cells are united and protrude large lobes into two intestinal cells (Figs. 2j, 17); after hatching, the protrusions are absent. Perhaps the germ cells are nursed by the intestine until their attachment to the somatic cells, for it is known that the latter are essential for their survival and division in larvae (Kimble and White, 1981).
CELL INTERACTION EXPERIMENTS
This section describes some investigations using the technique of cell ablation by means of a laser micro-beam (Sulston and White, 1980). Although the number of experimental animals is small, the invariance of the wild-type lineages ensures that any abnormalities observed are highly significant.
Ablation is more difficult in eggs than in larvae. Small cells can be killed satisfactorily (although it is difficult to avoid damage to their neighbours), but attempts to kill the large cells present early in embryogenesis frequently cause death of the entire embryo. Damage to these young eggs can be minimised by mounting them on 1% agar in an isotonic medium (see Light Microscopy: T. aceti), since heavy pulses seem sometimes to cause the shell to leak transiently. In this way the nucleus of any given cell can be destroyed, or at least prevented from dividing; although the cytoplasm inevitably persists, it is often displaced from its usual position. The technique used is to pulse the target cell repeatedly at an energy level sufficient to produce refractile debris in its nucleus; whenever the cell appears dangerously weakened (low refractility of the cytoplasm, excessive Brownian motion) it is rested for a minute or two before pulsing is continued. After a successful operation the cell, heavily loaded with refractile debris, appears largely to have lost contact with its neighbours and remains visible as an undivided blob.
After ablation of P1, AB continued to cleave and in due course generated a large number of cell deaths. There was no recognisable organogenesis or morphogenesis, and no particular cells could be identified by light microscopy, though nuclei characteristic of hypodermal and neural tissue were visible.
In a series of animals, the two daughters and four granddaughters of AB were ablated in turn. The remaining cells divided in a superficially normal way, and intestinal, dorsal hypodermal, and germ line cells became recognisable. The arrangement of tissues, however, was defective, and all these embryos arrested at the comma stage (see figure 4).
Ablation of P4 led to the production of larvae with no germ line. The somatic gonads of these animals grow more or less normally but are devoid of gametes: P4 is not replaced by any other cell.
Although these results are superficially consistent with cell autonomous development, they are inconclusive because the presence of the dying cells may inhibit potential replacements (see Discussion).
Body Muscle from C and D
The following cells were ablated in a series of animals: Da, Dp, Capa, Capp, Cppa, Cppp. In each case, the resultant larva lacked approximately the quota of muscles which the dead precursor would normally have made (accurate muscle counts, especially in damaged animals, are difficult). The missing cells left a gap, and the remaining muscles did not spread out much to fill it. The experiments are subject to the caveat given for the early ablations.
A number of experiments were carried out at the MS4 to MS16 stages. The great advantage of this group of cells is that they are gastrulating at the time of ablation; in favourable cases, the target cell can be damaged sufficiently to cause it to detach from its neighbours and to remain at the surface of the embryo for some time, although eventually it, too, becomes enclosed within the body cavity. This circumstance should permit the remaining cells to interact regulatively, if they are capable of doing so.
Various derivatives of MS(p)p were ablated at MS4 or MS8, and the following cells were scored in the L1 and later larval stages: Postembryonic mesoblast (M), right intestinal muscle (homologue of M), coelomocytes, somatic gonad (Z1, Z4: regarded as equivalent), head mesodermal cell (difficult), body muscle complement (approximate). In all cases the survivors appeared to generate those cells, and only those cells, which they would have produced in an intact embryo.
This result is particularly striking in the case of M and the right intestinal muscle, which are homologous and migrate along the midline in contact with one another (figure 16). M is a large cell with a characteristic Postembryonic lineage; it is therefore easy to score, and the result is very clear-cut (five animals). This experiment and the following one are important counterexamples to the regulative interaction shown by two pairs of AB cells (see below).
The head mesodermal cell (hmc) is more difficult to score, so the finding in this case was confirmed by watching for the death of its homologue. When MSapp or MSappa was ablated (three animals) the death was seen and hmc was absent from the larva; when MSppp or MSpppa was ablated (three animals) the death was not seen and hmc was present in the larva. Therefore, in spite of the similar embryonic appearance and position of hmc and its homologue, the latter is programmed to die even in the absence of the former.
Extension of these experiments to MS(a/p) leads to pharyngeal damage, as predicted from the lineage. Not surprisingly, these animals feed poorly or not at all. Detailed analysis, which would require electron microscopical reconstruction, has not been undertaken.
In the first two sections below, experiments which produced viable animals are described. In (a) the resulting animals were scored solely by light microscopy; in (b) the ultrastructure of the anterior sensilla was determined by electron microscopy. A third section (c) lists some experiments in which the animals died.
(a) The following cells were ablated at the AB32 stage: ABplapa (three animals), ABplaaa (one animal), ABarppa (one animal). The resulting larvae were scored, by Nomarski microscopy, for the postembryonic blast cells and certain neurons; although the blast cells were sometimes displaced in experimental animals they could still be accurately identified by their division patterns. With one exception (see below), the only blast cells missing were those normally generated by the ablated precursor. Furthermore, the surviving blast cells behaved normally during Postembryonic development, even to the extent of respecting the equivalence group boundaries (Sulston and White, 1980) in the usual way.
Because of the presence of the dying AB cells on the surface of the embryos these results do not unambiguously demonstrate cell autonomy, though they are consistent with it. The best evidence is that from ABplapa, in that the progeny of this precursor would normally move to two separated regions in the embryo, and it is unlikely that a dying blob of cytoplasm could make appropriate contacts in both places.
The exception was that, after the ablation of ABplapa, W was missing and G2 was present. In these animals ABprapaapa, which normally becomes W, presumably became G2 in place of the missing ABplapaapa. This example of regulative interaction was confirmed by the direct ablation of ABplapaapa (one animal) and ABprapaapa (two animals) at 260 min, before the cells moved ventrally: all three animals subsequently formed G2 and lacked W.
A second example of regulation was revealed by the ablation of ABplpaaaap (two animals) and ABprpaaaap (one animal): all three animals subsequently formed the excretory duct (normally ABplpaaaapa) and lacked G1 (normally ABprpaaaapa).
It appears that G2/W and duct/G1 are a forward extension of the P-cell regulative system. The latter is a series of six pairs of lateral cells (Fig. 13), which move to the ventral midline during postembryonic development. The members of each pair are equivalent, but in certain cases they compete for a particular "primary" fate (Sulston and White, 1980). The presumptive G2/W and duct/G1 move to the ventral midline and compete for a primary fate in the same way but at an earlier time.
(b) The following cells were ablated at the AB32 stage: ABalaaa, ABalaap, ABalapa, ABalapp, ABalppa, ABalppp, ABpraaa, ABprpaa. The pattern of cell deaths was scored at 270 min, certain neurons (RMED, ALA, RID, CAN) and the postembryonic blast cells were scored in the young L1, and finally the anterior sensilla of the L4 larva or adult were reconstructed by serial section electron microscopy. The results concerning the anterior sensilla are collected in Fig. 18.
In each case, those components which would normally have been generated by the ablated precursor were not seen in the resulting animal, indicating that no replacement of one cell by another had occurred (figure 18). This result strongly suggests that the selection of asymmetrical precursors for symmetrical roles in the head (see Discussion: Sublineages) is a cell autonomous process, because, according to the lineage, the time of selection is mostly later than the time of ablation: in this interval the dying cell acquires a position and a shape very different from that normally occupied by its progeny.
FIG. 18. Laser ablations of precursors of anterior sensilla. Intact. Anterior sensilla are represented by ovals, enclosing symbols representing neurons and supporting cells (see Ward et al, 1975, for realistic representation). In inner labials (IL), triangle represents two neurons. In amphids (AM), "wing" cells (AWA, AWB, AWC) are not scored; remaining amphid neurons are split into three groups: AFD ("finger" cell, scorable), a group of five cells sending seven ciliated processes, and a group of three cells sending three ciliated processes into the amphid channel (the two neurons with double processes -ADF and ADL- are included in the seven process group, but otherwise the designations are purely numerical. Ablations. Ablated precursor is shown at top of each figure. Empty symbols indicate that the designated cell was not found; stippled symbols indicate uncertainty; X's indicate the cells which the ablated precursor would normally have produced. Note that, without exception, X's cover empty symbols. In some animals neurons became associated with inappropriate sockets; since sockets tended to reamin in their usual positions, these changes are indicated by arrows leading from the neurons (and their sheaths: see below) to the sockets. Criteria used to identify particular cell types included: striated rootlets in IL1 and OLQ neurons; square linked array of four filled microtubules in OLQ neurons; dark deposit and supernumerary microtubules at tip of CEP neurons; prominent lips generated by Il sockets. In the region reconstructed there are no criteria for distinguishing between types of sheath; where a neuron is transferred to a different socket, so that the sheath designation is ambiguous, the enruon is arbitrarily assumed to move with its own sheath.
In most cases there were a few unpredicted deficiencies in the sensilla. These
can be accounted for in various ways: the difficulty of recognising supporting
cells in the absence of their neurons, displacement of unsupported neurons
beyond the zone of reconstruction (in several cases such neurons were found
lying beneath the hypodermis and well posterior of their usual positions), and
perhaps failure of expression as a result of displacement. Since these missing
cells are not numerous, they do not seriously weaken our conclusion.
Although replacement regulation at the cellular level was not seen, these experiments provided some interesting examples of anomalous assembly of sensilla (see arrows in figure 18). One clear conclusion is that socket cells are not restricted to association with neurons of a particular type.
(c) A number of ablations in the AB lineage repeatedly yielded animals that failed to hatch. Many of the precursors for which this was true (ABplaaa (two animals), ABarpap (five animals), ABpraap (two animals), ABplapp (seven animals), ABprapp (one animal) are normally responsible for generating large patches of hypodermis. After ablation of any one of them, overall morphogenesis proceeded as usual until the start of elongation at 400-450 min, and then the cells inside the embryo oozed out through the missing area. Elongation ceased when this happened, suggesting that it is driven by circumferential contraction in the anterior part of the embryo.
Ablation of ABplpap (three animals) and ABprpap (three animals) also caused problems. Of these animals, only one of the former hatched, apparently because the dying precursor interferes with closure of the ventral cleft. However, before the embryos burst, it was possible to see that the excretory cell was made only when ABplpap was present. The one animal that hatched lacked both the excretory cell and the rectum, as predicted, and failed to grow.
Other Late Ablations
Various other cells were ablated at 250-300 min. The object of these experiments was primarily to deduce the function of the cells or of their progeny (e.g., interneurons of the ventral cord, rectal cells, postembryonic blast cells). No further replacement regulation was revealed, but many cells remain untested. Three examples of mosaic assembly of the nematode are of interest: after ablation of ABplpapppa (parent of K and K', figure 17) the rectum was blocked; after ablation of ABprpapppp (parent of the intestino-rectal valve cells, figure 17) the posterior end of the intestine was not connected to the rectum; after ablation of ABalpaappp and ABalpapaaa (parents of hyp2) the buccal cavity was formed in the normal way at the anterior end of the pharynx, but was not connected to the mouth.
Two cases of regulative interaction were found. Both occur in late embryogenesis and involve the confrontation of similar cells at the ventral midline, a situation which is already familiar from Postembryonic studies. In other respects the results are consistent with cell autonomous development, the evidence for autonomy being strong for the MS and late AB lineages. Interactions before the 50-cell stage are difficult to analyse by laser ablation.
Adapted by Yusuf KARABEY for WORMATLAS, 2003