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In this paper we have described the cell lineage that gives rise to the newly hatched larva of Caenorhabditis elegans. Since postembryonic development has already been followed (see Background Information for references), our knowledge of the cell lineage of this organism is now complete. The embryonic cell lineage is drawn out in Fig. 3, a complete list of cells is given in the Appendix, and a summary of cell types appears in Table 1.
The main value of this information is as a basis for more detailed studies of development, though certain features of it seem of more general interest and are discussed below. There is, however, little point in thinking about the details of the cell lineage unless it has some function beyond mere cell proliferation; we shall therefore ask at the outset the question: To what extent are the fates of cells constrained by their ancestry?
Invariance, Cell Autonomy, and Cell Interaction
The embryonic cell lineage is essentially invariant. The patterns of division, programmed death, and terminal differentiation are constant from one individual to another, and no great differences are seen in timing. At any given moment in development, each blastomere not only has a predictable future but also has a reproducible position and a defined group of neighbours.
Although invariance is consistent with cell autonomous development, it is not in itself diagnostic, because it might be the result of either the absence or the reproducibility of cell-cell interactions; in order to distinguish between these possibilities it is necessary to perturb the system experimentally. Two approaches have been fruitful.
Note. Cell count is less than nuclear count because of cell fusions and Postembryonic divisions in the intestine (+) which are of nuclei only. Main entries are for hermaphrodite; male counts, where different, are shown as superscripts. Postembryonic blast cells (column 8) are included in L1 totals (column 7). Hypodermis includes XXX's, Q's, and hermaphrodite vulva. Rectum becomes cloaca in adult male. Sex muscles and body muscles are placed in a single category. Rectal cell Y becomes a neuron during Postembryonic development of hermaphrodite, indef: indefinite.
Blastomere isolation. The first approach is the isolation of early blastomeres in a supportive medium and the observation of their subsequent development. Using this method, Laufer et al. (1980) demonstrated that the unique characters of the founder cells (see General Description) are internally determined and do not depend upon interaction with neighbouring cells. They found that the progeny of isolated blastomeres do not undergo normal morphogenesis (for which, therefore, cell interactions are required), but did not examine later cell lineages in detail; it is thus an open question whether determination to follow a particular division pattern is dependent upon extrinsic factors or not.
However, an isolated daughter of AB can give rise to a group of cells in which unequal divisions and subsequent deaths are seen (J.E.S., preliminary results), so at least some differentiation can take place within an isolated founder cell lineage. The absence of morphogenesis in such cell groups means that there are few parameters for the identification of cell types: we do not know whether cells are undetermined or are simply failing to express their determined characteristics. The same difficulty arises with embryos in which early blastomeres have been ablated.
Cell ablation. The second approach is to destroy particular cells and to observe the behaviour of the remainder. Numerous experiments of this kind have been carried out in the past, usually by ultraviolet irradiation but also by crushing or ligation (for bibliography, see Nigon, 1965; Seek 1938). The first method has the advantage of precision, and is reasonably effective in preventing the nucleus of a cell from dividing, but leaves the cytoplasm intact and potentially able to interact with other cells. All these researchers (with the exception of Pai (1927), whose data were subsequently discredited, e.g., Seek (1938)) found that the founder cells were generated and maintained their individual characters regardless of the fates of their neighbours.
More recently, a laser microbeam apparatus has been built by J.G.W.; this instrument is effective in killing, and causing the disappearance of, entire cells, and has provided useful information about cell-cell interaction in developing larvae (Sulston and White, 1980; Kimble, 1981). Its application to eggs, however, is problematical because, except at relatively late stages, cells cannot be killed outright without causing death of the organism. Nevertheless, earlier blastomeres can be prevented from dividing and displaced; their relationships with other cells are thereby presumably altered, and the presence of an abnormally positioned large blob of cytoplasm probably distorts the relationships between the surviving cells as well. In this way a reasonable test for cell-cell interaction can be applied from about the 50-cell stage onwards. In a series of experiments covering this period, no examples of cell-cell interaction affecting embryonic lineage, and only two examples of interaction affecting fate, were found. As explained above (Cell Interaction Experiments: AB lineage), the latter two cases can properly be regarded as part of a postembryonic regulative episode in which pairs of homologous cells confront one another at the midline.
It is worth emphasising two areas in which regulative interaction has not been seen. One is the mesoderm, in which the behaviour of early blastomeres allows cells to be ablated relatively cleanly, and in which a variety of differentiated cell types can readily be scored by light microscopy. The absence of detectable regulation in either lineage or fate is therefore striking, particularly in the case of two cell pairs which confront one another at the midline (in the same way as the AB pairs just mentioned). The other area is the anterior sensory system, which is rich in pairs of precursors that are analogous but not homologous (see Symmetry and Asymmetry).
Another technique for killing individual cells is treatment with psoralen followed by irradiation with an ultraviolet microbeam. In this way James Priess (personal communication) has shown that removal of E or Ea precludes normal morphogenesis of the head, and that certain late divisions and deaths are dependent upon the presence of unrelated cells. The source and specificity of the implied inductive influences remain to be discovered.
The possibilities for regulation of cell shape seem to be rather limited. For this reason, ablation of a blastomere which generates a substantial patch of hypodermis usually leads to a burst embryo. Individual muscles and Postembryonic blast cells lie in approximately their usual positions even when a number of their fellows have been lost. At the ultrastructural level, however, anomalies can be caused in the assembly of neural components (see Fig. 18).
Early divisions of the founder cells. None of the experiments so far described have shed much light on this intermediate period. The initial divisions of MS, C, D, and P4 are more or less equational in terms of their subsequent lineages; indeed, although these divisions are approximately anterior-posterior, they give rise ultimately to left-right symmetrical groups of cells. (This pattern is more apparent in Turbatrix and Ascaris, because the initial divisions of MS, C, and D are left-right.) AB and E are different, in that they do not generate elements of left-right symmetry until their second rounds of division. Nevertheless, their initial divisions may be equational. In particular, the first anaphase of AB is transverse across the midline, and only at telo-phase do its daughters skew into an anterior-posterior configuration; this progression is seen more clearly in Ascaris, in which the transverse arrangement persists for much longer.
Classical attempts to investigate the cell autonomy of the early AB lineage in Ascaris, by the study of squashed eggs and giant eggs, led only to a mass of circumstantial data and heated controversy (see Seck, 1938). Blastomere isolation experiments would seem to offer the best hope of settling the matter, though this approach runs into further difficulties, as explained above. For the present, the bases for the difference between ABa/ABp, as well as those for the more subtle distinctions between MSa/MSp, Ea/Ep, and Ca/Cp, remain unknown.
Summary. In our limited survey, no replacement of any embryonic precursor by another was observed, but the postembryonic behaviour of two pairs of cells was found to be decided by regulative interaction in midem-bryogenesis. There is evidence for inductive effects upon morphogenesis, cell divisions, and cell deaths during the second half of embryogenesis. The period between the generation of the founder cells and the 50-cell stage has not yet been satisfactorily investigated.
Embryonic Germ Layers and Cell Fate
In their literal sense, the terms ectoderm, mesoderm, and endoderm refer to the three distinct layers of cells formed at gastrulation in triploblastic embryos -the so-called germ layers. By extension, the same terms are applied, in mature animals, to the groups of tissues typically derived from the germ layers. To avoid confusion, we shall qualify the terms with "embryonic" when using them in the former sense.
It has long been known that the correlation between germ layer and tissue type is not perfect. In vertebrate embryos, for example, part of the embryonic ectoderm, known as the neural crest, contributes cells to structures (such as muscle and cartilage) which are more typically derived from embryonic mesoderm (reviewed by le Douarin, 1980). Is this also true for the nematode?
The ancestry of various cell types in C. elegans is summarised in Fig. 9. From the behaviour of their progeny at gastrulation the founder cells can be classified as follows: -embryonic endoderm: E; embryonic mesoderm: MS, D, part of C; embryonic ectoderm: AB, remainder of C (though the progeny of AB which contribute to the pharynx might also be classified as embryonic mesoderm). Thus neurons can be derived from embryonic mesoderm and muscles from embryonic ectoderm.
The mingling in ancestry is most extensive in the pharynx. For example, there are three cases of neurons which are sisters to muscles (strictly, myo-epithelial cells), showing that divergence between these two fates can occur as late as the terminal division. Conversely, a consideration of symmetry and patterns of cell fusion in muscle rings m3, m4, and m5 (see Alimentary Tract and Appendix) indicates that a particular cell type may be generated by more than one developmental pathway.
The pharynx may perhaps be regarded as atypical, in that it is a remarkably self-contained organ with its own nervous system and highly specialised myo-epithelial cells. The very late derivation of four muscle cells from the AB lineage is a more straightforward example. The parents of these cells remain on the outside of the embryo until 290 min. Two of the muscles are associated with the rectum and are unique, but the other two are identical with cells derived from MS: one is an intestinal muscle and the other is a body muscle.
The conclusion is that, although there is a broad mapping of particular cell types onto particular blast cells, absolute distinctions are not laid down at an early stage. One reasonable explanation for this observation is that the broad categories correspond to a primitive ancestral condition, and that in the course of evolution their boundaries have occasionally been transgressed; such an event may well be improbable, in that it would involve reprogramming a partially committed cell, but may nevertheless occasionally be selected.
Lineal Boundaries and Functional Boundaries
Two identifiable tissues are generated as single exclusive clones; i.e. in each case one precursor generates all the cells of the tissue and no others. These tissues are the intestine (founder cell E) and the germ line (founder cell P4).
Such coincidence of lineal boundaries with functional boundaries is the exception rather than the rule. Examples of partial clonal derivation can be recognised: some precursors generate only, but not all, cells of a given type (e.g., D, Cap, and MSpppp all yield exclusive clones of body muscle); other precursors generate all, but not only, cells of a given type (e.g., MS yields all the coelomocytes and the entire somatic gonad). However, throughout most of the lineage cell types are intermingled even at the terminal divisions.
As a result of this heterogeneity of ancestry with respect to cell fate, many lineal boundaries cross the various somatic structures in an apparently meaningless way. For example, the neurons and supporting cells of a given embryonic sensillum never arise as an exclusive clone, and the boundary between AB and MS meanders through binucleate cells.
It is interesting that exactly the same conclusion is reached with regard to polyclones in the epidermis of insects (Garcia-Bellido et al, 1973; Lawrence, 1981). Here, the reproducible lineal boundaries surround the progeny not of single cells but of groups of cells called compartments; the cell lineages within a given compartment are indeterminate. Like the clonal boundaries in the nematode, compartment boundaries do not necessarily circumscribe recognisable functional units and yet are reproducibly positioned from one individual to another.
Nematodes are not usually considered to be segmented animals. However, the lateral hypodermis of the newly hatched L1 larva is divided into periodically repeated units (Fig. 13). The repeat unit is most obvious in the middle of the body, where it comprises a ventral cord blast cell (P), a lateral hypodermal blast cell (V), and a syncytial hypodermal nucleus (hyp7); the head and the tail can plausibly be regarded as containing additional degenerate units. How do these units arise?
Inspection of the embryonic lineage reveals no corresponding periodic repeat. The three types of cell are generated semiclonally (Fig. 13) and the repeat units are formed by reproducible but piecemeal recruitment. Presumably the regularity of the structure depends upon cell affinity and perhaps upon packing constraints in the embryo. There appears to be no correlation between the repetition in the hypodermis and that in the endoderm, muscle, or juvenile nervous system. During the Postembryonic expansion of the ventral nervous system (Sulston and Horvitz, 1977), the regularly repeated array of neurons derived from the P cells is simply superimposed upon the different, and less regular, repeat pattern of juvenile neurons.
This mode of limited "segmentation" partially parallels the true segmentation of the leech, which also develops by an invariant cell lineage (Stent and Weisblat, 1982). The leech embryo has five stem cells (four ectodermal and one mesodermal) on either side; every segment is founded by a group of cells comprising one or two daughters from each of the stem cells. Consequently, as in the nematode, a given cell in the mature animal is more closely related to its homologues in other segments than it is to most of the cells in its own segment.
We shall use the term "sublineage" as an abbreviation for the more descriptive, but cumbersome, phrase "intrinsically determined sublineage" - namely, a fragment of the lineage which is thought to be generated by a programme within its precursor cell. At present there is no direct evidence for such inherent programming, and so strictly the concept is hypothetical; nevertheless, in certain cases a variety of circumstantial evidence can be adduced to support it. The notion has been discussed elsewhere (Chalfie et al, 1981; Sternberg and Horvitz, 1982) and need not be further elaborated now, but it is convenient to begin by listing two of the available criteria for postulating the existence of a sub-lineage: (1) the generation of the same lineage, giving rise to the same set of cell fates, from a series of precursors of diverse origin and position; (2) evidence for cell autonomy within the lineage, obtained from laser ablation experiments or the study of mutants.
By the first criterion, two sublineages can be found in the head of the embryo (Fig. 19). The sixfold inner labial sublineage generates the neurons of the six identical inner labial sensilla. The outer labial sublineage can be regarded as either sixfold or (four + two)fold; this ambiguity is of interest in the light of the morphological difference between the OLQ and OLL outer labials (Fig. 19 legend; White et al, in preparation).
In postembryonic development, parts of the lineage which are simply bilaterally symmetrical do not usually qualify as sublineages by the first criterion above, because their precursors are of symmetrical origin. However, in the case of the embryo it is possible to take the analysis a step further.
In the lineage chart (Fig. 3), pairs of precursors which give rise to similar or identical division patterns and groups of cells on the left and right sides are indicated. In the posterior part of the animal, bilateral symmetry arises at an early stage, as a result of equational divisions of precursors, and the groups are large. In the anterior part, however, symmetry arises late, in a piecemeal fashion, and the groups are small; for example, identical lineages arise from ABalaappp and ABalapaap, which are not themselves of identical origin. There are therefore grounds for supposing that these symmetrical pieces of lineage are Sublineages, particularly since their precursors are frequently not disposed symmetrically and do not have identical neighbours on the left and the right.
FIG. 19. Labial Sublineages; see text. OLL resembles OLQ in its radial position (Ward et al, 1975) but differs in having neither a square array of four microtubules nor a striated rootlet. OLL sublineage is related equally closely to OLQ and inner labial Sublineages.
The latter observations suggest further that the precursors are determined, not by interaction with their neighbours, but by their ancestry. Evidence in favour of this conjecture was obtained from a series of laser ablation experiments, in which all tested precursors behaved autonomously. Some implications of this finding are discussed below (Symmetry and Asymmetry).
The assignment of sublineages is often a matter for subjective judgment, in that two sections of the lineage may be alike in some respects and discrepant in others. Thus, ABalpaaa and ABarapaa give rise to sublineages identical except for the fate of one cell, and it is reasonable to suppose that identical programmes are being used by the two precursors; the fate of the one anomalous cell may be determined either by position or by additional intrinsic factors. A more extreme example is provided by the pair ABalapap and ABalappp; in this case a dividing cell in one version of the sublineage is replaced by a cell death in the other, but there are still enough common features to suggest a shared programme.
Programmed Cell Death and Sexual Dimorphism
The large number of programmed cell deaths, and their reproducibility, is evident from the lineage. The most likely reason for the occurrence of most of them is that, because of the existence of sublineages, unneeded cells are frequently generated along with needed ones (Sulston and Horvitz, 1977; Horvitz et al, 1982).
Direct support for this point of view is provided by the sexually specific deaths seen in the embryonic lineage. In the hermaphrodite the putative CEM cells die but the HSN cells survive, whilst in the male the situation is reversed; before the deaths, however, each cell begins to differentiate in the same way regardless of sex. Rather than generate two different patterns of cell division, the nematode uses the same sublineages in both sexes and kills those cells that are not required. Sexually specific deaths are also seen in hermaphrodite larvae, but in other cases no surviving homologues of the dead cells are available for study. However, by means of mutants in which cell death does not occur (recently isolated by H. Ellis, personal communication; Horvitz et al., 1982), it should now be possible to exhume all of them.
There are few obvious restrictions upon the patterns of cell deaths. They are found in ectoderm, mesoderm, and (in Turbatrix and Panagrellus) endoderm. Several precursors give rise to two cell deaths in successive divisions, but none give rise to two in a single division. Presumably there is some selective pressure on the organism to dispense with programmed deaths, which must represent a waste of time and materials. Logically, sister deaths could be readily eliminated (by programming the death of their parent) whereas single deaths might require more extensive rearrangement of control elements. Sternberg and Horvitz (1981) have reported sister deaths in the Panagrellus gonad, but suggest, on account of their variability, that they are of recent evolutionary origin.
In addition to bilateral symmetry (see Sublineages) parts of the nematode display two-, three-, four-, and sixfold rotational axes of symmetry. The embryonic lineage, on the other hand, is in part bilaterally symmetrical and in part asymmetrical; the ways in which it generates the more elaborate symmetries of the mature nematode will now be discussed.
Twofold At 300 min the gonad is a bilaterally symmetrical structure consisting of two identical subunits, each comprising two cells of different types (Fig. 16). It subsequently turns to lie obliquely and acquires a twofold rotational axis of symmetry. This behaviour can be understood if, during late embryogenesis, the two subunits interact specifically with one another but not with nongonadal tissues, the oblique position being a consequence of packing. The autonomy of the gonad was pointed out by Kimble and Hirsh (1979) who found that, apart from the initial events in the male, post-embryonic gonadogenesis is reproducibly oriented with regard to the axes of the gonad rather than to those of the body.
Threefold The muscles and structural cells of the pharynx
have a precise threefold rotational axis of symmetry, whilst the nervous system
of the pharynx is bilaterally symmetrical. Broadly speaking, two of the three
identical sets of mechanical elements are generated by bilaterally symmetrical
lineages, and the third set is assembled by piecemeal recruitment. The threefold
symmetrical arrays of the arcade, hypl, hyp4, and parts of the valves are produced
in a similar fashion. This plan of development contrasts with that found for
the vas deferens (formed in the larval male), in which structures having a threefold
rotational axis of symmetry are generated by three equivalent precursor cells
which give rise to identical sublineages (Kimble and Hirsh, 1979; Kimble, 1981).
Four- and sixfold At the tip of the head the various classes of sensilla are arranged in rings with bilateral (amphid), fourfold (cephalic), and sixfold symmetry (inner labial); the outer labial sensilla can be regarded as having either sixfold or (four + two)fold symmetry. Various authors have speculated that one of the higher modes of symmetry may represent the fundamental plan of the nematode head (discussed by Ward et al, 1975). If this were the case, the plan should be detectable as a corresponding pattern of precursor cells. However, apart from the sublineages giving rise to the labial neurons (see Sublineages), no mode of symmetry other than bilateral is detectable in the embryonic lineage. Even the origin of the labial neurons does not support the idea that sixfold symmetry is fundamental, because the sublineages for the inner and outer labial sensilla are generated by unrelated precursors and do not include the supporting cells.
Symmetry and Asymmetry
It is clear from the events already discussed that, at numerous points in the lineage, symmetry is broken. This is obviously a necessary step on the pathways to unique cells and threefold symmetrical structures, but it can also occur in the generation of bilaterally symmetrical structures (see Sublineages).
More than one symmetry change can occur during the ancestry of a single cell. An extreme example is provided by a sequence from the complete (embryonic and Postembryonic) lineage of the male; in it, symmetry is generated three times and broken twice, as follows. Cells B and U are produced by identical right and left lineages; they break symmetry and arrange themselves respectively posterior and anterior to the anus; during larval development, the anterior daughter of B divides symmetrically and gives rise to identical lineages on the left and right sides; two pairs of its progeny break symmetry again and arrange themselves anterior-posteriorly; finally, all four of these progeny generate symmetrical groups of cells on the two sides of the animal (Sulston et al, 1980).
In addition to symmetry changes involving precursors, which are indicated in Fig. 3, certain new symmetries appear at the level of terminal differentiation (Fig. 20). These "phase shifts" in otherwise symmetrical patterns of fate assignments are reminiscent of those seen in the postembryonic development of male-specific muscle (Sulston et al, 1980; Sulston and White, 1980). They may have arisen as a result of selective pressure to produce either unique cells or threefold groups, and various hypothetical schemes can be constructed to explain their evolution. Sternberg and Horvitz (1981) have termed such events "altered segregations," to express the observation that two cells may acquire the same developmental potential by different routes.
FIG. 20. Phase shifts in patterns of fates of symmetrical cell pairs. Members of each pair are judged to be homologous partly by ancestry and partly by the assignments of their relatives. Chain 4 goes some way to account for the strange form of hyp2, a cylindrical syncytium which arises by the fusion of a ventral cell and a dorsal left cell; the corresponding dorsal right cell, anticipated on grounds of symmetry, is absent -perhaps sacrificed in the past for some greater goal. Chain 5 is unimpressive, but is included because apart from DB5 and DA8 pairs of homologous neurons always belong to the same functional class; it is thus remarkable that ABprpppaaaa, rather than ABprpapappp, becomes PDA/Y. In chain 1 the unique cell which may once have justified the phase shift has itself been eliminated: a striking illustration of the accretion of logical debris to the lineage.
How are the symmetry decisions taken? Laser ablation experiments show that two cases of symmetry breakage (G2/W and duct/G1, see Cell Interaction Experiments) involve competitive interaction between the homologous partners, which confront one another at the midline. In all other cases tested the behaviour of the cells seems to be independent of their neighbours and of their exact position. We are therefore forced to conclude that the various symmetries arise during development largely from a series of piecemeal decisions, each individually selected in the course of evolution. The precise bilateral symmetry of the head, for example, is not determined by a single global influence (such as a symmetrical morphogenetic field) but is the end result of many small decisions. Bilateral symmetry is evidently a favourable asset for an animal, and is selected even at the level of individual neurons and sensilla.
Perhaps the most striking findings are firstly the complexity and secondly the cell autonomy of the lineage. As regards the former, many of the guesses that have been made in the past about the ultimate fates of embryonic cells are correct in general but wrong in detail: the assignment of cell function follows certain broad rules to which there are numerous exceptions. As regards the latter, there are two examples of specific interaction between cells affecting their fates, and there is evidence for induction late in embryogenesis; doubtless other instances of cell-to-cell signalling remain to be discovered, but much of the detailed control of division pattern and fate, including such niceties as the generation of precise bilateral symmetry, seems to be vested in autonomous programmes within individual cells.
With hindsight, we can rationalise both this complexity and this
rigidity. The nematode belongs to an ancient phylum, and its cell lineage is
a piece of frozen evolution. In the course of time, new cell types were generated
from precursors selected not so much for their intrinsic properties as for the
accident of their position in the embryo. Perhaps the mutations responsible
for these novel developmental events acted directly upon the intrinsic behaviour
of the precursors, but equally they may have operated via extrinsic regulative
mechanisms. In the latter case, however, cell-cell interactions that were initially
necessary for developmental decisions may have been gradually supplanted by
autonomous programmes that were fast, economical, and reliable, the loss of
flexibility being outweighed by the gain in efficiency. On this view, the perverse
assignments, the cell deaths, the long-range migrations -all the features which
could, it seems, be eliminated from a more efficient design- are so many developmental
fossils. These are the places to look for clues both to the course of evolution
and to the mechanisms by which the lineage is controlled today.
PARTS LIST: Caenorhabditis elegans (Bristol), NEWLY HATCHED LARVA
This index was prepared by condensing a list of all cells in the adult animal, then adding comments and references. A complete listing is available on request to MRC, Cambridge. References: (1) Ward et al, 1975; (2) Ware et al, 1975; (3) White et al, 1976; (4) Albertson and Thomson, 1976; (5) Singh and Sulston, 1978; (6) Kimble and Hirsh, 1979; (7) Sulston et al, 1975; (8) Chalfie and Sulston, 1981; (9) Sulston and Horvitz, 1977; (10) Sulston et al, 1980; (11) White et al, 1978; (12) Horvitz et al, 1982; (13) Wright and Thomson, 1981; (14) Gossett et al, 1982; (15) Nelson et al, 1983.
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Adapted by Yusuf KARABEY for WORMATLAS, 2003