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The number of nongonadal nuclei in the hermaphrodite of C. elegans increases from about 550 at hatching to about 810 in the mature adult (Table 2). Essentially invariant cell lineages generate a fixed number of progeny cells of rigidly determined fates. These postembryonic cell lineages range in length from one to eight sequential divisions.
A number of these lineages produce the accessory sexual structures of the hermaphrodite: the vulva (through which eggs are laid), vulval and uterine muscles, and neurons which innervate these muscles. New neurons are also added to the motor nervous system. Almost one-third of all postembryonic progeny nuclei become part of the hypodermal syncytium of the nematode body wall. The number of intestinal nuclei almost doubles. There is a small increase in the number of body muscles. Few cell divisions occur in the head; both the anterior sensory nervous system and the pharynx appear to be complete at hatching.
In the development of the hypodermis, eight blast cells on each lateral side (H1, H2, V1-V6) follow similar programs of cell division to generate a total of 92 syncytial hypodermal nuclei and 28 seam cells. (Seam cells form a distinct cellular band embedded in the lateral hypodermal ridge.) These hypodermal cell divisions occur around the time of the first three larval molts. Two lateral tail blast cells (T) produce two seam and six syncytial nuclei. Twelve other syncytial hypodermal nuclei are produced in the ventral cord by precursors (P1-P4, P8-P12) primarily responsible for neuronal development. The other three ventral cord precursors (P5-P7) generate the 22-cell vulva, the hypodermal structure through which eggs are laid. In the young larva, the ventral cord precursors form an integral part of the hypodermal body wall.
Most of the neuronal development occurs in the ventral nerve cord and its associated ganglia. Identical patterns of division during the first larval stage of 12 precursor cells (P1-P12) and 1 neuroblast (P0.a) followed by a fixed program of cell deaths add 56 neurons to the ventral motor nervous system. Two of the lateral hypo-dermal blast cells (V5) produce small ganglia as well as the hypodermal nuclei mentioned above. Two other lateral hypodermal blast cells (T) and the lateral neuro-blasts (Q1, Q2) also generate neurons and or glial cells.
Mesodermal development derives exclusively from a single cell (M) present in the newly hatched larva. A series of divisions during the first larval stage produces 14 body muscles, 2 coelomocytes, and 2 myoblasts. These myoblasts migrate anteriorly until they reach the position where the vulva will form. They divide shortly before the third molt to produce 16 sex muscles, which function in egg-laying.
Most of the intestinal nuclei (In) divide once, before the first molt.
A number of other cell divisions also occur in the hermaphrodite.The postembryonic development of the male differs from that of the hermaphrodite. In the male, the number of nongonadal nuclei increases from about 550 to about 970 (Table 3). Most of the additional, male-specific cells are located in the specialized structures of the tail used by the male during copulation. Many cell lineages are similar in the male and hermaphrodite; the two sexes display virtually identical changes in the lateral hypodermis, body musculature, and intestine. Some male-specific cells are produced by lineages which are initially identical to those seen in the hermaphrodite. For example, extra divisions in the lateral hypodermal lineages (V5, V6, T) produce the 18 rays of the male tail. The mesodermal lineage (M) generates the male-specific sex muscles instead of the vulval and uterine muscles of the hermaphrodite. The development of the male ventral nerve cord involves extra cell divisions as well as fewer programmed cell deaths. Two posterior ventral cord hypodermal cells (P10.p, P11.p) divide to form part of the male tail, whereas the three cells homologous with those which produce the vulva in the hermaphrodite (P5.p, P6.p, P7.p) do not divide in the male. The other male-specific structures (the cloaca, spicules, and associated ganglia) are derived from cells (B, C, E, F) that are present in all L1 larvae but divide only in males.
The postembryonic development of C. elegans involves the sequential addition of new structures onto a preexisting larval edifice; no destruction of larval-specific cells occurs. However, there are structural modifications of larval cells: The "dorsal D" motoneurons of the ventral cord reverse their polarity during postembryonic development; in the L1, they receive innerva-tion from the dorsal cord and synapse onto ventral body muscles, whereas in the adult they receive innervation from the ventral cord and synapse onto dorsal body muscles (J. G. White, personal communication).
(B) Functions of Postembryonic Cell Divisions
A number of the postembryonic cell lineages are related to sexual maturation. At hatching, the hermaphrodite and the male are almost identical with respect to cell number, position, and (presumably) function. Yet, by the adult stage, gross anatomical differences are obvious in nongo-nadal tissues. Most of the cell divisions which give rise to these sex-specific structures occur shortly before the third molt. In the hermaphrodite, ectodermal lineages produce the 22 cells which form the vulva, the structure through which eggs are laid. Mesodermal lineages generate the 16 muscle cells which control the vulva and uterus during egg-laying. Neurons which innervate at least some of these muscles (White et al., 1976) are also formed post-embryonically from the neuronal lineages of the ventral cord. In the male, related ectodermal, mesodermal, and neuronal lineages generate some of the structures of the tail which function during copulation. The other male-specific structures are produced by lineages derived from cells which in the hermaphrodite do not divide.
Some of the postembryonic cell divisions may relate to growth. For example, four rounds of hypodermal cell divisions during the first three larval stages substantially increase the number of hypodermal nuclei. The increases in the numbers of intestinal nuclei and body muscle cells may similarly reflect growth, although it is unclear why there are cell divisions in these tissues only during the first larval stage.
Explanations for other postembryonic cell divisions are not obvious. For example, the L1 moves quite adequately using its juvenile locomotory ventral nervous system; yet, the number of neurons in this system is almost tripled during early larval development. Perhaps for mechanical reasons the larger adult requires a more intricate nervous system to control its locomotion. Alternatively, the adult may be capable of behaviors more complex than those of the L1.
The postembryonic somatic cell lineages of C. elegans are generally invariant, with a fixed pattern of cell divisions and a fixed developmental program defined for every daughter cell. However, essentially five types of variations in the details of this developmental program have been observed, as follows. (1) Variation in the pattern of cell divisions: The greatest inconstancy appears in the single round of divisions of the intestinal nuclei; the four posterior most nuclei sometimes divide and sometimes do not. Slight variations occur in the patterns of divisions of the hypodermal cells of the ventral cord. Among males, P9.p occasionally divides; among hermaphrodites, P3.p sometimes fails to divide. Similarly, E.la occasionally divides in males. (2) Variation in the pattern of cell deaths: Q2.pp, which normally appears to die, has once been observed to survive into the adult. (3) Variation in which of two alternative lineage programs a given cell will follow: These cases appear to involve positional effects and are discussed below (see Mechanisms of Determination). (4) Variation in the precise order of specific events: For example, in mesodermal development, sometimes M.vla divides before M.vra, whereas sometimes M.vra divides before M.vla. Similar timing variations occur between lineages as well as within a given lineage. Migrations and divisions also are not rigidly ordered, because sometimes a cell will migrate and then divide, whereas sometimes it will divide and its daughters will migrate (see Results, Mesoderm). (5) Variation in the precise positions of cell nuclei: For example, the development of both the ventral nerve cord and the somatic musculature leads to variable intercalation of new progeny cells with the juvenile cells present at hatching. For this reason, our diagrams cannot be used to identify all cells in an animal which has already developed.
(D) Patterns of Cell Divisions
Two distinguishable types of cell divisions occur during the postembryonic development of C. elegans. Some divisions are symmetrical, producing daughters which are equivalent in morphology and, often, in subsequent development. Other divisions are asymmetrical, producing two distinctly different types of daughter cells; in at least some instances, asymmetric divisions generate a posterior daughter that is morphologically like its mother cell and an anterior daughter of a new cell type. Both types of divisions can be seen in the development of the lateral hypodermis (Fig. 10); for example, Vn.p divides symmetrically, whereas Vn.pa and Vn.pp divide asymmetrically. Asymmetric divisions almost always occur along an anterio-posterior axis and could reflect an anterio-posterior asymmetry of developmental determinants located either inside or outside the parent cell. That transverse divisions are symmetrical (see below, Symmetry) suggests that the distribution of such determinants may be bilaterally symmetric. The few dorsal-ventral divisions appear to be asymmetrical. However, the examples in the male specific B, C, and F lineages are difficult to interpret, because divisions in these lineages generally fail to conform to the orthogonal coordinate system seen elsewhere. The only other example (the first division of the mesoblast, M) produces progeny which follow identical lineages for three further divisions; the subsequent different fates of a few of the lineally equivalent dorsal and ventral cells (e.g., M.dlpa and M.vlpa) could arise from local environmental influences.
Comparison of the various cell lineages reveals that there is no standard type of cell lineage utilized for generating groups of new cells. Although most lineages involve both symmetrical and asymmetrical cell divisions, two exemplify extreme types of lineage logic. In the mesodermal lineage (Fig. 25), a series of symmetrical divisions adds a relatively large number of new cells of a particular type at one time. In the lateral hypodermal lineages (Fig. 10), a series of asymmetrical divisions adds sequentially individual progeny which differentiate into types distinct from the parent cell; in such stem cell lineages, one daughter always maintains the morphology of the original mother cell.
A variation of this "stem cell logic" is thought to produce the ventral nervous system of Drosophila melanogaster (Poulson, 1950; Seecoff et al., 1973) and may also be used by C. elegans. In Drosophila, each of a set of neuroblast stem cells generates another stem cell and a "predifferentiated" cell that divides once before differentiating. The lineage of the C. elegans ventral nervous system (particularly in the male; Fig. 17) is similar. Perhaps this type of lineage is common in neuronal development.
Another type of lineage that has been observed in insects may be utilized by C. elegans. In a number of insects, similar cell lineages generate scales, hairs, bristles, and sensilla (Lawrence, 1966); many of these insect lineages involve programmed cell death. In C. elegans, we find possible analogs in the development of the rays (probably mechanosensilla), the posterior lateral ganglia (associated with the postdeirid sensilla), and the lumbar ganglia (partially associated with the phasmid sensilla) ( Fig. 27). Although there are difficulties in interpretation (see the legend to Fig. 27), we feel that the similarity of these lineages may reflect a fundamental subprogram of development.
Frequently, several blast cells follow the same asymmetric program of divisions. Thus, 13 neuroblasts of the ventral nervous system produce 5 daughters each, 10 lateral hypodermal stem cells produce 9 daughters each, 4 sex myoblasts produce 4 daughters each, and 18 ectodermal blast cells in the male tail produce 5 daughters each. In all of these cases, the lineally equivalent progeny of different blast cells differentiate into functionally equivalent cells. In effect, each precursor cell generates one of a number of repeating "units" present in, in these examples, the ventral cord, lateral hypodermis, sex musculature, and male tail. The logic utilized in these lineages may be contrasted with an a priori plausible alternative in which functionally equivalent cells are all produced from a given blast cell and then repositioned with respect to the daughters from other lineages to form an appropriate final configuration.
The relationship between lineage history and developmental fate suggests that specific types of cells may arise only from an appropriate series of divisions of a particular type of blast cell. Hence, when only some progeny cells of a given lineage are needed, it may be necessary to generate and later destroy the other daughters. Such a mechanism provides one explanation for programmed cell death, common in both the nematode (see Results) and other developmental systems (Saunders, 1966).
FIG. 27. Comparison of lineages found in insects and C. elegans. Insects: Generalized from Lawrence (1966). The lineage tree does not reflect the division axes. Ray: The neuron assignment is based on the identification of dopamine in this cell. The assignment of the structural element is based upon experiments with the laser (see Results, Cell Lineages). Postdeirid: Assignments were made by comparing nuclear morphology and arrangement as seen with Nomarski optics with that observed in serial-section electron micrographs. Because individuals of known lineage have not been examined by electron microscopy, the assignments of the sheath and socket cells (defined by Ward et al., 1975) conceivably could be switched. Lumbar ganglion: assignments were made by tracing nuclei observed with Nomarski optics through to the adult stage and identifying these nuclei in serial-section electron micrographs. The wing-shaped accessory cell and the socket cell are associated with the phasmid. However, the three neurons apparently are not closely associated with this sensillum. Furthermore, the phasmid has another associated accessory cell which is present in the newly hatched larva. In this case, we would propose that the lineage defines the general functions of cells which subsequently assort into different sensory elements. The fates of some sister cells in the lumbar ganglion lineage are reversed compared to the fates of lineally equivalent cells in the ray and postdeirid lineages, possibly reflecting local environmental influences (see Mechanisms of Determination).
(E) Mechanisms of Determination
The strong correlation between lineage and function could arise in essentially two distinct (but not necessarily exclusive) ways. On the one hand, the ultimate differentiation of a cell could be determined extrinsically according to its position; alternatively, its fate could be determined intrinsically according to its lineage history. Our observations are consistent with the hypothesis that much of the development of C. elegans is based upon a lineal determination of cell function. The high degree of invariance of these lineages suggests such a mechanism. Furthermore, experiments with the laser microbeam system (see Results) suggest that the fates of specific cells in the posterior lateral ganglia, the ventral cord, and the rays of the male tail are autonomously determined; these fates remain invariant despite the fact that the undamaged cells sometimes differentiate in positions which normally would be occupied by cells with different fates. Preliminary experiments (J. White, J. Sulston, N. Thomson, E. Southgate, and R. Horvitz, unpublished results) utilizing mutants with abnormal cell lineages have also confirmed these observations; blocking one branch of the cell lineages of the posterior lateral ganglia or the ventral cord does not alter the developmental fates of cells generated by another branch.
There are, however, some developmental events in which position appears to have an influence. In the male tail in each of two cases (Balpha and Bbeta; Bgamma and Bsigma) two cells of different lineage histories appear to be equally competent to follow either of two alternative developmental programs; their subsequent lineages are correlated with their relative positions and not with their previous histories. Similarly, ventral cord hypodermal blast cells P5.p and P6.p follow distinct programs in the development of the vulva, despite the fact that either of two cells present in the young L1 can become either P5 or P6; in other words, the developmental program that each of these cells follows is correlated with the position it assumes in the ventral cord. Two observations suggest that the gonad may be responsible for this positional influence on P5 and P6: (a) The lineages of the ventral hypodermal cells Pn.p are arranged essentially symmetrically around the midpoint of the developing gonad (Fig. 18), as if this point were acting as the source of a positional determinant, and (b) the Pn.p cells which divide are those immediately adjacent to the gonad. Other developmental events -e.g., the Pn.aap cell deaths in the ventral cord and the differentiation of the sex muscles also occur symmetrically around the developing gonad of the hermaphrodite and similarly may reflect its influence.
Other aspects of the postembryonic development of C. elegans possibly may be affected by positional influences: (1) Those lineages which utilize sets of "equivalent" blast cells show apparent end effects; such effects include logical inversions (in which the fates of anterior and posterior daughters are reversed) and/or positional inversions (in which daughters migrate past one another), as well as modifications of the pattern of cell divisions itself. For example, such alterations are apparent in the H and T lineages (the head and tail equivalents of the V lineage; Fig. 10) and in the divisions, deaths, and ultimate cell fates at the ends of the ventral cord (Fig. 17 ; Table 1). These various modifications of cell lineages in the head and tail regions may reflect local environmental influences. (2) Six apparently equivalent developmental "regions" can be defined longitudinally along the lateral hypodermis of the nematode. In five of these regions, identical lineages are produced by the hypoder-mal blast cells. However, hypodermal blast cell V5 follows a modified developmental program to generate part of the posterior lateral ganglion. This region of the nematode is also unique in that it contains the lateral neuroblasts (Q) as well as the single mesoblast (M). Perhaps positional factors possibly defined by one or more of these cells determine the unique developmental aspects of this area of the nematode.
Although the examples described above indicate that cell lineages may be affected by positional influences, we do not know if the nematode is capable of regulation per se. In experiments involving the posterior lateral ganglia, the rays of the male tail, and the ventral cord, those structures normally derived from a cell which had been eliminated either by the laser or by mutation, were unequivocally absent in the adult; no regulatory processes induced extra divisions to compensate for the laser-or mutation-induced deficiencies. The unique observation of an abnormal death in the intestine followed by a compensatory abnormal division (see Results, Intestine) is a possible example of regulation.
Although the young L1 is essentially bilaterally symmetrical, there are a variety of minor differences between its left and right sides which can be distinguished in the light microscope: (1) The gonad pri-mordium lies obliquely, with its anterior portion displaced to the right and its posterior portion displaced to the left. In the region of the gonad primordium, the intestine is reciprocally displaced (anterior, left; posterior, right). (2) The mesoblast (M) is on the right. (3) The two coelomo-cytes on the right are anterior to those on the left. (4) There is an extra lateral hypodermal nucleus on the right side near the head and another on the left side in the tail. (5) There is an extra ventral body muscle nucleus on the right side posterior to the gonad primordium. (6) The lumbar ganglion on the right contains one more nucleus than that on the left. (7). The anal sphincter muscle nucleus is located on the left. (8) The excretory cell is displaced left of the midline.
Bilateral symmetry is reflected in the transverse divisions which occur during postembryonic development. Virtually all transverse divisions produce morphologically identical progeny which follow identical programs of development. The only indications of exceptions to this rule(1) B.alapaav dies, B.arapaav does not; (2) Bgamma. ald dies, Bgamma.ard does not; (3) E.ra divides, E.la sometimes does not may well arise from positional influences at the end of identical lineage programs.
The left and right lateral neuroblasts, Q1 and Q2, are bilaterally symmetrical in their initial positions and in their division programs. However, their progeny migrate differently. Q1 produces three progeny in the posterior half of the left side and Q2 produces three (or four) progeny in the anterior half of the right side.
The obliquely oriented gonad primordium of the young L1 shows twofold rotational symmetry around a dorso-ventral axis. The subsequent development of the hermaphrodite gonad, vulva, and sex muscles is also symmetrical about this dorso-ventral axis through the midpoint of the gonad.
Cellular migrations occur frequently during the postembryonic development of C. elegans. Because Nomarski optics does not allow clear visualization of cellular boundaries, we cannot always be certain that whole cells as opposed to nuclei are migrating. However, in most migrations, nucleus and cytoplasm do appear to move together. The migrations of the precursor (P) cells into the ventral cord are distinctive; a cytoplasmic extension moves into the cord and is followed by the nucleus and, finally, by the remaining cytoplasm. Some migrations such as those in the posterior half of the ventral cord appear to be passive events that depend solely on the activities of neighboring cells; often such movements are variable in extent from individual to individual. Other migrations are rigidly determined, with cells traversing relatively long distances from reproducible starting and stopping sites. The most striking of the migrations involve progeny of the lateral neuroblasts (Q1 and Q2) and the mesoblast (M): (1) Three of the progeny of Q2 (Q2.a, Q2.p, Q2.ap) travel rapidly along the left lateral hypodermis; they move about 50 micrometer (about 16 nuclear diameters) in 2 hr. Q1.ap shows a similar, but somewhat shorter, migration. (2) The two sex myoblasts of the hermaphrodite (M.vlpaa, M.vrpaa) migrate from their origin among the posterior ventral body muscles to the anterior-posterior center of the developing gonad; they move about 65 micrometer (about 16 nuclear diameters) in 6 hr. (3) Most of the daughters of the dorsal anterior sex mesoblasts of the male (M.dlpaa, M.drpaa) migrate from the region of the dorsal body muscles around to that of the ventral body muscles; they move about 20 micrometer (about 5 nuclear diameters) in 1 hr. The movements of these male-specific mesoblasts occur independently of the concomitant divisions of these cells. As described above (see Results, Mesoderm), sometimes a given cell will migrate and then divide; sometimes it will divide and then both daughters will migrate; and sometimes it will start to migrate, divide, and then the two daughters will complete the migration.
The postembryonic development of C. elegans offers a variety of opportunities for further exploration: (1) This development is rigidly determined; specific cells follow precise developmental programs at well-defined times. This system can now be used to obtain a detailed analysis at the fine-structure level of such developmental events as cell migration and synapse formation as well as other types of cellular differentiation. For example, the morphological changes associated with programmed cell death could be studied for a specific cell; the sequence of events could be traced by examining individuals at different stages from the time that cell is born to the time it disappears. (2) The laser system developed by J. G. White will allow exploration of possible intercellular communications during the processes of cell divisions, migration, death, and differentiation. In addition, this laser system can be used to determine additional cell assignments [as described above (see Results) for the posterior lateral ganglia and the rays in the male tail] and cell functions. (3) Mutants which affect the cell lineages of postembryonic development may similarly be used to examine regulative effects and to determine cell assignments and functions. Furthermore, such mutants might provide clues concerning the logical bases of the genetic program for the postembryonic development of C. elegans.
Adapted by Yusuf KARABEY for WORMATLAS, 2003