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The development of the nematode was observed by light microscopy, using Nomarski differential interference contrast optics. This technique is non-destructive, can be applied to the animal under reasonably natural conditions, and, at least in the L1, enables every nucleus to be resolved. Various methods of restraint were tried, without success, and it was finally found best to allow the animal to move freely, though confined beneath a coverslip by the attraction of a bacterial lawn. The presence of an agar layer slows movement somewhat, but does not impede feeding.
Although photographs are useful for illustrative purposes, direct observation is the only satisfactory method for following cell division and migration. Since the cells move in three dimensions, large numbers of photographs would be needed for a complete record, and, in any case, much potential resolution is lost when photographing the low contrast Nomarski image with its shallow depth of field.
Cell boundaries cannot always be visualized by Nomarski optics, but nuclei are resolved clearly and different types can be distinguished (figures 8-14, plate I). Nerve cells have granular nucleoplasm and generally no visible nucleolus in the L1: they are indistinguishable from the supporting cell nuclei which lie anterior to the nerve ring (Ward, Thornson, White & Brenner 1975), and will be termed compact nuclei. Muscle nuclei in the L1 are ovoid, with granular nucleoplasm surrounding a spherical nucleolus; during L2 the nucleoplasm becomes smooth so that they can no longer be distinguished morphologically from ventral hypodermal nuclei. The body muscle nuclei lie in four longitudinal rows, subventrally and subdorsally. Lateral hypodermal and stem cells have large nuclei and nucleoli. Gut and gonad nuclei can be identified both by morphology and by position. The designation of the various cell types depends upon tracing them through to the adult, in which they have been identified by reconstruction from serial section electron micrographs.
In the young L1, the ventral cord contains fifteen compact nuclei in a practically invariant arrangement (figure 1a). At the anterior end lies the retro-vesicular ganglion (r.v.g.), comprising twelve compact nuclei and one large nucleus with a nucleolus. The cells of the r.v.g, lie within a basement membrane (White et al. 1976); this membrane, and particularly the constraint which it imposes upon relative movement of the cells, helps to mark out the r.v.g. as an identifiable unit. At the posterior end of the ventral cord lies the pre-anal ganglion (p.a.g.); it contains six compact nuclei. Unlike the r.v.g., the p.a.g. does not have a well defined boundary, and, since it has not yet been studied by electron microscopy, there is no independent check upon the number of nerve nuclei in the vicinity. It will be seen later that the r.v.g. contains cells of the types found in the cord as well as more specialized cells; the same may also be true of the p.a.g.
In all these areas, the number of cells and their approximate arrangement are invariant. The compact nuclei found in the ventral cord, r.v.g. and p.a.g. of the young L1 will be termed juvenile (J).
FIGURE1. Development of the ventral cord in the L1 larva. The ventral aspect, shown here for clarity, is purely diagrammatic; the living nematode is constrained by the dorso-ventral undulation of its body to lie on its side. Although the number of cells is invariant, their arrangement and the timing of divisions are not precisely constant. (a) 10 h before the moult, showing the positions of the juvenile and precursor (P) cells. (Magn. ca. x 700.) (b) The ventral cord: 8 h before the moult. The precursors have migrated into the cord between the juvenile (J) cells. (Magn. ca. x 1000.) (c, d, e) The ventral cord anterior to P6.2: 6, 4 and 2 h before the moult. The progeny of the neuroblast P1.1 have migrated into the r.v.g. (Magn. ca. x 1500.)
In the young L1, six symmetrically placed pairs of precursor nuclei lie ventro-laterally, between the subventral muscle nuclei and the lateral hypodermal nuclei (figure 1a). By mid-Ll there is an indication of discontinuity between the precursor cells and their hypodermal neighbours (figure 12), and at about eight hours after hatching, their nuclei begin to migrate into the ventral cord and p.a.g. The migration is preceded by the slow growth of characteristically striated areas of cytoplasm in the ventral cord; presumably, an extension of each precursor cell grows round into the cord some time before the nuclear migration. The most anterior pair migrate first, followed roughly in order by successively more posterior pairs: the same general anterior-posterior ordering applies to every phase of the development. In the case of the pairs P3/P4 and P5/P6, the left-hand precursor may finally lie either anterior or posterior to its right-hand partner, at random. Elsewhere, however, there is at least a strong tendency to a particular configuration. Thus P1 has been seen eleven times to come from the right and once possibly to come from the left. PI2 normally comes from the right. The final positions of the precursor nuclei among the juvenile nerve nuclei are not precisely constant, but a typical arrangement is shown in figure 1b.
Every precursor divides as shown in figure 2. The first anterior daughter is a neuroblast, while the first posterior daughter is a ventral hypodermal cell. The axes of all the divisions are approximately parallel to the longitudinal axis of the nematode. In order to label the various cells, an anterior daughter is given its parent's number with the suffix 1, and a posterior daughter its parent's number with the suffix 2. In addition, the differentiated cells are labeled by the letters a to f as shown.
White et al. (1976) have demonstrated cytoplasmic continuity between the ventral and lateral hypodermal ridges in the adult. It is now known that the precursors are separate cells whose posterior daughters ultimately fuse with the ventral hypodermal ridge (J.G. White, personal communication).
There is one additional neuroblast, which is already present in newly hatched larvae: it is the nucleolated cell which lies at the anterior end of the r.v.g. It divides in exactly the same manner as the other neuroblasts, and will be regarded as the daughter of a hypothetical precursor P0; its actual ancestry, however, is as yet unknown.
During the period of division, the neuroblasts and their progeny migrate freely past the juvenile cells and the hypodermal cells. Except in the r.v.g., however, the progeny of one neuroblast do not migrate past those of another; nor, except at the anterior end of the r.v.g. and the posterior end of the p.a.g., do the progeny of a single neuroblast migrate past one another.
The direction of movement of each nucleus at a given time is quite reproducible; the extent of movement, however, is variable and seems to depend upon local pressures. It is also noteworthy that the entire process has a fixed hand. For example: J3 always moves far to the right; J1 and J2 move slightly right and ventrally; all hypodermal cells move gradually to the left. This asymmetry may be determined by the previous existence of a hypodermal ridge to the left of the L1 ventral cord, and the consequent restriction of the muscle synapses to the right of the cord. Typical stages in the development of the anterior part of the ventral cord are shown in figure 1 and figures 8-11, plate 1.
Towards the end of the period of division, certain cells die. Their death is signalled by a loss of contrast in the granularity of the nucleoplasm, followed by a sudden increase in the refractility of the nucleus (figure 13, plate 1). This stage persists for a few minutes, and then the refractility fades with erosion of the nuclear outline. In crowded regions other nuclei completely obliterate the site, but in some places a permanent gap remains; in either case, no trace of the dead cell's nucleus can be detected by Feulgen staining. The pattern of cell death in the hermaphrodite differs from that in the male (table 2), but no individual variation has been seen.
Cell deaths can be visualized in another way, by using the mutant E1392 (nuc-1, X) which lacks the principal endodeoxyribonuclease. In this mutant, high molecular weight DNA is degraded much more slowly than usual, and, after Feulgen staining, the approximate site of each cell death is marked by a 0.5 Ám dot (figure 15).
With one exception, the ventral hypodermal cells do not usually divide in the L1. The exception is P12f whose division is followed after a short time by the death of its posterior daughter.
A typical timetable for the division of three of the precursors is shown in figure 3; the division times in the rest of the cord lie between these extremes. As indicated in figure 3, the steady pumping of the pharynx ceases rather abruptly some 2 h before the L1 moult. About 15 min later a variable number of gut cells divide; the most anterior six never normally divide, and any of the most posterior four may also fail to divide.
In the hermaphrodite, no further division or rearrangement of the ventral nerve nuclei has ever been noticed. In particular, two individuals have been followed through to the adult stage, with observations at sufficiently frequent intervals that any changes should have been detected; the only development is a Iongitudinal stretching, so that some nuclei which are initially beside one another become separated. At first sight P6c and P7c appear exceptional in that they move past their neighbours towards the developing vulva. The reason for this behaviour is that they alone enter the left-hand branch of the ventral cord as it forks around the vulva. Their consequent lateral displacement means that they no longer have a defined position relative to the other nuclei. The remaining nuclei of the ventral cord eventually become linearly arranged, but in the r.v.g. and the p.a.g. some lateral stacking persists into the adult. The hypodermal nuclei, on the other hand, move to the left of the line of nerve nuclei, and in some places migrate freely past them. In the L3, three hypodermal nuclei (P5f P6f and P7f) divide as shown in figure 4, and their progeny form the vulva. The number of free hypodermal nuclei is maintained by division of P3f, P4f and P8f.
FIGURE3. A typical timetable for the development of the ventral cord. ' x ' indicates cell death.
Events in the male are quite different. Shortly before the L3 moult the c nuclei (with the exception of P2c and P12c) divide once; the divisions take place to the left of the ventral cord, but afterwards the daughters insert themselves between the other nerve nuclei giving a linear array once more. The hypodermal cells in the middle of the cord do not divide, but P10f and P11f divide in the L3 and participate in forming the specialized structures of the male tail.
Figure 5 indicates the extent of variation in the arrangement of nerve nuclei in the adult hermaphrodite ventral cord. It will be seen that the differences are restricted to the positioning of the juvenile cells in the fixed pattern of late cells. Examples of cell arrangements in the r.v.g. and p.a.g. are shown in figures 6 and 7.
FIGURE 4. Cell division in the formation of the vulva.
FIGURE 5. Nerve cell sequences in the mature ventral cord. J, juvenile; a-e, cell ancestry as defined in figure 2. Each row represents the sequence determined for a single nematode. ( A )Anterior ventral cord. (B) Posterior ventral cord.
FIGURE 5. Arrangement of the nerve nuclei in two adult r.v.gs. Ventral aspect, schematic. (Magn. ca. x 3000.)
FIGURE 7. Arrangement of nerve nuclei in an adult p.a.g. Ventral aspect, schematic. (Magn. ca. x 3000.) Plla is arbitrarily included in the p.a.g., because of its ancestral association with this ganglion.
Web adaptation, Chris Crocker, for Wormatlas, 2008