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Summary We have described how the nongonadal cell lineages of the Caenorhabditis elegans male eventually lead to the secondary sexual apparatus of the adult. The cells were followed by using their nuclei as markers under Nomarski differential interference contrast optics, and late stages were reconstructed from electron micrographs of serial sections. In muscle and structural elements, the final positions of the cells are reproducible; in parts of the nervous system this is not the case, and the reconstruction had to be carried out on an animal of known lineage. The three most posterior hypodermal seam cells on each side (V5, V6, T) are responsible for generating the fan and the sensory rays (Figs. 1 and 17). Two ventral hypodermal cells, P10.p and P11.p, give rise to neurons and supporting cells in the preanal ganglion (Fig. 22). Of the juvenile cells which do not divide in the hermaphrodite, three (B, E, and F) form the union between the gonad and the alimentary tract, the copulatory spicules and associated neurons; the fourth (C) forms the postcloacal sensilla (Fig. 6). Three mesoblasts on each side (SM1, SM2, and SM3), which are formed from the juvenile mesoblast (M), produce the copulatory muscles and a coelomocyte (Fig. 26). Development culminates in a mass movement of cells which abruptly changes the overall shape of the tail (Fig. 2).
Cell Fate Inspection of the cell assignments reveals some pattern in the correlation between cell lineage and cell fate. Thus, the sensory rays share a common terminal lineage; the repetitive formation of groups of neurons is reminiscent of the ventral cord lineage (Sulston and Horvitz, 1977). Elsewhere, there is some clustering of related cells into particular sensilla
On the other hand, cells which appear quite similar in the adult are often drawn from disparate lineages. For example, the spicule socket cells, which are sufficiently alike to fuse together in the adult, come from seemingly nonhomologous branches of the B lineage in different parts of the proctodeum (cf. Sulston and Horvitz, 1977, Fig. 21). Similarly the dorsal and ventral spicule protractor muscles arise from diverse lineages at diverse locations. The fact that analogous cells can be well separated spatially at first, and only subsequently assemble at the same location, suggests that their commitment is not an accident of position but rather is programmed into them at birth.
Sensilla Inspection of Figs. 6 and 22 shows that lineages of sensilla can take a wider variety of forms than those proposed by Sulston and Horvitz (1977). Different tail sensilla contain from one to three supporting cells (in contrast to head sensilla, which invariably contain two). Furthermore, neurons and supporting cells do not necessarily occupy discrete branches of the lineage.
Nevertheless, it is worth searching for common features, because data presented previously (Sulston and Horvitz, 1977) and in the following paper indicate that the terminal branches of a number of lineages are determinate. One generalization that can be made is that sheath cells are more often sisters of neurons than are socket cells. Socket cells are sometimes formed later in development than the other components of sensilla, their function being at first performed by the seam cells from which they eventually arise; this is true of the phasmid and probably also of the deirid (John White, personal communication).
Comparison of the hook and ray sensilla reveals more specific similarities. The pair of neurons which innervate the hook sensillum (HOA and HOB) markedly resemble the pair (RnA and RnB, respectively) which innervate each ray (other than ray 6). It is therefore interesting that the lineage of the hook sensillum can be seen as a permutation of the ray lineage. On this view the programmed cell death in the ray lineage removes the potential ancestor of the sheath and motor neuron formed by the hook sensillum lineage; the ray structural cell would then be the homologue of the hook socket cell.
The cephalic sensilla may perhaps fit into the same pattern. Ward et al. (1975) showed that, in the male, each cephalic sensillum contains a pair of sensory endings: one lies beneath the cuticle, like the solitary, dopaminergic neuron found in the hermaphrodite, whilst the other penetrates to the exterior. The male has four swollen cell bodies, which are absent from the hermaphrodite, near the nerve ring (Sulston and Horvitz, 1977); these "cephalic companions" are thought to be the cell bodies of the four male-specific cephalic processes. Their morphology in the electron microscope is similar to that of RnB and HOB. There is thus an analogy between the cephalic neuron and RnA on the one hand, and between the cephalic companion and RnB on the other. However, the cephalic neuron does lack the striated rootlet typical of RnA. At present the lineages of the cephalic sensilla, which are embryonic, are unknown.
Comparison with Hermaphrodite Development With few exceptions, all the nongonadal cells of the hermaphrodite are found also in the male, and most of them have similar functions in two sexes.
The only significant absence is that of the vulva and its associated musculature. The blast cells which generate the vulva in the hermaphrodite (P5.p, P6.p, P7.p) do not divide in the male, in which they form part of the ventral hypodermis. The hermaphrodite sex myoblasts (M.vlpaa, Mvrpaa), on the other hand, are also male sex myoblasts (renamed SM3), but their division pattern and the functions of their progeny are different in the two sexes.
The remaining male blast cells divide less, or not at all, in the hermaphrodite, but nevertheless become differentiated cells with welldefined functions. In the male, one or more of the progeny of a blast cell may take on the function which their parent performs in the hermaphrodite. Thus, C becomes a neuron in the hermaphrodite and its anterior daughter becomes a similar neuron in the male; the mesoblast Mdlpap (SM2) becomes a coelomocyte in the hermaphrodite whilst M.dlpappp (SM2.pp) does so in the male; B, E, F, and the various lateral and ventral blast cells are all hypodermal cells in the hermaphrodite and except for E each produces hypodermal progeny in the male as well.
For the most part, then, the secondary sexual apparatus of the male overlies the existing hermaphrodite anatomy. This contrasts with the development of the gonad (Kimble and Hirsh, 1979), in which the division patterns of the hermaphrodite and the male diverge at an early stage.
Changes in Cell Function during Development A number of cells play more than one role in the course of the development of the male, as the following examples show:
(1)The anal depressor muscle acts to open the anus during defaecation of larvae.During the final moult its myofilaments turn through 90°, and the muscle then acts as an accessory to the spicule protractors.
(2)The spicule retractor muscles are required during L4 lethargus for morphogenesis of the proctodeum. This function may not be related to their subsequent muscular nature; probably they simply act as couplers at this point.
(3)The seam cells, which lay down the lateral cuticle at each moult, are also blast cells in both the hermaphrodite and the male. In addition, the most posterior seam cell (T) is the socket cell of the juvenile phasmid, and segregates this function to two of its daughters during its subsequent division.
(4)Cells B, C, E, F, and K are responsible for forming parts of the rectum in larvae. At the same time they divide repeatedly to yield both structural cells and neurons.
Another striking example of changing cell roles is the reversal in information flow along certain neurons of the ventral cord at the L2 stage, described by White et al. (1978).
Symmetry In the Postembryonic lineages of the hermaphrodite, the two daughters of a transverse division across the midline have equivalent fates (Sulston and Horvitz, 1977). The lineages of an individual male contain several exceptions to this rule, but when several animals are examined it is found that in all but one case the two cells have equal potential.
Thus, in both cases of asymmetric cell death (B.al/rapaav and Bgamma.al/rd) either the left or the right cell can die (Fig. 6). The decision may be due to a position effect, the left and right cells being ordered at random as in the case of the Balfa/Bbeta and Bgamma/Bdelta pairs (Sulston and Horvitz, 1977). Similarly, we now know that the left and right daughters of E both have the potential to give rise to neuroblasts. This is not to say that all possible outcomes are equally probable: indeed it would be surprising if that were the case, since the animal is inherently asymmetrical at the cellular level. At present, though, not enough individuals have been watched for the probabilities to be estimated.
In a similar way, each individual is asymmetric while the population as a whole is symmetric with regard to the gonadal lineages (Kimble and Hirsh, 1979). The possible exception to the rule, the development of the mesoderm, is intriguing and unexpected. In fact, because this development is difficult to follow, it seemed likely at first that the observations were in error. However, the cell assignments have proved to be perfectly reproducible on each side.
Cell Death The gonadal linker cell is engulfed by E.lp or E.rp, and dies soon afterwards. That these events are causally related is shown by the ablation of blast cell E, after which operation the linker cell does not die. It is confirmed by the observation that the linker cell survives in gonads which fail to reach the proctodeum, due to genetic defects or to ablation of other gonadal cells (J. Kimble, personal communication).Thus E.lp and E.rp seem to be killer cells directed against the linker. In the same way P12.pa appears to be responsible for engulfing and killing either B.alapaav or B.arapaav, and the fusion product of F.ld and F.rd may be involved in the death of Bgamma.ald or Bgamma.ard. It is possible that all programmed cell deaths in the nematode involve killer cells. Alison Robertson (personal communication) has found that ventral cord cells which are programmed to die are surrounded by the syncytial hypodermis before cytokinesis is complete; since overt signs of cell death follow only after some 30 min or more, it is likely (though not proved) that the hypodermis is killing them.
Developmental Variability The development of the male is more prone to variation than that of the hermaphrodite.
(1)A common error is the loss of one
or more rays, the most vulnerable
being 3, 8, and 9; this appears to be
due to the failure of the structural
cell to form a process. Frequently,
in place of ray 3, a thin process can
be seen by Nomarski microscopy,
suggesting that at least one of the
neurons has been laid down in the
(2)A less common error is the fusion of rays 8 and 9.
(3)The lineage of cell E is variable: either one, or both, or neither of its anterior daughters may move for ward into the preanal ganglion and there divide into two neurons.
None of these variations, or errors, is known to influence mating behavior significantly, but they do show that the male copulatory apparatus is constructed in a less precise way than other parts of the nematode.
Moulding of the Cuticle During the final moult, the adult cuticle is shaped into a membranous fan encircling the ventral surface of the tail. The animal achieves this by secreting a layer of cuticle at a time when the tail is expanded: subsequently, the tail shrinks, and the oversized cuticle collapses in a welldefined way to form the fan (Fig. 4). This stratagem of collapsing a single layered surface into a double layer is analogous to the formation of an insect wing (Seligman et al., 1975), except that the layer is acellular rather than cellular. The collapse might be caused either by withdrawal of fluid from the subcuticular space or by an active contraction of the middle layer of cuticle. Contraction of cuticle secreted by the body seam has been implicated in the maturation of dauer larvae (Singh and Sulston, 1978). With this in mind, the tail seam was ablated in four animals; however, although the fan cuticle was somewhat attenuated by the loss of hypodermis, it still folded correctly.
Another apparent example of the use of cells as temporary moulds for the cuticle is found in the formation of the spicules. In the late L4, the bundle of processes from the socket, sheath, and neuronal cells of each spicule grow along a U-shaped channel (Fig. 8) and sclerotic cuticle is laid down around the bundle. Subsequently, the cells protruding into the channel shrink, leaving the resulting U-shaped spicule free to move.
Adapted by Yusuf KARABEY for WORMATLAS, 2003