The organisation of processes in the ventral nervous system is established during a short period of little more than an hour, at the same time as the animal is elongating in the eggshell from a stubby ¬tadpole' to a worm. Electron microscope reconstructions of varying lengths were made from a series of four embryos at different developmental stages during this period (figure 3.1). Figure 3.4 shows a schematic picture of the state of the ventral nervous system in each of the reconstructions, which will be referred to by the letters A to D. During the period covered by these reconstructions the embryo increases by a factor of about two in length, being about one and a half fold in the egg (100 microns) at the time of the A reconstruction, and three and a half fold (220 microns) in the D reconstruction. At the beginning of the period under consideration here the nerve ring contains the majority of the final number of processes. Uncoordinated muscle activity has already started before the time of the A reconstruction (the onset of twitching is at about 430 mins). Movement becomes more organised around the stage of the final, D reconstruction, although since the embryo is restricted inside the egg shell it is not possible to assess fully the degree of coordination.
Growth cones are generally extended flattened lamellar structures that also
have long thin filopodial extensions. In C. elegans the most extensive
growth cones are seen on the growing tips of the motor neuron commissures.
Typically they are a flattened sheet a few tenths of a micron thick and of
variable shape and size in the plane of the sheet (figures 3.2, 3.5). The
absence of normal looking filopodia may be due to the small scale (2-5
microns across); a vertebrate tissue culture growth cone could extend right
round the C. elegans embryo. However stubby finger-like extensions
are seen in many cases, and these may perform an equivalent function.
Figure 3.2 shows a three dimensional reconstruction of the complete cell DB4
from the B reconstruction, in which the thin sheet-like nature of the
growth cone can be clearly seen.
Extended growth cones like those seen on commissures were not seen on processes growing along the ventral or dorsal cords, although some tips do have swollen or spread out endings (e.g. PVCL in figure 3.7). This corresponds to observations made in other organisms that process growing along pre-existing nerve bundles do not have such extended growth cones as those growing over virgin territory (Lopresti et al., 1973).
The quality of the cytoplasmic fixation in the embryos used for reconstruction was poor, since primary fixation is with OsO4 follwed by tannic acid, which fixes membranes well but leaves little cytoplasmic structure. Therefore neither actin microfiliaments nor microtubules are preserved. However in some cases it is possible to see vesicles in growth cones, as for example in a commissural growth cone in the C reconstruction (figure 1.1). Studies by de Cino (1981) have indicated that transmitter is sometimes released by growth cones. An alternative explanation for the vesicles is simply that they may be a source of new membrane for insertion at the leading edge of the growth cone.
This shows the approximate ages of the embryos used in this study from which long series were reconstructed completely, and the parts of them that were reconstructed. Ages were determined as described in Chapter 2.5. The bracket below the embryo indicates the part of the ventral cord shown in figure 3.4.
A three-dimensional reconstruction of the motor neuron DB4 from the B series. The cell body is on the right. Out of this extends a growing commissure, terminating in the flattened extended structure at the left, which is the growth cone. This diagram was made with the aid of a 3-D reconstruction program written by J.G. White. The growth cone is approximately 5 microns across.
The growing DB5 commissure (Com) is forced to choose whether to pass the lateral neuron body of CANL (Neur Bod) on the side of the hypodermis (Hyp) or on the side of the basement membrane (BM). It passes on the hypodermal side, as do all motor neuron commissures in similar situations. This suggests that commissural growth cones attach to and move over cell surfaces rather than basement membrane. From the C reconstruction. Scale is 1 micron.
Growth cones have been seen in vitro to extend very well over artifical
substrates made of basement membrane components, such as fibronectin and
laminin (Baron van Evercooren et al., 1982). This has led to the
suggestion that basement membrane may provide a favoured substrate for
growth cones to grow over. It is possible in at least one case in C.
elegans to determine the substrate on which the growth cone moves.
The motor neuron commissures grow out sandwiched between hypodermal cells
and the basement membrane. There are several lateral neuronal cells that
also lie between the hypodermis and the basement membrane, in the way of
the growing commissures. Whenever a commissural growth cones reaches a
lateral cell body it leaves the basement membrane and passes between the
hypodermis and the lateral neuron (figure 3.3). There has never been
observed an exception to this rule. Thus it seems that the growth substrate
for these commissures is the surface of hypodermal cells, not the basement
A couple of similar results are provided by ablation experiments (Chapter 4) in which in one case DD5 moves from the right side of the cord to the left (after removing AVG), and in another case PVQL moves from the left to the right (after removing PVPR). In each case the process changing sides passes under a motor neuron cell body, rather than over it, again maintaining contact with the hypodermis rather than the basement membrane. There are many other examples where processes grow between cell bodies and other processes, well removed from the ectodermal basement membrane. During later development several posteembryonic processes grow the length of the ventral cord in the middle of the main bundle of embryonic processes. The embryonic reconstructions presented here show processes from the lumbar ganglia growing forward through the middle of the cluster of cell bodies in the preanal ganglion. In general wherever there is evidence on the subject of neuronal growth cone guidance in C. elegans it suggests that the substrate for growth is the surface of other cells, rather than a basement membrane. However this does not rule out the possibility that the basement membrane is important in certain cases.
The first nerve process to grow along the ventral cord belongs to the interneuron AVG. AVG has its cell body in the retro-vesicular ganglion at the front of the ventral cord; it is an unpaired cell, and is the most posterior interneuron in the front of the animal to send a process back along the cord. The cell body and process were identified in both the A and and B reconstructions. By the A reconstruction the process has already grown back along the cord. At this stage the DD ventral cord processeshave also grown out on the right side of the cord (figure 3.4). However inspection of a younger embryo revealed that there was a single continual process in the ventral cord at a stage at which DD processes had not grown out (not shown). In the B reconstruction the AVG process grows the full length of the cord and up out of the pre-anal ganglion into the dorso-rectal ganglion, where it stops by the DVC cell body. IT reaches no further than the DRG in all the latter embryonic reconstructions (B to D), although in the adult it is seen to extend right back into the tail spike (White et al., 1986). Therefore there must be a second period of extension postembryonically or during late embryogenesis.
The next event after the appearance of AVG is the growth of
processes from the DD motor neurons forward alongside the AVG
process on the right side of the ventral cord. In the A
reconstruction these extend until they nearly touch the next DD
cell body (figure 3.4). In the adult, adjacent DD neurons overlap
for a short stretch and are linked by gap junctions, but in all
the embryonic reconstructions there are small spaces between them
of the order of a micron in length (figures 3.4, 3.5). It is of
course possible that contact has been made and the processes
subsequently withdrawn. Another possible correlate of DD
extension in the ventral cord, discussed further later, is that
all the DD ends of DD processes are by DB cell bodies. Ventral
cord processes from the DA and DB motor neurons do not grow out
until later, after the commissures and dorsal cord processes are
The first signs of commissure outgrowth can also be seen in the A reconstruction. All the motor neurons have lamellar extensions poking laterally under the ventral musculature at the site where their commissural growth cones will leave the ventral cord (figure 3.4). These nascent growth cones leave from the DA and DB cell bodies, and from near the anterior end of DD axons. Outgrowth of commissures from the motor neurons of all classes is synchronous; in the B series, only about 20 minutes older than the A series, they have all reached the lateral hypodermis, and in the C series they are just about to reach the dorsal hypodermal ridge (figure 3.4). RID, the only process to grow along the full length of the dorsal cord, is not present at this time, and the commissures apparently turn of their own accord, DA ones anteriorly, DB posteriorly, and DD in both directions, and link up to form the dorsal cord. Although they are reproducible, there is no regular anterior/posterior order to the positions of the commissures from the different classes of cells (figures 1.3, 3.5), so the direction in which they turn cannot be simply determined on the basis of the classes of neighbouring processes (e.g. DA's and DB's towards each other). Since the dorsal hypodermis is syncytial and does not contain apparent landmarks it seems that the direction must be intrinsically determined. In addition, if we assume that the direction is determined in the same fashion for all the members of a class, then it must be specified in terms of the anterior and posterior of the animal, rather than whether to turn left or right, since some of the DB axons go round the left hand side of the body and turn right when they reach the dorsal midline, while others go round the right side and turn left.
Schematic diagrams of the part of the body indicated in figure 3.1 from the A, B, C and D reconstructions (1), b), c) and d) respectively). The diagrams are cylindrical projections in the same basic form as figure 1.3, with the positions of the muscle bands being shown as hatched areas. The DA3, DB4 and DD2 cell bodies and the DA3, DB4 and DD3 commissures are shown in each case. Initially only the AVG and DD processes in the ventral cord are present (a). Then the commissures grow out simultaneously from all three motor neuron classes (b, c), followed eventually by the ventral cord processes of DA and DB motor neurons and other ventral cord interneurons (d). ALML/R and CANL/R are lateral neurons, which migrate back from the front and then send processes forward.
A schematic diagram of the entire ventral cord region from the C reconstruction. The motor neurons and lateral neurons are shown on the left in the same form as in figure 3.4. (HSNL/R are postembryonic neurons that grow out processes in the L4 larva to innervate vulval muscles). The positions reached by all the interneurons that have grown substantially into the ventral cord are shown on the right. In addition to the PVT, PVCL and PVCL processes have also grown just past the front of the preanal ganglion in this reconstruction.
There appears to be a possible correlation between the position of DA
commissures and the location of hypodermal cell boundaries. The embryonic
ventral hypodermis consists of 6 left/right pairs of cells, known as P
cells, which are joined at front and back to the main body hypodermal
syncytium, called hyp7. DA3
lie on the boundaries between adjacent P cells, and they send solitary
commissures to the left directly from their cell bodies out along the P/P
cell boundary. In contrast DA2,
which is on the boundary between the most anterior P cells and hyp7, sends
its commissure forward and out to the right together with those of DB3
none of which are near a P/P cell boundary, send their commissures together
with processes from other cells (DB2
in the case of DA1,
the lumbar commissures for DA8
There are no corresponding visible cues for DB commissure guidance,
and only weak ones for DD commissures. One
possibility is that DB cells are involved;
commissures all leave the cord from approximately opposite DB cells (figure 3.5,
note especially DD3,
whose commissure exit point is quite a long way behind the DD2
cell body, but opposite DB4).
It is hard to tell whether a DD commissure is
created by diversion of the growth cone that generated the ventral cord
process, or by a genuine branching. The commissure always comes from near
the anterior tip of the ventral cord process, the extension of which is
essentially complete when the commissure starts growing, suggesting that
only a single growth cone is used. However there is a definite T junction
in the final structure, and the DD process can make
branches, since one is certainly made when the commissure reaches the
dorsal cord (figure 1.3). The side of the animal that the DD commissures go round
is easier to explain. They all go round the right side of the animal,
which is consistent with the position on the right side of the cord of
their ventral cord processes, from which the commissures diverge or branch.
It is only after the dorsal cord processes have extended for some distance that we begin to see growth of the ventral cord dendrites from DA and DB neurons, (D reconsruction, figure 3.4). This coincides with the growth back along the cord of some of the ring interneurons, possibley including the motor circuitry interneurons that innervate the DA and DB ventral cord processes. However there are no visible synaptic connections between the interneurons and the growing DA and DB dendrites.
The dorsal cord, in the other hand, does show signs of synaptic activity in the D reconstruction. The DA and DB neurons are already making small, but clear, neuromuscular junctions from their dorsal cord axons (figure 3.6). As in the final adult version these involve a joint synapse onto muscle and a DD process. No corresponding DD neuromuscular junctions in the ventral cord have been seen in this reconstruction. The dorsal cord processes have not reached their final length in the D reconstruction, and in fact are at a rather interesting stage: each DA axon stops where the next one arrives at the dorsal cord and turns. Later the axons overlap considerably, but the regions of neuromuscular output do not; instead they correspond closely to the regions where the axons are present at this stage. It is possible to speculate that there is a pause in axon extension while the zones of neuromuscular activity are being set up, but more data would be required to provide respectable evidence!
A neuromuscular junction from the DD reconstruction. Although small this shows all the characteristics of normal neuromuscular junctions in the developed nervous system. The dorsal cord process of the motor neuron DA4 is synapsing jointly onto muscle (mus) and the DD3 process. There is also a DB4 process present in this section. Scale bar is 1 micron.
While the motor neuron commissures are growing out, a set of interneurons
are growing forward along the ventral cord from the pre-anal ganglion (PAG)
at the back (figure 3.5). The most advanced of these are two pairs of
and PVQL on the left side of the ventral cord and PVPL
and PVQR on the right side. These are followed by DVA
which are both unpaired neurons that run on the right. Figure 3.7 shows a
cross section of the posterior cord from the C reconstruction, after these
posterior neurons have reached the front of the cord, that we see other
anterior interneurons growing back along the cord (figure 3.4). PVPL/PVPR
neurons are the most anterior cells in the lumbar ganglia (PVQL in the left
lumbar ganglion, PVQR in the right, figure 1.3); their processes descend
the lumbar commissures and then run forward through the PAG and along the
have cell bodies in the PAG, where they are the only bilateral pair of
interneurons. Their processes cross over when they leave the bodies,
joining up with the PVQL/PVQR
process on the opposite side, and then run forward along the cord. The
structure of PVPL/PVPR
decussation is discussd below together with that of three pairs of neurons
in the retrovesicular ganglion.
PVQL and PVPR pioneer the left hand ventral cord; this appears to be a joint action, since their anterior tips are never more than a few tenths of a micron apart in any of the reconstructions (e.g. figure 3.5). The tips of the PVQR and PVPL are similarly close to each other, but there is no such tight relationship between the left hand pair and the right hand pair (figure 3.5). The fact that the left hand pair are often more advanced than the right hand pair suggests that the prior presence of AVG and DD processes on the right side of the cord has little effect on PVPL and PVQR outgrowth. The two processes in each pair are tightly associated all the way along the cord back to the point where the PVPL/PVPR processes cross over in the PAG. This association is also seen in the adult reconstructions of the ventral cord; the processes diverge only when they reach the nerve ring (White et al., 1986, unpublished data). Together these observations suggest that PVPL/PVPR and PVQL/PVQR growth in the ventral cord might be cooperative, and a number of ablation experiments were performed to test this hypothesis (Chapter 4). In general the PVPL/PVPR process is on top of the PVQL/PVQR process, i.e. there is a ventral to dorsal order of: hypodermis, PVQL/PVQR, PVPL/PVPR, basement membrane (figure 3.7). DVA and DVC: DVA and DVC are the two embryonic neurons with cell bodies in the dorso-rectal ganglion, above the rectum (1.3). They both grow forward along the right hand side of the cord behind PVPL and PVQR, but their tips are not close together like those of a PVPL/PVPR/PVQL/PVQR pair. DVA grows down around the right side of the rectum back along the track of AVG, and in all cases keeps to the outside of the main right hand bundle of processes in the ventral part of the PAG (figure 3.7). DVC, on the other hand, grows down the left side of the rectum and crosses from dorsal left to ventral right, in the same place that two PVPL/PVPR processes cross over (3.8). Neither DVA nor DVC appear to be particularly tightly associated with any other process in the ventral cord. In the adult cord DVA is always at the ventral right hand exremity of the main right hand bundle, whilst DVC runs in the middle of the bundle, in association with DVB, which is the postembryonic dorso-rectal ganglion neuron (White et al., 1986). Other lumbar commissure processes: In addition to the DA8, DA9 and PVQL/PVQR processes the lumbar commissures contain processes descending into the pre-anal ganglion from the following lumbar ganglion cells: PHAL/PHAR, PHBL/PHBR, LUAL/LUAR, PVCL/PVCR and PVR. Of these the PVCL/PVCR cells and PVR eventually grow the full length of the cord; the others stop at the front of the pre-anal ganglion. There is a very characteristic pattern at the back of the pre-anal ganglion where the lumbar processes from the two sides meet, which has been seen whenever the region has been reconstructed (figure 3.9). The processes from each side meet slightly to the right of the midline and "zip up", each contacting its contralateral analogue. The exceptions to this rule are the PVQL/PVQR processes, which are at the top of the row one each side but stay at opposite corners of the structure and do not make contact. The dorsal to bentral order of this structure is PVQL/PVQR, PHAL/PHAR, PHBL/PHBR, PVCL/PVCR, with the unpaired process of PVR wrapping around the ventral side of the whole group. This suggests that the contralateral pairs other than the PVQL/PVQR's have a strong affinity for each other, and it is probably significant that the PVQL/PVQR's are the only lumbar processes to run up the cord with one process on each side of the cord, as opposed to the more normal pattern of both processes being on the right. Anterior interneurons: Anterior interneurons other than the AVG are only seen growing back along the cord in the D reconstruction. As described in the section on cell identification in Chapter 2 it is not possible to identify these processes. However it is clear that there are at least PVPR and PVQL on the left side. There is one process growing part way back along the cord on the left, which may be RMEV or AVKR, and there are 11 interneurons on the right, some of which probably come from the back (e.g. PVPL and PVQR), but others of which are from the front; in particular 4 from the front stop within the anterior D reconstruction. The anterior interneurons seen here may include (some of) the major interneurons that innervate the ventral cord motor neurons.
Typical transverse sections through (a) the preanal ganglion and (b) the back of the ventral cord (from the C reconstruction). In (a) the star indicates the main group of processes that will become the right hand ventral cord. The other cells and processes are individually labelled. This section comes from just posterior to the point where the PVP processes and DVC will cross over (shown in figure 3.8). In (b) the independent growth of PVPR and PVQL along the left hand cord is clear. This section comes from very close the the preanal ganglion, and the PVCL/R and PVT processes are near their front tips, and are a little swollen, particularly PVCR, which is showing signs of wrapping around other processes. Scale bar is 1 micron in each case.
The PVPL/PVPR processes cross over in the pre-anal ganglion where they leave their cell bodies and then grow forward on the opposite side of the cord. This crossing over is at the back of PVT and above DD6, at the same place that the DVC process crosses from left to right. PVQL, DVC and, to a lesser extent, PVQR flatten out on the surface of DD6; DVC is always between the PVPL/PVPR crossover and DD6 (figure 3.8). The PVPL/PVPR cross over was observed in the adult reconstruction (White et al, 1986), but it is much less clear there since there are extra cells in the adult preanal ganglion (6 postembryonic motor neurons) and the arrangement of cells and processes is much less well organised. This loss of symmetry and order is already visible in the difference between the B and D reconstructions. In the B reconstruction the cells in the left/right pairs (PVPL/PVPR and DA8/DA9) are nearly opposite one another, while in the D reconstruction there is a definite tilt to each pair and the whole preanal ganglion is becoming more linear. This is probably caused by a lateral constriction due to elongation of the embryo and an increase in the amount of space taken up by the muscle cells as they mature. In the adult the symmetry and order present in the early embryo when processes first grow out is almost entirely lost.
The crossover of PVPL/R processes in the preanal ganglion (from the C reconstruction). (a) to (d) are a posterior to anterior series, each being separated from the next by about 0.5 microns. (e) to (h) are tracings of (a) to (d) showing the positions of significant processes. The sequence of events is as follows: at the front of its cell body (a) PVPR sends a process across to the left side (b), in front of which DVC and PVPL cross from left to right (c, d). The PVPL process invariably crosses in front of the PVPR process. Scale bar is 1 micron.
The place at the back of the preanal ganglion where the two lumbar commissures meet (from the C reconstruction). All the processes are labelled: QL/R are PVQL/R, AL/R are PHAL/R , BL/R are PHBL/R , CL/R are PVCL/R , and PVR , DA8 and DA9 are all the correct full names. The PHAL/R , PHBL/R and PVCL/R processes all line up against each other, to some extent wrapping around their partners and thus increasing the area of contact (particularly the PHAL/R and thus increasing the area of contact (particularly the PHAL/R processes). In contrast the PVQL/R processes appear to have no mutual affinity. More anterior to this, the PHAL/R , PHBL/R and PVCL/R processes all grow along the right side together, while the PVQL/R processes split, one growing on he left and one on the right. Scale bar is 1 micron.
The decussation in the retrovesicular ganglion (from the B series). The RIF processes have both just crossed over posterior to this section, and we can see the back of the SABVR cell body and its process also thrusting across the midline. In contrast to the preanal ganglion decussation here processes cross on the surface of the neuropil. Scale bar is 1 micron.
A phenomenon equivalent to the PVPL/PVPR crossover is seen in the
retrovesicular ganglion at the front of the ventral cord, which is
once again more symmetrical in the embryo than in the adult.
There are three bilateral pairs of neurons in the embryonic
retrovesicular ganglion: RIGL/RIGR, RIFL/RIFR and SABVL/SABVR, all of whose
processes run forward. The RIFL/RIFR processes have grown out in the A
and B reconstructions and once again they are seen to cross over
by their cell bodies (figure 3.10). Following this observation,
careful comparison of the positions of cell bodies in embryonic
reconstructions and the embryonic lineage study (Sulston et al.,
1983) with those in the adult reconstructions (White et al., 1986)
confirmed that the RIFL/RIFR processes did indeed cross over in the
adult reconstructions, and that the RIG and SABV processes do the
same. The Processes from all three classes cross in nearly the
same place in the adult reconstruction, by the SABV cell bodies.
The SABV processes are just beginning to grow out and cross in the
B reconstruction (figure 3.10), but the RIG processes have not yet
Therefore crossing over is seen in all the embryonic left/right pairs of interneurons in what might be termed the extended ventral cord, i.e. everything on the ventral hypodermal ridge between the excretory duct and the anus. During postembryonic development another interneuron pair, AVFL/AVFR, is added in the retrovesicular ganglion, but their processes are not bilaterally symmetrical; they are bipolar, running back together down the right side of the ventral cord and forward also together to the left of the excretory duct and round the left side of the nerve ring.
The crossing over, or decussation, of processes to the opposite side of the body from the soma is a property of many nerve types in higher animals, and the cases observed here may provide extremely simple examples of the same event that are susceptible to experimental manipulation of both the cells involved and of their environment. Several cell ablation experiments were performed to investigate factors involved in the PVPL/PVPR cross over in the preanal ganglion (Chapter 4).
An observation previously made in other animals is the insertion of thin
processes from growth cones into other neuronal processes or target tissues
that might be important for guidance (Bastiani and Goodman, 1983). The
same phenomeno9n has been observed in the developing C. elegans
nervous system, and there are two cases in particular where it is
especially noticeable and correlates with possible guidance decision
The first case is when the motor neuron commissures reach the dorsal midline. In one reconstruction (wild type but not on of A to D), a single commissure has just reached the dorsal hypodermal ridge, on emerging from underneath the left dorsal muscle quadrant. The growing tip of this commissure inserts two stubby finger-like processes about 0.1 « 0.2 microns in diameter and 0.4 « 0.8 microns long into the dorsal hypodermal ridge (figure 3.11). It is at this stage that the growth cone must turn through a right angle and grow along the ridge.
A finger from the DA6 commissural growth cone inserting into the dorsal hypodermal ridge as it comes out from under the left dorsal muscle quadrant. The particles to the left of the finger are not vesicles (determined by viewing at higher resolution).
An insertion of an extension at the front of the DD5 ventral cord process into the cell body of DB6 . DD5 stops growing forward along the cord at about the point that it reaches DB6 . Both DD5 and DB6 send commissures out to the right around the same place. A part of the DB6 commissure can be seen. From the C construction. Scale B 1 micron.
The second case concerns what happens when DD processes meet
DB cell bodies. In all cases that have been reconstructed the
anterior tips of DD6 and DD5 insert into the bodies of DB7 and DB6
respectively. Insertion of DD4, DD3 and DD2 into DB5, DB4 and DB3
also takes place but less frequently and in a less pronounced
fashion. An example of DD5 insertion into DB6 is shown in (figure
3.12). These DD insertions into DB cells reinforce the suggestion
made above that DB cells might be involved in DD morphology.
Where the insertions are most pronounced (DB6 and DB7), the DB
commissures grow to the right next to the DD commissures.
There are occasional other insertions into the ventral hypodermis from growth cones of processes growing along the ventral cord (data not shown), but no particular pattern is discernible. Also I have seen no cytological correlates of the insertions, such as vesicles clustering around the insertion in the cell into which the insertion is made, which have been seen in the corresponding phenomenon in insects (Bastiani and Goodman, 1983).