The last two chapters have described the results of a number of electron microscope reconstructions of the developing ventral nervous system in both normal and experimental C. elegans embryos. In a short space of around an hour the first nerve process grows back along the ventral cord from the front, the motor neurons in the ventral cord grow commissures around the body of the animal to form the dorsal cord, and a number of additional processes grow forward from the preanal ganglion at the back of the animal. The short time taken in laying down the skeleton of the ventral nerve cord and preanal ganglion reflects the rapid development of C. elegans embryos (13 hours total). The small number of cells present, and the simple morphologies of the nerve cells, allow precise suggestions to be made about the roles of individual cells during process outgrowth. Several possible intercellular interactions were investigated by killing the parents of specific cells with a focussed laser beam. Before discussing the pattern of process outgrowth in the ventral nervous system, and how it might be controlled, I will first consider the reliability of the observations on which the work is based.
The approach of reconstruction from serial electron micrographs precludes
the examination of a large number of individual animals, either in the wild
type time series or in any particular experiment. It is reasonable to ask
whether reliable conclusions can be drawn from the necessarily small number
of reconstructed animals that have been presented: there are two possible
sources of error or variation: experimental "noise" created by variability
in the observational and experimental techniques, and natural variability
of the phenomena themselves.
As far as the determination of process disposition is concerned the
technique is very reliable; each individual reconstruction provides a large
amount of information at a very fine level of detail, so that essentially
all the nerve processes present can be positively identified and a complete
picture of the relevant parts of each neuron determined (the exceptions are
discussed in Chapter 2). The technique of laser ablation of individual
identified cells is also very specific. It is unlikely that the killed
cell has any residual influence, because, except for the DVC
parent ablation, when no subsequent change was seen in any other cells
anyway, the dead cell was observed to be excluded from the embryo when the
hypodermis closed up (figure 2.2). It is in principle possible to damage
neighbouring cells at the time of ablation, and in fact one of the embryos
sectioned in the DD3/DD5
set showed signs of general morphological disorganisation, presumably due
to such damage. However in all the cases discussed, except the AVG
experiments, any changes that were observed were confined to a small number
of neurons normally associated with the particular missing cell. Although
control experiments in which random neighbouring cells were ablated were
not performed, altogether six different cells all near together on the
ventral surface of the 270 minute embryo (figure 2.1) were ablated without
there being any overlap in the observed consequences.
As regards intrinsic variation, all the reconstructions are consistent with
a fixed time sequence of normal axonal outgrowth. When a change in this
pattern was seen in laser ablation experiments then, again excepting the AVG
experiments, it was clean and restricted in its extent, was generally
observed in at least two cases, and was consistent, never being seen in one
case but not another. The situation with respect to the removal of AVG
appeared to show variability and is discussed more fully in the next
section. However, taking all the results together and in conjunction with
the known fixed adult anatomy, there is sufficient evidence to indicate
that the developing C. elegans nervous system is simple and
reproducible enough for the techniques used here to provide an accurate
picture of events.
The high level of reproducibility and the generally restricted, fixed
effect of removal of individual cells are typical of C. elegans
development and anatomy. The cell lineage and the disposition of somatic
cells at all stages of development are known to be nearly invariant
(Sulston and Horvitz, 1979, Kimble and Hirsh, 1979, Sulston et al.,
1983) the final antomy is equally stereotyped (White et al., 1986).
Although a number of cases of adjustment in cell lineage after individual
cell ablations are known (e.g. Sulston and White, 1980, Sulston et al.,
1983) they are the exception rather than the rule, and in no case do they
result in complete regulation back to the native form. All the results of
ablation experiments performed here are consistent with only the daughters
of the ablated cell being missing, and with there being no change of
identity of any other cell. In addition they confirm the relevant cell
assignments in the embryonic lineage, since in each case only the expected
cell or cells was or were missing.
One of the striking features of the C. elegans ventral nervous
system is the almost, but not quite, complete asymmetry of the ventral
nervous cord, which has around 55 neurons on the right side and only 4 or
so on the left. If all the processes were together on the right hand side
then it could be regarded as a single fused nerve that was displaced to one
side for steric reasons, but since a small number of left/right pairs of
processes are arranged symmetrically (PVQL/PVQR,
PVPL/PVPR,
AVKL/AVKR
and in the adult, HSNL/HSNR)
the question arises of why not all the others? In fact the arrangement is
essentially symmetrical anterior to the RVG; the cord splits into two to
pass the excretory duct on both sides, with each bilateral pair of
processes being split so there is one member on each side, an stays
symmetrical throughout the ventral ganglion and into the bottom of the
nerve ring.
Most animals with a symmetrical body plan have a symmetrical ventral
nervous system, often consisting of a chain of ganglia linked by paired
nerves, which are sometimes fused but clearly retain their symmetrical
character. There are in fact some nematodes that have symmetrical paired
ventral cords (Martini, 1916). Chitwood and Chitwood (1974), in discussing
the differences amongst nematode species, state (p. 162):
Differences in the central nervous system lie chiefly in the degree of
subdivision of the lateral ganglia, the form of the ventral ganglia, and
the degree of fusion of the ventral nerves.
They go on to state that in many species both around the RVG and for some
distance anterior to the PAG there are symmetrical paired nerves, though in
most cases these are fused for the main part of the length of the body.
They continue (p. 163):
The apparent doubleness in both anterior and posterior ends of the ventral
nerve caused Meissner and many later authors to conclude that the entire
nerve was at one time double. (we) subscribe to the primitive double
ventral nerve hypothesis.
Several observations that have been made in this study are relevant to the
origin of cord asymmetry. Perhaps I should start with AVG.
AVG
is a unique neuron with its body in the RVG, it is the first neuron to send
a process out along the ventral cord, and it sends it along the right hand
side. When AVG
was removed by ablating its parent the cord was seen to be disrupted in two
ways.
First, as seen most clearly in the embryonic AVG
reconstruction, the organisation of the embryonic ventral cord motor
neurons was disturbed. In particular a DD process was seen to
switch across to the left side of the cord and send its commissure round to
the left rather than the right, and the DA and DB cells near this
point also sent their commissures round the opposite side to normal. The
switch of the DD process to the left
cord confirms that AVG
must normally grow out before the DD ventral cord
processes. In the adult AVG
reconstruction the DB3
and DD2
cells show abnormal process organisation. These effects would seem to be a
direct consequence of the absence of AVG,
because the outgrowth of DD processes and motor
neuron commissures follow directly after the outgrowth of AVG.
The postembryonic motor neurons do not seem so badly affected as the
embryonic neurons, although the VD3
process in the adult reconstruction is switched from being on the right
side to the left.
The second effect of removal of AVG,
seen in the adult reconstruction, is a general disorganisation of the cord
in which instead of a large ordered bundle on the right side and a very
small one on the left there are several intermediate sized bundles at
various positions on the left and right sides (figure 4.1). This indicates
that AVG
is ultimately necessary for correct organisation of the interneurons as
well as motor neurons, whose disarray appears earlier. However AVG
does not seem to be necessary for outgrowth of processes, since the total
number of processes in a cross section of the experimental adult cord is
within the expected range, and all the fully reconstructed cells send out
processes in the correct direction, if not on the correct side.
It is also clear that AVG
is not the sole determining influence for the left/right organisation of
the ventral cord, because in the embryonic experimental reconstruction all
the early interneurons from the back were growing forward correctly PVQR,
PVPL,
DVA
and DVC
on the right, and PVQL and PVPR
on the left). Also in the adult AVG
reconstruction the majority of processes was at all times on the right,
including the motor control interneurons (AVAL/AVAR,
AVBL/AVBR,
AVDL/AVDR
and AVEL/AVER).
In the only positively identified case of interneurons growing on the
wrong side, both AVFL/AVFR's
were seen to grow on the left (they are normally both on the right, figure
4.1).
The fact that removal of AVG
leads to no major behavioural defect suggests that it has no critical
function of its own. In the adult reconstruction, although it is a fairly
large cell, it has been seen to make very few connections to other neurons,
the only consistent ones being large gap junctions to the two RIFL/RIFR
interneurons and a small amount of synaptic input from the PHAL/PHAR
phasmid neurons (probably chemosensory) (White et al., 1986). It
has been postulated to be a sensory receptor itself on the basis of its
adult extension beyond the dorsorectal ganglion into the tail, although no
ultrastructural specialisation is seen there (ibid.). One might instead
speculate that its main function is developmental. If one considers that
it is just as important for a nervous system to be able to build itself as
to function correctly in the end, it makes sense that there be selective
pressure for neurons important in development even if they serve little or
no purpose in the final circuitry. Another candidate for such a cell in
the C. elegans nervous system is PVT.
This is a large cell demarcating the front of the preanal ganglion and
forming the most anterior link between the rectal epithelium and the
ventral ectoderm, which has no observed synaptic output and only a couple
of possible inputs. However no experiments have been performed to test the
suggestion that it too may be primarily involved in developmental
organisation. Of course one should beware of suggesting that every neuron
must have a major function; it is quite likely that there are also
redundant cells present that are not particularly important at any time.
The disarray seen in the ventral cord of the adult AVG
reconstruction is very reminiscent of that seen in a reconstruction of a
mutant in the gene unc-3
(e151)(figure 4.1, J G White, E Southgate and N Thomson,
unpublished results). In that case too there were several subbundles,
looking very similar to those of the AVG
reconstruction; the majority of processes were on the right, including the
identifidable cluster of major motor interneurons which again retained
their internal organisation, but not their relative position in the bundle.
The defect appears to be restricted to the ventral cord since the nerve
ring was correctly organised according to several electron mircoscopic
criteria, bu the phenotype of unc-3
mutants is much more severe than that after ablation of the AVG
parent, and indeed in the reconstruction of the mutant it appeared that
some postembryonic motor neurons might be missing or not properly made.
There are two other uncoordianted genes for which mutants show relevant
defects. The DD and VD commissures can be
visualised by immunocytochemical staining with antibodies against the
neurotransmitter GABA (helping to confirm that the DD and VD classes are probably
GABAergic and inhibitory); they normally all grow to the right. However in
mutants for unc-71(e451)
and unc-73(e936)
a significant proportion of the commissures grow round the left side of the
animal (25% and 35% respectively; S McIntire, pers. Comm.). The ventral
cord is also seen to be disorganised, in that in some places in the cord
the VD
and DD
processes, which normally run so close together that they are inseparable
by light microscopy, are clearly separated. It would be interesting to see
what happens in early ventral cord development, particularly to AVG,
in all of these mutants.
The suggestion derived from the reconstructions of the adult AVG
animal and the unc-3
mutant that left/right pairs of processes tend to stick together may be
significant. When the lumbar neuronal processes meet in the PAG at the
bottom of the lumbar commissures they "zip" together, each process in
contact with its homologue, except for PVQL/PVQR which remain apart (figure
3.9). Eventually PVQL/PVQR end up on separate sides of the cord, while the
others all stay together on the right side. This affinity of a process for
its opposite homologue provides a simple mechanism to ensure that processes
stay together. Then perhaps only a slight bias is needed to send the pair
to one side rather than the other. The experiments in which the parents of
PVPL/PVPR
cells were ablated reveal an underlying preference for the right side in at
least one case. When PVPR
was removed PVQL crossed to the right side rather than grew along the left
side of the cord by itself, but when PVPL
was removed PVQR still grew along the right side. It may be that the
presence of preexisting fibres on the right rather than the left was the
determining factor in this particular case, but after AVG,
the DD
axons, DVA
and DVC
have grown out on the right side, which might prove sufficient to continue
to attract later arrivals.
To return to Meissner's suggestion the the primitive ventral nerve was
double, it may be worth discussing the advantages and disadvantages of a
fused cord over paired nerves. The obvious disadvantage of a single cord
like that of C. elegans is the loss of possible left/right control
over body movement. Although there are four bands of muscle in C.
elegans both ventral quadrants receive the same input from the right
hand ventral cord, as do both dorsal quadrants from the single dorsal cord.
Therefore the body of the animal moves only in the dorsal/ventral plane,
although the head can and does move freely in all directions. However there
are extra cross connecting motor neuron and interneuronal classes in the
head, and it is likely that in order to obtain reasonable left/right
coordination, something similar would be needed in the body. There is no
sign of this, even in vestigial form. On the other hand, if, as seems
likely, the putative primitive twin-nerved ancestor did not have the
capability for left/right body control (I have found no mention of any
nematode that does), then there is a strong case for bringing the motor
circuitry elements together in one nerve. First it allows an effective
halving of the number of motor neurons; with the system as it is in C.
elegans there is only one active motor neuron of each class at each
cross section of the body. Second it removes at source any loss of
synchrony between wave generation on the left and right sides of the body.
Third it provides back up in an extremely important part of the animal's
nervous system by having twofold redundancy of each motor circuitry driving
interneuron. However there is no obvious reason why the interneurons not
involved in the motor circuitry should join together or not, since they
serve no function in the cord but merely use it as a route from one end of
the cord to the other. Indeed this view is supported by the fact that a
minority of three apparently unrelated classes (AVKL/AVKR,
PVPL/PVPR
and PVQL/PVQR)
are still bilateral in C. elegans .
In conclusion I would like to speculate that the primitive nematode ventral
cord was double and symmetric, and that the selection pressure for the
currently more common asymmetric cord came from the motor circuitry. It
appears that AVG
plays a critical role in organising the left/right asymmetry of the motor
neurons. An important factor for the interneurons appears to be the mutual
affinity of left/right pairs (and of the motor circuitry interneuron
classes for each other, since they preserve their approximate relative
structure under perturbation by AVG
parent ablation and unc-3
mutation). The interneuron pairs of groups may then tend to go to the
right side either directly or under the influence of AVG,
the motor neurons, or other previously determined processes, such as that
of DVA.
If this picture is correct then the fact that so many left/right pairs of
non-motor circuitry interneurons also join up and grow together on the
right would suggest that, even in situations like this where all the cells
are individually distringuishable, neural guidance may be often controlled
by non-specific factors that affect a large number of neurons.
The preceding section described how the presence of AVG
appears to help determine the side of the cord that the DD processes grow
along. A second question concerns how the DD processes growing
along the ventral cord know where to stop and send out their commissures.
Although they have short posterior processes, the main DD ventral cord
processes extend forward from the cell bodies, eventually making contact
with the next DD cell along. However
there is a certain amount of evidence to suggest that the determining
factor for DD ventral cord growth
may not be the next DD cell, but the
position of the next DB cell body. First the
DD
commissures always exit from next to DB cell bodies, even
when these are not immediately behind the next DD cell (e.g. DD3/DB4
in figure 3.4). Second there often seems to be some sort of recognition
event involving DD process tips
inserting themselves into DB cells at the time of
and soon after process outgrowth, particularly at the back of the cord
(figure 3.12). Third, in Acaris, where distances are much greater, all the
DD
commissures exit opposite DB cell bodies together
with DB
commissures, which are all on the right hand side behind the RBG (Johnson
and Stretton, 1987). VD and AS commissures also
grow out together in Ascaris (ibid.). Neighbouring VD and AS cells are sisters,
but there is no lineal relationship whatsoever between DB and DD cells (in C.
elegans , and presumably also in Ascaris, whose early lineage is
identical to that of C. elegans , Sulston et al., 1983).
Fourth, after DD3
and DD5
were removed by ablating their parent, DD4
did not extend to fill the whole space left by DD3,
but instead stopped and began sending out a commissure at an only very
slightly anterior position to normal (figure 4.3). This experiment does
not prove DB involvement,
however, because it remains possible that the normal growth length is
intrinsically determined, as appears to be the case with the postembryonic
touch cells AVM
and PVM
(Chalfie et al., 1983). A more conclusive, but unperformed,
experiment would be to remove a DB cell.
The next event after DD process outgrowth is
the growth of the motor neuron commissures. All the commissures grow out
synchonously and reach the dorsal midline at the same time, well before any
other longitudinal process has grown along the dorsal cord (RID
will do so eventually). There is therefore a problem of recognising the
correct point at which to turn, and a subsequent problem of deciding the
direction in which to turn. Although adjacent to the basement membrance,
the commissural growth cones appear to grow on the surface of the
hypodermis, rather than the basement membrane (section 3.2). Similar
behaviour was inferred from experiments on early optic nerve outgrowth
(Krayanek and Goldberg, 1981). When the growth cones reach the dorsal
ridge they have been seen to insert finger-like extensions into the
hypodermis, indicating that some cell recognition event may have taken
place (figure 3.11). Therefore it seems that the best candidate for the
source of the required information is the dorsal hypodermal ridge itself,
and that the growth cone "tastes" the hypodermis as it advances, eventually
recognising the dorsal ridge.
The suggestion that there is a specific property of the dorsal hypodermal
ridge that is recognised, while simplifying the explanation of how the
dorsal cord is formed, creates problems of its own. The dorsal hypodermis
is a syncytium containing many nuclei and covering the dorsal side of the
animal from head to tail and from one lateral ridge to ther other (the
lateral boundaries can be seen in the section in figure 1.1). The
commissure therefore grows on the surface of this syncytium for some time
before it recognises a specific part of it. In so doing it crosses the
path of some later longitudinal nerves, such as the ALML/ALMR
process, and the sublateral bundle (SAAD,
SABD,
SIBDL/SIBDR,
SMDDL/SMDDR,
see figure 1.2). Hence it appears that some property of the membrane must
be localised to only that part of the cell surface covering the dorsal
ridge. The syncytium is formed in the embryo in a curious fashion by two
rows of cells passing between each other and then fusing. Mutations in two
genes, unc-83
and unc-84,
are known to affect this process (Sulston and Horvitz, 1982). Although
mutant L1 larvae move well, they have been seen in electron microscope
reconstructions to contain defects in the structure of the dorsal cord (J.
G. White, unpublished observation), which might be due to the failure in
the correct localisation of recognition components in the dorsal hypodermal
ridge.
Once the motor neurons have turned onto the dorsal cord, they seem to grow
out rapidly along it and, if they are DA or DB neurons, start
making neuromuscular junctions (D reconstruction, figure 3.6). It is only
at around this time or later that their dendrites grow out in the ventral
cord, so they start neuromuscular activity receiving organised synaptic
input. A system in which neurons generate synaptic activity before they
receive their controlling input would be expected to generate a lot of
random signals, but would allow the whole nervous system to be built
simultaneously instead of sequentially, starting with sensory neurons and
progressing along the processing pathway.
Decussation of nerve processes, in which an entire group of cell processes
cross the midline, is a standard phenomenon in most animal nervous systems,
and a scaled down version of the same type of behaviour can be seen in
C. elegans in the crossing over of processes from paired
interneurons in the preanal and retrovesicular ganglia. The PVPL/PVPR
processes cross in the PAG (figure 3.8) and the RIFL/RIFR,
RIG
and SABV processes cross in the RVG (figure 3.10). Since the general
property of decussation appears to be functionally unnecessary, it may give
some insight into general constraints on developmental organisation.
It is very clear in C. elegans that there is no ultimate functional
advantage to be gained from the decussation. The crossovers are not used
to facilitate transfer of information from one side of the nervous system
to the other by receiving input on one side and having output on the other,
since in almost every case all the synapses and gap junctions observed in
the adult wild type reconstructions are on the parts of the processes
beyond the cross over point. The exception is that the RIFL/RIFR
cells both make gap junctions to AVG
on their cell bodies, but this also would not be logically different if the
cell body positions were reversed. It is not even the case that the
symmetrical body positions of the neurons involved are preserved into later
development; in fact the cell bodies in both the PAG and the RVG get
squashed into a single row as the muscles mature.
This situation is different from that in most vertebrate decussations, in
which the cells remain on the opposite side from their axonal termini, and
have some functionality on both sides. However, even there it is clear
that, considering the whole organism, there is more crossing over than is
necessary. An engineer would have the right side of the brain receive
information from, and control, the right side of the body. Some
communication between the two dies is certainly necessary, and this is seen
for example in the corpus callosum between the two hemispheres of the
cerebral cortex (and in the C. elegans nerve ring). However such
connections are inherently different from the general sensory and motor
decussations, for which the argument can still be made that they are
functionally necessary, and are more likely to reflect developmental than
functional constraints.
One common factor between the four miniature examples of decussation in the
PAG and RVC of C. elegans is that they are all between pairs of
neurons touching across the ventral midline. It might be suggested that
their mutual affinity causes their processes to grow towards the opposite
cell, and therefore cross over. However after either PVPL
or PVPR
was removed by ablating its parent the other stayed in position and still
sent its process across the midline and along the opposite side of the cord
as normal. In addition there are three pairs of cells in the ventral
ganglion in front of the excretory duct which are also adjacent across the
midline (AIAL/AIAR,
SMBVL/SMBVR
and SAAD)
and none of them cross over. Instead the simplest unifying property of the
decussating pairs is regional: they comprise all the left/right pairs of
interneurons associated with the ventral hypodermal ridge between the
excretory duct and the anus. This, however, suggests neither a mechanism
nor a reason for the crossing over.
One possibility is that the crossing is ballistic: both processes are
attracted to some point or region on the midline and once they get there
they keep on growing in the same direction and thus cross over. Nerve
processes in vitro tend to grow in straight lines (Bray, 1979). The
attraction of the ballistic hypothesis is that it permits there to be no
intrinsic distinction between the two cells. The fact that all the
decussating pairs in the RVG cross in the same place supports the
hypothesis. Also the PVPL/PVPR
crossing point in the PAG seems to be special, since the DVC
process crosses from top left to bottom right in the same place, on its way
forward through the preanal ganglion. The change in position of DVC
does not define the site, because removal of DVC
by ablating its parent had no effect on the PVPL/PVPR
processes and their crossover. Neither did removal of PVQL,
which normally contacts PVPR
as soon as it crosses to the left and grows forward with it.
If we accept the ballistic hypothesis then it seems likely that PVT
defines the site in the preanal ganglion, since the PVPL/PVPR
processes cross between PVT
and the processes of DVC
and PVQL/PVQR
neurons, which are flattend out over the surface of DD6,
partially separating the PVPL/PVPR
cells from DD6
(figure 3.8). Alternatively it may be that the site is defined by the DVC
and PVQL/PVQR
processes in a redundant manner, so that removal of any one of them makes
no difference. The affinity of these three processes for the DD6
cell body is striking; they spread over its surface whereever it is
available, and when DVC
was removed the PVQL/PVQR
processes spread further to mostly fill the gap (figure 4.7). Further
experimentation removing either PVT
or DD6
might prove illuminating.
A variant of the ballistic hypothesis is that the initial directions of
outgrowth of the processes are both intrinsically towards the midline, and
so the processes simply cross over before turning forward. All the cells
involved migrate ventrally from lateral positions as the hypodermis closes
over the ventral surface of the embryo. It might be that the growth cones
start out continuing the direction of migration of the cell and thus cross
the ventral midline. This argument would apply equally well to the ventral
ganglion cell pairs that do not cross, and it is certainly not necessary
for an axon to leave a cell body in the same direction that the cell has
been migrating. For example the ALML/ALMR
cell bodies are seen migrating backward along the lateral hypodermis in the
C and D reconstructions, and in the E reconstruction they are sending axons
forward along the same path they have just followed but in the opposite
direction (figure 3.4). However, even if this does not provide a complete
explanation, it does suggest how intrinsic opposite polarities of the two
cells in each pair may be established.
I have already suggested that AVG
helps organise the ventral cord by providing a preferential parth for
growth of, at the least, the DD axons. The wildtype
outgrowth of PVPL/PVPR
and PVQL/PVQR
processes from the back of the cord, in which their tips always were found
very close together along the cord (section 3.5), suggested that there
might be some interaction involved. Therefore a series of ablation
experiments were performed to investigate PVPL/PVPR
and PVQL/PVQR
outgrowth (section 4.3).
PVPL/PVPR
and PVQL/PVQR
processes grow on both sides of the ventral cord. The left hand cord
contains only three processes at hatching, PVPR,
PVQL, and AVKR
(plus RMEV
at the front, see fig. 1.3). The normal sequence of events is that PVPR
and PVQL grow forward together, and AVKR
was only seen to be growing back after they had reached the front. It
appears that PVPR
is needed for the other two to grow on the left side, because when it is
removed no processes are seen n either the embryonic or adult left hand
cords (figure 4.4). If PVQL is removed then PVPR
still grows forward along the cord by itself. Therefore, although PVQR is
not a unique pioneer in normal development because the PVQL growing tip is
parallel with its own, it does appear to have a primary role in
establishing the left hand cord. When VPR is removed the PVQL process
still grows forward along the cord, but on the right side rather than the
left, and apparently somewhat delayed compared to PVQR and PVPL
which normally grow on the right. In this case therefore the ability to
grow and the basic directionality of growth are preserved, although the
actual path taken was altered, as when AVG
was removed. This corresponds to what is seen when guideposts are removed
in the insect PNS (Berlot and Goodman, 1984), or motor neurons in the
chinck embryo (Landmesser and Honig, 1986). It is not known whether the AVKR
process also extended along the right hand cord in the absence of PVPR
and PVQL on the left side.
These results are not symmetrically reproducible on the other side of the
ventral cord, since PVQR still grows forward along the right side in the
absence of PVPL.
However, as discussed above, the cord is not symmetrical. While PVQR and
PVQL are the first processes to grow along the left side of the cord, there
are other preexisting processes on the right at the time when PVQR grows
forward (AVG
and DD
axons) which might provide some degree of non-specific affinity that
assisted PVQR in growing along the right side. This could in principle be
tested by removing PVPL,
AVG
and DD6.
Although the removal of PVQL had no effect on the outgrowth of PVPR
along the left hand ventral cord, it did appear to affect the growth of
other processes down the lumbar commissure from the left lumbar ganglion to
the preanal ganglion (see figure 1.3 for a schematic plan of the normal
situation). In neither of the reconstructed embryos in which PVQL had been
removed did any of the left lumbar processes grow down lumbar commissure,
although they had done so on the right side. As well as containing
processes descending from the lumbar ganglion, the lumbar commissures
contian a DA motor neuron process
ascending from the preanal ganglion. This was present in both the PVQL
reconstructions.
These results suggest that there is a specific need for PVQL in order for
the other lumbar ganglion cells to grow correctly in the right direction.
Similar behaviour is seen in the developing grasshopper CNS, where in
several cases it has been shown that an identified neuronal growth cone
normally fasciculates with a specific preexisting fascicle, in the absence
of which it fails to grow in any organised fashion (Raper et al.,
1984, Bastiani et al., 1986, duLac et al., 1986). In one
case it was shown that a specific subset of the processes in the
preexisting fascicle is required (Raper et al., 1984). This
corresponds to the observation that the DA process in the
lumbar commissure is not sufficient to promote growth of other processes
down the commissure.
If PVQL provides guidance for the left lumbar processes by some process of
selective fasciculation, then this fasciculation does not last for long.
When processes from the two lumbar commissures meet in the preanal ganglion
all the cell types other than PVQL/PVQR
immediately form contact with their bilateral homologues, "zipping up" with
each other (figure 3.8). The other left lumbar processes then leave PVQL to
join their right hand homologues and PVQR on the right hand side.
Therefore it seems that there is a hierarchy of affinities that applies the
left lumbar processes other than PVQL; first they follow, and in fact
require, PVQL, then they leave PVQL in order to join their right hand
homologues.
These observations all fit the "labelled pathways" hypothesis (Ghysen and
Jansen, 1979, Goodman et al., 1982), that growth cones are
programmed to recognise a sequence of surface labels on fascicles, possibly
in some adhesive hierarchy, and that this determines their path through the
developing nervous system. The situation when the left and right lumbar
processes meet is somewhat novel, in that then two equivalent sets of
processes fasciculate together, and must decide which of the two PVQL/PVQR
neurons to follow. There is no good clue as to what determines this
(discussed earlier in the section on cord asymmetry.
The observations about lumbar commissure formation contrast with those made
in the ventral cord that, even if normal cues are missing, processes tend
to keep on growing in the correct direction. A plausible explanation of
this difference is tat there is a non specific property of the ventral cord
which permits or promotes neuron growth along it. Apart from the presence
of other processes, at the relevant time there is a continuous line of
motor neuron cell bodies along the ventral midline, which may act as
general guideposts in the same way as neuronal cell bodies that have been
proposed to facilitate neuron outgrowth in the insect PNS (Bentley and
Keshishian, 1982).
There are a number of uncoordinated mutants that are known to be defective
in outgrowth of processes from the lumbar ganglion cells, on the basis of
fluorescent staining of the PHAL/PHAR
and PHBL/PHBR
phasmid sensory neurons by direct uptake of fluorescein isothiocyanate
(Hedgecock et al., 1985). Mutants in unc-33,
unc-44
and unc-76
all show the same phenotype. Rather than growing forward into the preanal
ganglion the phasmid axons stop abruptly where they meet at the bottom of
the lumbar commissures, often with swollen endings. This is at the point
where the resorting of the fibres takes place, with the majority of the
left lumbar processes leaving PVQL to grow forward with their contralateral
homologues. The fact that there are several genes with both this phenotype
and also defects in movement is interesting in relation to a suggestion
made earlier (in the discussion of ventral cord asymmetry). This proposed
that the mutual affinity of ventral cord bilateral homologues may be a
basic general mechanism whose biological purpose is to bring together the
motor circuitry interneurons, and which affects other neurons incidentally.
A prediction of this hypothesis would be that the anterior motor circuitry
interneurons would also be afected by the mutations. In mutants for unc-6
(referred to as unc-106
in Hedgecock et al.), the PHAL/PHAR
and PHBL/PHBR
axons normally fail to grow down the lumbar commissures, but instead wander
forward along the lateral hypodermis. This is reminiscent of the defect
seen in the left lumbar commissure when PVQL was removed. However the
defect in unc-6
mutants is more general than that following PVQL removal, since axons from
the postembryonic PVD
neurons on the lateral hypodermis also fail to reach the ventral cord, and
motor neuron commissures are also disrupted (S McIntire, personal
communication).
In the introduction to this part of the dissertation it was proposed that a
number of different mechanisms could be used to influence neuronal
guidance, often concurrently, and a list of possible types and sources of
influence was provided. The behaviour of outgrowing neurites in both
normal and experimental C. elegans embryos that has been described
here has suggested new examples of several different types of influence.
The formation of the dorsal cord could be explained by the presence of a
preexisting preferred pathway along the dorsal hypodermal ridge. This would
essentially be an epidermal blueprint, as proposed by Singer et al.
(1979). DD growth along the
ventral cord may be limited by some inhibitory effect of DB cells, although from
the observations that are available the inhibition seems more likely to be
caused by selective recognition accompanied by membrane insertion than by
the retraction of growth cones as seen by Kampfhammer et al. (1986)
in vitro. The decussation of processes in the preanal and retrovesicular
ganglia may be due to the tendency of growth cones to grow in straight
lines, as discussed by Bray (1979). In the lumbar commissures and the
determination of which processes grow along the left and right nerve cords
there appear to be several examples of selective fasciculation, similar to
that proposed in the labelled pathways hypothesis (Ghysen and Jansen,
1979). There also appeared to be a general directionally or premissive
property of the ventral cord region that meant that, even when specific
cues were removed, processes still grew out along the cord.
All these proposed interactions fall broadly into some class of interaction
that has been suggested previously. Further experiments of the same type
as described here, some of which I have mentioned in the discussion, could
be carried out to define more precisely the characteristics of particular
interactions. The other possible approach to furthe rwork is to use the
existing picture as a basis for an investigation of the genetic factors
controlling neural outgrowth, eventually uncovering the critical molecular
mechanisms involved using molecular genetic techniques (Greenwald, 1985).
In this discussion I have mentioned a number of mutants that affect neural
guidance in the ventral nervous system, in some cases in ways that are
partially interpretable in terms of the mechanisms proposed here. The
genetic approach is discussed further in the final conclusion after part
II.