The previous chapter described a times series of embryonic reconstructions that allowed a picture to be drawn of the course of normal nerve outgrowth in and around the ventral nerve cord. This chapter describes a set of cell ablation experiments in which chosen cells were removed by ablating their parents with a focussed laser beam, using a system developed by J. G. White (Sulston and White, 1980). Chapter 2 describes the protocol used. The advantage of killing the parent cell is not only that it unequivocally prevents production of the cell of interest, but also that the remains of the dead cell are excluded from the embryo when the ventral hypodermis closes up, removing them also from any possible influence. The chapter is organised with a section for each set of experiments.
AVG
is the first process to grow along the ventral cord
(Chapter 3). It grows back along the right hand side, and later
the adult cord is remarkably asymmetrical, with over 90% of its
processes on the right hand side (there are 3 to 5 on the left,
depending on anterior/posterior position, as against about 50 on
the right). To what degree is AVG
involved in establishing this
asymmetry, and which cells, if any, depend directly on AVG
to
correctly determine the positioning of their processes? To answer
these questions I removed AVG
by ablating its parent cell,
Abprpapppa. The sister of AVG,
which is also removed by this
ablation, is RIR,
a ring interneuron whose cell body and processes
all lie some distance anterior to those of AVG,
and whose synaptic
connections in the adult are not closely related to ventral cord
circuitry (White et al., 1986).
Nine experimental
animals
were permitted to develop and hatch in order to test whether motor
control was affected, a crude test of ventral cord function. Six
of these showed a very mild uncoordinated phenotype as newly
hatched L1 larvae, in some cases only clearly visible when the
worm was made to swim in water, in which case sections of their
thrashing bodies looked stiffer than normal. Those worms that
were uncoordinated as larvae were similarly uncoordinated as
adults.
One of the adults showing an uncoordinated
phenotype was sectioned through the front part of the ventral cord
and the retrovesicular ganglion. Eileen Southgate reconstructed
this series, since she and John White were interested in another
question, concerning regulation in the circuitry.
The
reconstruction confirmed that AVG
was absent sine: (1) there were
one too few cells in the RVG, and (ii) there were only two cells
in the RVG with posteriorly directed processes that extended back
through the complete series (AVFL
and AVFR,
they and AVG
are the
only ventral cord interneurons in the RVG). Almost all the cells
in the RVG were identifiable, but there were some ambiguities
concerning motor neuron identification both here and in the
anterior ventral cord. This is because the spatial organisation
of many nerve processes, especially those belonging to motor
neurons, was abnormal. Cell body positions tended to be slightly
displaced from normal, but the general order was preserved.
Figure 4.1
Adult ventral cords from (a) a normal animal, (b) an animal in which
AVG
had been removed, (c) an unc-3 mutant. The hypodermal ridge is
raised in adults compared with embryos. Process bundles are outlined
in
dashes. There is inly one on each side of (a), but there are four
bundles
in each of (b), (c), with many more processes (labelled with stars)
are on
the right, but in (c), (c) there are also motor neurons on the left.
There
is a neuromuscular junction on the right in (a), and one from DB3
on the left, abnormally, in (b) (thick arrows). The motor circuitry
interneuron processes are labelled A for AVAL/R
, B for AVBL/R
, and d for AVDL/R
and AVEL/R
, which are indistringuishable in this part of the cord. They are
abnormally rotated, with motor neuron processes on the hypodermal side
of
them in (b). In (a), (b) the two AVFL/R
neurons are labelled F. They are on the left in (b), which is not
normal.
The thin arrow in (a) points to a hypodermal extension, rather than a
neuronal process. Scale bars are 1 micron in each case.
The most striking aspect of the process bundle
disorganisation can be seen in a random cross section of the cord
behind the RVG (figure 4.1): instead of a large bundle of about 50
processes on the right and one of 4 or 5 on the left there are
several smaller bundles, including two on the left hand side.
There is no fixed arrangement of these small bundles as one
progresses along the cord: processes occasionally transfer between
bundles, and sometimes bundles fuse to form a larger grouping or
split to form two smaller ones. However there are always
significantly more processes on the right than on the left. The
total number of processes appears normal (this comparison can only
be made approximately, since the number of motor neuron processes
present at any particular point is variable).
Several of
the motor neurons are disrupted. These are motor neuron cell
bodies associated with both sides of the cord (VD2
and DB3
are to
the left), and also motor neuron processes on both sides (all of
VD3
and parts of DB3,
VD2
and VB2
processes are on the left). In
addition DB3
is lacking a commissure, and DD2's
commissure is
severley misplaced or missing (the DD2
cell body is off the
posterior end of the reconstruction, but its commissure should
come out from the cord with that of DA2,
some 150 sections
anterior to the end of the series). Instead of a commissure, the
DB3
process has a branch that crosses to the left hand side and
shows some characteristics of the normal dorsal branch, in that
first it runs backwards from the crossover point, and second it
contains three neuromuscular junctions (figure 4.1). Normally all
ventral cord neuromuscular activity is from the right hand cord,
and all DB3's
neuromuscula routput is from its backward dorsal
branch.
Many of the interneuron processes cannot be
identified because they make no synapses and their cell bodies are
outside the bounds of the reconstruction. Among those that can
are the two AVFL/AVFR
neurons, which have cell bodies in the RVG, and
which both send their processes back down the left hand cord in
this recosntruction, as opposed to the right normally (figure
4.1). It is also possible to identify the 8 main motor circuitry
interneurons by class (2 each of AVAL/AVAR
and AVBL/AVBR,
and the 4 AVEL/AVER
neurons, figure 4.1), because of their patterns of synaptic output
and gap junction formation with the motor neurons. In most cases
where they are accessible to the motor neurons the normal synaptic
connections are made. Normally these motor circuitry interneurons
run in the central left side of the main (right hand) ventral
cord, with a regular internal order: AVBL/AVBR's
on top, AVAL/AVAR's
on the
bottom, and AVDL/AVDR's
and AVEL/AVER's
loosely sandwiched in between. In
this reconstruction they all keep together in the main right hand
bundle, and amongst themselves they roughly preserve their normal
order, but the whole group is often displaced from its regular
position and orientation (figure 4.1). Thus it appears that, as a
group, their internal organisation remains, but that they have
lost the external cues that give the group as a whole a fixed
position relative to other processes, some of which, indeed, are
separated by being in other bundles.
In addition to this
adult reconstruction I also looked at an embryo in which the
parent of AVG
had ablated, fixing it at the stage when the motor
neuron commissures are normally just growing out from the ventral
cord (around 500 minutes). In this case I recosntructed the back
part of the ventral cord and also the preanal ganglion (PAG).
Figure 4.2
A schematic illustration of the reconstruction of the ventral cord
from the
embryo in which AVG
had been removed. The illustration has the same form as the central
region
of the diagrams in figure 3.4. There is no continuous interneuron
process
in the cord, indicating that AVG
was both correctly identified and correctly removed. The positions where
commissures are leaving the ventral cord are indicated by arrows. The
DD6
, DA7
and DB7 neurons look normal, but the DD5
ventral cord process switches from right to left, and all the commissures
from DD5
, DB6
and DA6
are leaving the cord from the wrong side (compare with figure 3.5).
Although the AVG
process was clearly missing, there was no
other alteration to the organisation of the PAG and the early
posterior interneurons that grow forward along the cord from it
(the PVPL/PVPR,
PVQL/PVQR's
DVA
and DVC),
every process following its normal
trajectory. However, as in the adult, the ventral cord was
disorganised. In this case the most posterior three motor neurons
(DD6,
DA7
and DB7)
looked normal, but DD5's
anterior process in
the ventral cord, although leaving the cell body on the right side
as normal, switched sides from right to left and sent out its
commissure on the left. DB6,
whose commissure usually goes to the
right with that of DD5,
sent it s commissure to the left also.
DA6's
commissure, which is usually on the left, went to the right
instead (figure 4.2).
In summary, it seems that, in the
absence of AVG,
the motor neurons in the ventral cord are variably
disorganised in terms of process growth. Some examples look
normal, while others send processes on the wrong side of the cord,
or fail to form commissures, etc. This applies to both embryonic
and postembryonic motor neuron classes, although the postembryonic
neurons look much less affected. A second, possibly related,
consequence of AVG
removal is a splitting of the ventral cord into
several bundles, some of which are on the left hand side of the
cord. Some interneurons are also split off into these alternative
bundles, but in the adult example that was reconstructed the main
motor circuitry interneurons look fairly normal. Many of the
motor neurons are able to make correct synaptic contact both with
their innervating interneurons and with muscle. This probably
explains why the observed behavioural phenotype of removing AVG
was only minor uncoordination, when a difference was noticeable at
all.
Figure 4.3
A schematic illustration of the same form as figure 4.3 of the ventral
cord
reconstruction of the embryo in which the DD3
/DD5 parent had been ablated. DD3
is missing (see figures 3.4, 3.5 for the comparable region in normal
animals). The DD2
process has grown slightly back but has not grown beyond the front of
DB4
, while DD4
has not grown forward beyond DB5
. There is thus a gap of an entire cell between the DD processes.
However since this is a young embryo (approx. 175 minutes) one cannot
say
if the gap will be filled later.
In the wild type embryo, after the growth of AVG back along the right hand cord, the DD motor neurons grow out processes in the ventral cord next to AVG. These processes
grow forward until they meet or almost meet the next DD in the sequence (in the various
wild type embryonic series they were often separated by a gap of about 1 micron, Chapter
3). Then commissures grow out to the right from near their front tips. By removing a
DD cell and examining whether the processes would extend further along the cord to fill
in the gap, I was able to test whether process growth is terminated solely by contact, and
if so, whether the position of the commissure also changed. Does Does it always leave from the
front of the ventral cord process?
In fact it was easy to
remove DD3
and DD5
together, creating two gaps in a single animal,
since they are sisters. As with AVG
it was checked that their
dead parent (Abplppappp) was excluded from the embryo after laser
ablation, and that the relevant DD cell was missing in the subsequent reconstruction.
I have reconstructed the front part of one embryo from the seven that were fixed and
sectioned. In this animal, which is the same age as the wild type A series (around 480
minutes), the gap left by removing
DD3
has not been
filled by DD4.
Instead the DD4
process stops at the front of DB5,
only very slightly further forward, if at all, than normal (figure
4.3). There is a short posterior extension from DD2,
which is not
unusual (figure 3.5), but this stops around DB4,
leaving a gap
with noDD processes along the whole
extent of the DA5
cell body.
The third set of ablation experiments concern the four PVPL/PVPR and PVQL/PVQR neurons. To summarise briefly: these form the first group of interneurons to grow forward from the back of the ventral cord. The PVQL/PVQR cell bodies are in the lumbar ganglia; they send processes down the lumbar commissures through the preanal ganglion (PAG), wheter they pick up contact with the PVPL/PVPR processes, and then forward along the ventral cord, one on each side. The PVPL/PVPR bodies lie in the PAG; their processes leave their bodies heading towards the midline, cross over, and then grow forward on the opposite side of the ventral cord. So PVPR runs with PVQL on the left hand side, while PVPL runs with PVQR on the right. The growing tips of each PVPL/PVPR/PVQL/PVQR pair, either on the left or right, are always very close (within 0.5 microns). The only processes apart from PVPR and PVQL to grow down the left side of the ventral cord in the embryo are AVKR and RMEV, both of which grow back from the front, RMEV stopping part back. In the adult the left vulval motor neuron HSNL also grows forward on the left side of the cord from the vulva half way along the body. In the oldest wild type embryonic series (D series) only one of AVKR and RMEV was seen growing back in the anterior cord (it is not knows which one), and this was only after PVPR and PVQL had reached the front.
The questions that can therefore be asked concerning possible
organising
roles for PVPL/PVPR
and PVQL/PVQR
processes are:
1. Are one or both of a PVPL/PVPR/PVQL/PVQR
pair needed for the other to grow along the cord?
2. Are PVPR
and PVQL needed for growth of the other processes down the left cord?
3. Are the PVQL/PVQR
processes, or the other PVPL/PVPR
cell, necessary for crossing over of the PVPL/PVPR
processes in the preanal ganglion?
4. Is the growth of a PVQL/PVQR
process down a lumbar commissure necessary for other lumbar ganglion cell
processes on the same side to reach the preanal ganglion?
Experiments were carried out in which PVPR,
PVPL
and PVQL were independently removed. As with AVG
and DD3/DD5,
a block of fixed experimental embryos was completely sectioned for each of
the sets of ablations (7 embryos for PVPR,
5 for PVPL,
and 5 for PVQL). In addition 5 adult PVPR
experimental animals were cut at 3 random sites in the posterior half of
the cord to help answer the second question. Again the parent cell
was
ablated in each case and only embryos that excluded the dead cell on
closure of the hypodermis were considered further. In the case of
PVQL the
parent is Abplapppa and the sister cell normally undergoes programmed
cell
death and engulfment soon after being born; therefore there is no
additional cell missing in experimental animals at the time of process
outgrowth. The sisters of PVPL
and PVPR
(parents AB.plppppa
and AB.prppppa)
are left and right ventral rectal epithelial cells (repVR and repVL).
Together with repD these form a ring of rectal cells that lie above
and
forward of the PAG; they are sufficiently distant to be unlikely to be
important in nerve process guidance in the PAG. In the reconstructions
of
PVPR
and PVPL
experimental embryos the correct repV cell was seen to be missing; in each
case the rectum had resealed by extension forward of one of the
posterior
neighbouring pair of rectal epithelial cells (K and K') rather than
circumferential filling in by the other rep cells.
I will consider the four questions posed in above in turn:
1. It is easier to observe the presence or absence of PVPL/PVPR/PVQL/PVQR
processes on the left side of the cord (PVPR
and PVQL) than on the right side, since during the stages under
consideration those are the only nerve processes on the left side. I
first
considered the embryos in which PVPR
had been removed. Of the four embroyos in which it was possible to
identify a region of the ventral cord anterior to PAG where the PVPL
and PVQR processes were visible on the right side of the cord, none had
any
processes on the left side (figure 4.4). The PAG and posterior cord
of one
of these embryos was reconstructed; in this case PVQL grew forward as
normal through left side of the PAG past the point where it would
normally
have picked up contact with PVPR
and then, at the front of DD6,
which was displaced slightly anterior to its normal position, it switched
sides from left to right and ran forward for a short distance with PVPL
and PVQR (figure 4.5). Its anterior tip, however, was more than 2.5
microns posterior to the tips of PVPL
and PVQR (which were off the anterior end of the series, 54 sections from
the PVQL tip). Therefore it appears that PVPR
is necessary for growth of PVQL along the left side of the cord, and that
in its absence PVQL is retarded somewhat, but grows forward along the
established path of PVPL
and PVQR.
However, when PVPRL (erratum in the manuscript) was removed, in each
of
the three embryos for which the same region anterior to the PAG was
identified, a solitary process was seen on the left side (figure
4.4). The
PAG region of two of these embryos was reconstructed; in each case
PVQL was
missing and PVPR
grew as normal along the left side of the cord. In the younger of the
series (about 470 minutes) it stopped about 1.7 microns (34 sections)
posteriorly to the point where the PVPL/PVQR
processes on the right stopped; the older series did not contain the
anterior tips of any of the processes. Therefore, in contrast to
PVQL, it
appears that PVPR
is competent to grow by itself to the left side of the cord.
Finally, I considered the consequences of removing PVPL,
the bilaterally homologous experiment to that of removing PVPR.
In this case PVQR stayed on the right side, rather than crossing to join
PVPR
and PVQL (two animals: one was reconstructed completely and one animal had
two long processes but no third process in the left cord, even near
the
PAG, figures 4.4, 4.5). However it must be remembered that, in the
absence
of PVQL when PVQR was removed, since event though the PVPL
process is absent on the right side of the cord there are still AVG
and PVPR
was removed there was nothing on the left side. The corresponding
reciprocal experiment of removing PVQR was not attempted, since it
seemed
unlikely that there would be an effect in the more populated right
hand
side of the cord, where there had been none when PVQL was removed on
the
left side.
In summary, PVPR
is necessary for growth of PVQL on the left side of the cord. In its
absence PVQL grows on the right side. However PVPL
is not necessary for PVQR to grow on the right side, presumably because
PVQR can follow the preexisting AVG
and PVPR.
2. To answer the question of whether PVPR
and PVQL are needed for growth of other processes down the left cord, I
ablated the patent of PVPR
in six animals and looked at the left side of the adult, rather than the
embryonic, so that all processes would have had the opportunity to
complete
growth. The fixed animals were cut at three random sites in the
posterior
half of the body, where AVKR
is normally present on the left side together with PVPR
and PVQL. One of the five animals was rejected because of poor fixation.
None of the remaining five had any consistent process showing on the
left
side of the cord (figure 4.4). In several cases there appeared to be
a
process visible at one of the sites. This was probably a fold or
finger of
hypodermis; such hypodermal extensions are common around the adult
ventral
cord (for example there are two in the section from the control
reconstruction in figure 4.1). Therefore both PVPL,
as expected from the previous result, and AVKR
were missing from the left side in all five cases, implying that the PVPR/PVQL
pair is necessary for AVKR
to grow down the left hand ventral cord. It is not possible to ascertain
whether AVKR
had switched to the right side of the cord in the experimental animals, or
had failed to grow back at all, without reconstruction of the complete
nerve ring.
3. The removal of neither a PVPL/PVPR
nor a PVQL/PVQR
cell affected the crossing over the opposite side of the PVPL/PVPR
processes when they leave their cell bodies in the centre of the preanal
ganglion (figure 4.5). The embryonic PAG reconstructions after
removing
either PVPR
or PVPL
show that the remaining PVPL/PVPR
cell sent its process across the midline in exactly the same location as
usual. This rules out an explanation of the chiasm being caused by
mutual
attraction of PVPL/PVPR
processes. The reconstructions after PVQL parent ablations also showed no
change.
4. However there did appear to be an effect on the left lumbar
commissure
when PVQL was removed. In neither of the two experimental animals
that
were reconstructed did any other processes come down the left lumbar
commissure into the PAG, although in each case the DA8
process had already grown dorsally via the same path out of the PAG. One
of
the reconstructed animals was young enough that the following
processes on
the right side had only just passed through the commissure; however in
the
second four of the right hand lumbar processes other than PVQR had
grown
half way through the PAG (figure 4.6).
Figure 4.4
The ventral cords of animals in which a PVPL/R
or PVQL/R
cell has been removed. In each case an arrow points to the left hand
cord.
For embryonic cords compare with figure 3.7 (b) for a control, and for
adult cords compare with figure 4.1 (a). Two examples of each
experiment
are shown. (a), (b) Embryonic cords after removal of PVPR
; there are no processes on the left side, but sufficiently many on the
right to show that PVQL would normally have been visible in these
situations. (c), (d) Embryonic cords after removal of PVQL
; there is one process on the left side. (e), (f) Embryonic cords after
removal of PVPQ (NB, typo); 2 processes on the left. (g), (h),
Posterior
adult cords after PVPR
removal; there are still no processes on the left side in the posterior
half of the animal. Scales bars in (a) for (a) to (f), and in (g) for
(g),
(h), 1 micron in each case.
Figure 4.5
Schematic diagrams of the front of the preanal ganglion in normal
embryos
and ones in which a PVPL/R
or PVQL/R
cell has been removed, based on complete reconstructions of the
preanal
ganglia in these animals. The ages of the reconstruction varied but
they
were all around 500 minutes. (a) normal, (b) after PVPR
removal; (c) after PVQL
removal, (d) after PVPL
removal. Neither of the last two experiments caused any effect on other
processes in this region.
One further ablation experiment was tried in an attempt
to
understand why the PVPL/PVPR
processes cross over in the preanal
ganglion. As described in Chapter 3, at the point where the
crossover takes place the process of DVC
spreads out into a thin
sheet that separates the cell bodies of PVT
and DD6;
the PVPL/PVPR
processes actually cross between PVT
and the DVC
sheet. It
therefore seemed possible that DVC
was essential for the
crossover. Therefore five embryos were fixed in which the parent
of DVC
had been ablated, two of which were later reconstructed in
the region of the PAG.
The sister of DVC
(parent Caapa)
normally undergoes programmed cell death before differentiating
and so is unlikely to be required for the development of the PAG.
Since the DVC
parent lies underneath the tail hypodermal cells at
the time of its ablation it is not excluded from the embryo as in
all other cases. However I checked that condensed nuclear debris
was visible about 20 minutes after the ablations, and the absence
of the DVC
cell body was confirmed in the two reconstructed
embryos.
No effect was seen on the PVPL/PVPR
crossover in either
reconstruction. Instead PVQR
and PVQL
flattened out somewhat and
met in the centre, partially replacing DVC's
role in separating
PVT
and the crossing over PVPL/PVPR's
from DD6
(figure 4.7). This
possibly suggests that PVQL, PVQR and DD6
have somewhat
interchangeable or redundant functions at this point in organising
the PAG. However the multiple ablations which might test this
suggestions have not yet been attempted.
Figure 4.6
The complete posterior nervous system in 500 minute embryos, shown as
in
figure 1.3. (a) Normal, (b) after PVQL
removal. Although there was no effect on the PVPL/R
processes in (b) (see figure 4.4), the other processes from the left
lumbar
ganglion have failed to grow down the left lumbar commissure. However
the
DA8
process, which also grows in the lumbar commissure, but in the opposite
direction looks normal.
Figure 4.7
The complete posterior nervous system in 500 minute embryos, shown as
in
The site of PVPL/R
process crossover in an embryo in which DVC
has been removed. The PVPL/R
processes still cross over (thick arrows). However the situation is a
little abnormal because PVQR
flattens out much more than normal (compare with figure 3.8). Scale bar
is
1 micron.
