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The first part of this dissertation describes an investigation into the outgrowth of nerve processes in the region of the ventral nerve cord of C. elegans during embryonic development. The course of normal development was deduced from serial section reconstructions of a set of embryos fixed at different stages. Then laser ablation experiments were performed to remove specific neurons whose processes grew out during these early stages, in order to test whether the presence of these processes was necessary for correct subsequent development of the nervous system. Chapter 2 gives materials and methods. The observations from the wild type reconstructions are given in Chapter 3 and the results of the ablation experiments are described in Chapter 4. The two sets of results are discussed together in Chapter 5. There are no previous direct results on the course of neural outgrowth in C. elegans, although disruption of the final arrangement of nerve processes has been observed in mutants (Hedgecock et al., 1985) and animals in which laser damage has prevented nerve cell migration (Chalfie et al., 1983). Below I first review previous work in other systems on neural guidance, and then give an introduction to C. elegans and its nervous system.
The building of a nervous system during development can be
divided into three phases: the generation of the correct cells in
the correct places, the outgrowth of nerve processes, and the
formation of synapses. All of these phases show a high degree of
specificity, which means that a large amount of information must be
expressed by mechanisms that on the whole we do not yet understand,
but would like to. In some ways the second phase, that of process
outgrowth is the most clearly defined. This is because all neural
branching structures are a consequence of a single phenomenon, the
migration of growth cones during development, a truth which Cajal
saw early and fought hard for (Hamburger, 1981), and which led
Harrison to develop the first tissue culture techniques in order to
follow outgrowing neurites directly (Harrison, 1910).
A growth
cone is a specialised structure at the tip of any growing neurite
that migrates through the animal, spinning out the nerve process
behind it. This is not the only means by which nerve processes can
be lengthened, since change in size and shape of the animal is
matched by addition of new material to already existing processes.
In many cases most of the length of nerve fibres is created in this
way, but it is almost entirely passive, having at most a very small
effect on the layout of the neurons axonal structure. For
instance, most nerve processes grow along the ventral cord of C. elegans
when it is only around 100 microns long, a tenth of its final
length. However some changes in overall structure do occur by
intercalary insertion; an example is the conversion of an initially
bipolar cell to one that is pseudo-monopolar, by retraction of the
cell body away from the branch (Kuwada, 1986 and with ventral nerve
cord motor neurons in C. elegans). Such small alterations during
subsequent development emphasise the importance of looking at
outgrowth as it takes place, rather than making inferences from the
finished pattern.
Growth cones are generally spread out, lamellar
structures, which often extend fine microspikes, or filopodia
(Letourneau, 1983). They mover over surfaces and as they move
thevarious lamellar and filopodial extensions are retracted and new
ones are extended out, so that the overall shape is continually
changing (Bray and Chapman, 1985). It is easy to study growth cone
migration in vitro using cultured neurons and a wide range of
factors that affect migration have been observed. In order for
motion to take place it appears that fairly tight adhesion to the
substrate is necessary (Bray, 1979), and this leads immediately to
the idea that differential adhesion may be important for growth
cone guidance. Letourneau (1980) has shown that growth cones do
indeed tend to grow along regions of higher adversity when faced
with a choice in vitro. Although this may support the common
suggestion that growth cones may in many cases be guided up an
adhesive gradient in vivo (Nardi, 1983, Berlot and Goodman, 1984),
it does not directly address that proposal, and there are several
severe problems with the idea. Growth cones show different
morphologies when migrating on artificial surfaces of different
adhesivities, but even though the range of morphologies seen on
different neurites in vivo is vast, any one growth cone does not
change shape as it migrates over a uniform surface. In addition
the strength of adhesivity would have to increase exponentially,
which would require an excessive magnitude range of adhesivity for
a gradient of any substantial length. In fact growth cones in
culture tend to grow in straight lines anyway, only changing
directions when they branch. Based n an elegant combination of
observations and experiments Bray has suggested that the neurite
leaving the back of the growth cone exerts a tension, and that the
growth cone always grows away from the source of tension (Bray,
1979). If the angle of the neurite is altered then the direction
of growth coordinately changes, and if the tension is relaxed, by
for example cutting the neurite, then the growth cones divides in
two, the two halves growing off in opposite directions and exerting
tension against each other. Together these results suggest that
direction changes and branches may occur in vivo either where a
path of higher adversity is crossed, or possibly at a point where
the growth cone becomes tethered, so that growth in the new
direction can pull against something.
Although there is a
strong tendency to think of attractive forces on growth cones as
being the principle tools of guidance control, it is equally
possible for repulsive forces to be influential, and there are
several examples that are known. There is a highly selective
inhibitory effect when the neurotransmitter 5-HT is released from a
micropipette near the advancing growth cone of an identified cell
from the mollusc, Helisoma (Haydon et al., 1984). This has been
proposed to have developmental significance in the detailed
development of the Helisoma buccal ganglion (meinertxhagen, 1985).
A retraction of the growth cone in vitro is also seen when rtinal
and sympathetic axons meet each other in culture (Bray et al.,
1980, Kampfhammer et al., 1986). Although retinal growth cones
will cross retinal axons, and sympathetic growth cones will cross
sympathetic axons, when one meets the other it shrinks back and
withdraws its filopodial and lamellar extensions. Similar
avoidance behaviour between different neurites of the same neuron
could possibly explain the marvellous space filling,
non-overlapping properties of many neurons' dendritic or axonal
arborisations. Experimental evidence for such avoidance has been
provided by studies of single sensory neurons in the leech, which
fill a planar surface from several points in an apparently
self-competitive fashion (Kramer and Stent, 1985). As yet there is
no experimental evidence of such mechanisms acting between
different neurons in vivo, but there are several cases in C.
elegans where neurons abut against but do not overlap other members
of their own classes; often there is a gap junction between the two
abutting processes (see Chaper 7).
It is convenient to make a distinction between directional,
tropic influences on neural guidance and spatially restricted,
contact mediated influences. Both appear to play an important
part. To oversimplify the situation, tropic influences are
directionally constraining, while differential adhesivities are
spatially constraining. There is also a division between specific
and nonspecific factors. By nonspecific factors I mean those that
would influence any of a large range of different neurons. Neither
of these divisions is totally sharp, and in particular specificity
is clearly a graded phenomenon.
The classical example of a
non-specific factor would be a gradient of positional information
(Wolpert, 1971), probably some chemical or surface marker, and the
classical experimental system where there is evidence for such a
gradient in neural development is in the establishment of a
topographical mapping from the retina onto the optic tectum of
lower vertebrates. A series of experiments in which an ordered
mapping reformed after parts of the retina and/or tectum were
removed or grafted back in abnormal orientations suggested that the
original chemo-affinity hypothesis of Sperry (1963), which proposed
specific matching between corresponding sectors of the retina and
tectum, was incorrect (see Gaze, 1970). More recently Bonhoeffer
and Huf (1982) have shown using an in vitro axon growth choice
assay that there is a gradient of affinity for temporal axons
across the surface of the tectum, with highest affinity for the
rostral part of the tectum, which is their normal target.
Progressively more nasal axons show less specificity. The overall
effect of these affinities would then be established by
competition. There are many other proposed sources of information
for the retino-tectal system, some also driven by competition (e.g.
Willshaw and von der Malsburg, 1979).
However the situation
during creation of the retino-tectal map on the surface of the
tectum is different from the early outgrowth of processes that
concerns the study of embryonic C. elegans outgrowth in this
dissertation, since the axons have already reached their target
tissue and are finding the correct place on it amongst a group of
equivalent cells. For the rest of this review I will focus on the
pathfinding properties of growth cones necessary to find their
targets from the cell bodies, rather than the final stage as
discussed here.
A very different function of a gradient is to specify a
direction up which axons can travel. There are several examples
where a general attraction that is not path specific has been
indicated experimentally. Harris (1980) has shown this type of
effect using the same retino-tectal projection in Xenopus menioned
above as an experimental system, but at the earlier stage of
development where the optic tract must be formed. Before axon
outgrowth he implanted whole eye primordia into abnormal places in
the brain, after which in most cases the retinal axons grew out and
took a nearly direct route to the tectum, usually via a pathway
totally different to the one they normally follow. If the implant
was sufficiently caudal then the retinal processes ran instead down
the spinal cord, in a particular dorsolateral tract, reproducing
previous observations that this part of the spinal cord attracted
displaced retinal axons (Constantine-Paton and Capranica, 1976).
These results suggest that there is a general attraction of retinal
axons to their target, and that this acts over a fairly wide zone,
but that the mechanism may not be uniquely used for retino-tectal
pathfinding; in the spinal cord, outside the normal range of
retinal axons the same attraction system may be used for another
set of processes.
A more specific attraction of neurons to
their targets has been observed in the vertebrate peripheral
nervous system (PNS). Lance-Jones and Landmesser (1981) showed
that after a short piece of chick neural tube was reversed the
motor neurons till largely found a way to the correct target
muscles, crossing over each other on the way. However if the
displacement is too great then they often grow to inappropriate
muscles (ibid. and Summerbell and Stirling, 1981). Again this
influence appears to be over a longer range than the reach of the
filopodia, though still reasonably localised (Landmesser, 1984).
There are also indications in the insect PNS that after the more
specific cues are removed there is still a tendency for sensory
neurons to grow proximally towards the central nervous system
(CNS), even along abnormal routes (Berlot and Goodman, 1984, Nardi,
1983).
One suggestion of a possible agent involved in the
general attraction of a whole class of nerve fibres is nerve growth
factor (NGF). Sympathetic fibres grow over abnormal territory
towards a site of NGF injection in vivo (Gunderson and Barrett,
1980). However in both cases the amounts applied are much larger
than the ovserved natural levels; NGF is much better known as a
trophic agent necessary for neuron survival and a general promoter
of neuron outgrowth, and the directional effect may be a subsidiary
non-physiological consequence of an overdose of these other
behaviours. In a careful set of experiments with explants from
embryonic mouse trigeminal ganglia and their target tissue,
maxiliary epithelium, Lumsden and Davies (1983, 1986) have shown a
clear directional tropic attraction of trigeminal fibres to their
target. This is diffusible through the colagen matrix in which the
explants sit and the axons grow, and is separable from NGF, which
appears to act later in development to preserve the connection. It
also has no effect on axons from comparable neighbouring ganglia.
Lumsden and Davies argue that NGF is active on too many cell types
to be sensible as a tropic agent. However it might be countered
that a general tendency for sympathetic axons to grow towards the
periphery could be useful.
All these results suggest that
there may be general directional (often homing) guidance mechanisms
that are not restricted to specific pathways, and apply to fairly
broad classes of neurons. Interestingly the range of all the
attractions is approximately the same, of the order of a few
hundred microns. In cases that are more specific, such as the
chick motor neuron guidance, the absolute size of the embryo is
larger. Such distances correspond to a fairly small number of
growth cone extensions, suggesting that a growth cone could detect
a gradient on this scale. Since some specificity is involved and
the directions of different sets of fibres can cross (as in the
chick limb motor neuron experiments), it seems unlikely that a
single gradient, such as a general adhesive gradient, provides the
best explanation for them. In at least one case (Lumsden and
Davies) the substrate if artificial and the factor is diffusible.
Before automatically explaining any experiment indicating a
directional effect by a gradient, it should be born in mind,
however, that there are at least two other ways in which a polarity
or directionality could be specified. The first is intrinsic to
the neuron, simply by the orientation in which it was created by
its final cell division. This may often be important for
initiating process outgrowth in the correct direction (Jan et al.,
1985). The second is by a repeated sequence of more than two
signals, in which case the direction can be determined by
inspecting neighbouring sequence elements, or equivalently by a
moving wave of some signal. This type of signal can operate over
very long distances if it is actively maintained, and is the method
is slime mould aggregation (Gerisch, 1982).
A different sort of nonspecific influence that is important
for neuronal outgrowth is the strong tendency of growth cones to
grow along other neurons, which leads to the fasciculation of nerve
processes. This is clearly one of the most important factors
determining the structure of the peripheral nervous system, which
is made of nerve bundles, and where closely studied it has also
been seen to be important in the early developing central nervous
system at stages where processes are not dense (e.g. the insect
CNS, Bate and Grunewald, 1981, Goodman et al., 1982). This has
been seen by immunofluorescence to be expressed on many neuronal
cell surfaces, and also on various epithelial and glial cells
(Silver and Rutishauser, 1984). It has been claimed that the
modulation of a single molecule such as NCAM could account for a
very large proportion of the control of neural outgrowth (Edelman,
1983), but this appears unlikely because of the degree of
specificity seen in many different but often adjacent and
simultaneous interactions. However there is a large part to be
played by fairly non-specific adhesion.
Almost a direct
consequence of general neuronal fasciculation is the concept of the
preservation of order within nerve bundles by a process tending to
stay stuck to its neighbours. Many nerve projections show a
general topographic order preservation, both in the central and
peripheral nervous system (e.g. the retinal-tectal and spinal cord
projections) and a simple method of correct guidance may be to
place neurons in positions corresponding to a topological map of
their targets and then to preserve the relative spatial arrangement
in the outgoing bundle of fibres and rely on non-specific cues to
spread the projection onto the target tissue(s). In fish
retino-tectal projections Scholes (1979) has shown that order is in
general maintained, but that there is a zone of active
reorganisation near the tectum, and in other cases where ordering
has been observed an active mechanism for correcting the final
projection has also been detected (e.g. Landmesser, 1984).
The observation that fasciculation is a significant factor
led to a realisation of the importance of the first nerve pioneers
to grow out, called "pioneers" by Harrison (1910) and to the
suggestion that they may be specialised in order to be able to lay
down new paths. The pioneers in a various part of different insect
peripheral nervous systems have been studied first by Bate (1976a),
and subsequently by many others (e.g. Ho and Goodman, 1982, Bentley
and Keshishian, 1982, Blari and Palka, 1985, Jan et al. 1985).
Although in certain cases outgrowing central neurons grow out over
new territory (Ho and Goodman, 1982), the majority of nerve bundles
are pioneered by peripheral sensory neurons that essentially always
follow a series of other neuronal cell bodies spaced out at
intervals on the way to the CNS. This observation led to the
"guidepost" hypothesis, that there are a class of specified cells
in the periphery that are guideposts (maybe all neurons) and that
pioneer growth cones search for and grow towards the nearest
guidepost cell within reach at each stage (Bentley and Keshishian,
1982). In this case it appears that no single pioneer is
essential, since various cell removal experiments resulted in
satisfactory correction or adaptation (Keshishian and Bentley),
1983, Blair and Palka, 1985).
Ho and Goodman (1982) argue
for a certain degree of specificity of fasciculation in the
grasshopper PNS, particularly for outward growing CNS axons which
must choose branches at points where afferent fibres have
converged. There appears to be a much greater amount of
specificity in the grasshopper CNS. Here again the earliest pioneer
fibres have been identified (Bate and Grunewald, 1981), and the
subsequent outgrowth of certain identified neurons has been
followed (Goodman et al., 1982). A large number of closely
adjacent fascicles are established and growth cones often cross a
number of them before fasciculating with a particular one. This
has lead to the "labelled pathways" hypothesis (Ghysen and Jansen,
1979, Goodman et al., 1982), that the fascicles are differentially
labelled by surface molecules and that growth cones are programmed
to recognise a sequence of these labels and grow along them, thus
defining a route through the developing nervous system. Ablations
of neurons that generate the pathways for identified cells in this
system have resulted in the stalling of growth cones (Raper et al.,
1984, Bastiani et al., 1986). This contrasts with what has been
seen in the PNS, and provides a genuine example of a specialised
pioneer, whose presence is necessary for later axons to follow.
The chick PNS experiments described earlier provide another
example of the requirement for a preexisting fascicle along which a
subsequent neuron type will follow. In the experiments in which
sections of neural tube, or limb buds, are displaced, sensory
neurons that innervate muscle only follow the correct pathways to
their muscles if the corresponding motor neurons do so (Honig et
al., 1986). Furthermore, if instead of displacing motor neurons
the whole motor neuron pool is removed before axon outgrowth, so
that later there is no motor innervation of muscle, then there is
effectively no sensory innervation of muscle either, and instead
cutaneous sensory innervation is increased (Landmesser and Honig,
1986).
Therefore, in addition tot he nonspecific general
tropism and fasciculation that were discussed earlier, there is
substantial evidence for specific interactions between neurons and
bundles of other neurons with which they will fasciculate. In the
case of the insect CNS the specificity appears to be almost
certainly mediated by contact; not only are the differing choices
too tightly packed for a longer range influence to be sufficiently
selective, but there have also been seen in the electron microscope
direct interactions of growth cone filopodia inserting themselves
deep into the surfaces of cells they wille ventually fasciculate
with (Bastiani and Goodman, 1984). Monoclonal antibodies have
recently been made that appear to recognise specific fascicles in
the grasshopper CNS, and the growth cones that will join them
(harrelson et al., 1986). Interestingly in each case several
different bundles stain with the same antibody. If the antigens
are involved in determining fasciculation then this would be
reminiscent of the observation with ectopic retinal implants that
there seems to be an affinity of retinal axons for an abnormal
target in the spinal cord, as well as the tectum.
Up until now the interactions between growth cones and their
targets, or other neurons, have been stressed, but clearly their
relationship to non-neuronal substrates may also be important,
particularly for pioneer neurons. In various different situations
growth cones have been proposed to migrate over basement membrane,
glial cells, epithelial cells, and mesenchyme. One of the strong
reasons for proposing basement membrane as a possible neuronal
substrate is that both raw basement membrane and several purified
basement membrane components, such as fibronectin and laminin, have
been shown to provide good surfaces for outgrowth in vitro
(Varon-van Evercooren et al., 1982). Also in vitro processes are
often found growing in spaces adjacent ot a limiting basement
membrane (e.g. the CNS pioneers in the grasshopped, Bate and
Grunewald, 1981, or the first fibres in the fish spinal cord,
Kuwada et al., 1986). However this region almost always also
contains a large number of glial processes, and at least in the
case of retinal axons, the nerve fibres seem to be particularly
strongly attached to these glial endfeet (Krayanek and Goldberg,
1981), which have been shown to stain early on for NCAM (Silver and
Rutishauser, 1984). The ordered outgrowth of retinal axons can be
disrupted by injection of anti-NCAM antibodies (ibid.). In
addition Silver and Ogawa (1981) have shown that a preformed glial
bridge is ncessary and sufficient for growth of neocortial fibres
across the corpus callosum.
On the basis of this type of
observation, Singer et al. (1979) proposed the blueprint
hypothesis, suggesting that there was a preformed meshwork of
favoured pathways established on the glial and neuroepithelial
external surface, which would channel growth cones in the same sort
of way as Letourneau's adhesive grid in vitro (Letourneau, 1980).
As with fasciculation, to which this type of concept is clearly
related, non-neuronal blueprints could come in a complete range of
specificities, from generally available for all axons to completely
specific for a particular growth cone. In the case of the
grasshopper CNS it has been possible to implicate a particular
glial cell, the segment border cell, as determining the exit site
for one of the main connectives to the periphery (Bastiani and
Goodman, 1986). It effectively acts as a specific labelled pathway
itself.
There is no case where the underlying mechanisms that control
a nontrivial outgrowth pattern for a particular neuron or type of
neuron have been determined in detail. One of the reasons for this
is that we still know too little about the molecular and cellular
basis of growth cone movement and guidance (Letourneau, 1983). On
a larger scale, there are a number of experiments suggesting
various sources of influence for process outgrowth. These
experiments normally involve perturbation of particular factors in
vivo and the results can sometimes be open to variable
interpretation, depending on the hypotheses being addressed by the
interpreter. One certain conclusion, however, is that a large
range of different mechanisms can be used to influence neural
guidance, usually in various combinations, and often in a redundant
fashion. The information necessay for determining the outgrowth of
any particular neuron will be expressed via a subset of these
factors, the relevant subset probably differing in different stages
of outgrowth.
Therefore the best that can be done at the
general level is to identify the basic forms of the different types
of relevant influence and interaction, and provide a list of tools
that are available to whatever program controls development. In
generating such a list I again restrict myself to outgrowth from
the cell to the target, rather than interactions on the target
tissue in which competition and neural activity may well play a
part. With this restriction there currently seems to be evidence
for the following list:
1. Much of the necessary organisation can be achieved by the initial
positioning and orienting of the neurons.
2. There is a general tendency for axons to extend in straight lines unless
otherwise influenced.
3. There can be local inhibitory influences on growth cone extension,
either humoral or contact mediated.
4. Adhesion is clearly important for growth cone migration, and it seems
likely that preformed generally adhesive pathways provide a set of
preferred highways for processes to grow along.
5. Also in the realm of general adhesivity, there is a strong tendency for
extending neurites to fasciculate together.
6. Both these last two influences can also act in a specific, as well as a
non-specific, fashion, for example when a growth cone joins one particular
fascicle out of several.
7. There can be a directional attraction of axons, normally from some
fairly broad class of neurons, to some target or region, and this can
function when a normal route is unavailable. At least in some cases this
attraction is mediated by diffusible factors.
For those elements of the list where there is specificity, as in
the last two cases, it seems that the same specificity mechanism
may be used in more than one place.
Even if this list were
complete, it would only provide a framework for two further lines
of inquiry. The first is to search for the molecular and cellular
mechanisms involved in each type of interaction, and the nature of
their possible diversity and specificity. The second is to
investigate how the consequent repertoire of available influences
intricate outgrowth patterns for the huge variety of different
neurons. One way to attack these problems is to choose an organism
where the types of interaction involved and the different levels of
specificity can be made as clear as possible, and then use the
experimental power of molecular genetics as a technique to probe
both the nature of the molecules concerned and the internal control
structure of the genome. A good candidate for that organism is C.
elegans.
So far in this introduction I have mixed examples
from invertebrate and vertebrate model systems fairly freely, since
many of the results can be directly compared, and it seems likely
that factors which control growth cone guidance at the cellular
level may well be analogous, if not identical, between even very
widely diverged species. The significant difference between
invertebrate and invertebrate nervous systems for the purposes of
experimentation on axon guidance is that, in addition to in general
containing orders of magnitude fewer cells than vertebrate ganglia,
many and in some cases all, neurons in an invertebrate ganglion are
reproducibly identifiable from one animal to the next. Often there
will be only one or a small reproducible number of cells with any
particular set of characteristics. Therefore repeatable
experiments can be undertaken concerning a known individual neuron
and the specific factors involved in controlling the outgrowth of
its processes. C. elegans contains only 302 neurons altogether,
all of which are identifiable, and for all of which the complete
audit anatomy is known at the electron microscope level (White et
al., 1986).
Finally, but not least importantly, we turn to
the use of genetic techniques to study neural outgrowth. The
primary reason for choosing C. elegans as a model organism for the
study of neural development was not the simplicity of its nervous
system, but that it is well suited to genetic analysis (Brenner,
1974). The reason that genetics has not been mentioned before this
point is that, although it can provide an extremely powerful tool
for studying biological function and control and has been
extensively used to study neuronal cell determination (e.g. Lehmann
et al., 1983, Hedgecock, 1985), it has as yet provided very little
insight into neural guidance. In vertebrates a few known mutations
affect neuronal branching patterns and guidance, such as mouse
mutants weaver, staggerer and reeler, which affect the structure of
various cell types in the cerebellum (Caviness and Rakic, 1978).
In Drosophila there are several mutations that have been used as
experimental tools to remove neurons, or produce them in abnormal
places (e.g. the homeotic mutants, Palka, 1982) but the only
published mutation that seems to directly affect neuronal guidance
is bendless, in which one of the neuron types involved in the
escape jump response fails to reach its target (Thomas and Wyman,
1982). However it is not known whether other processes are
affected, nor is the wild type development of the particular neuron
known. In fact the organism in which the greatest number of neural
guidance specific mutants are known is C. elegans (Hedgecock et
al., 1985, S. McIntire, J. White, E. Hedgecock, personal
communications, discussed further in the next section). In
addition to any intrinsic interest and possible significance, it
was in order to provide the developmental framework for further
characterisation of the molecular mechanisms involved in guidance
via this genetic approach that the study described in this thesis
was undertaken.
C. elegans is a small nematode, or roundworm, approximately
1mm long in the adult form. It has a simple body structure and a
small number of cells: 959 somatic cells including 302 neuons.
Development from egg to fertile adult takes only three and a half
days at room temperature. Wild type animals used in this study are
isogenic, since the egglaying sex is a self-fertilising
hermaphrodite, rather than a female, with the consequence that
strains are normally propogated asexually, forming clones. Males
occur naturally at low frequencies. Ther hermaphroditism also
facilitates genetic analysis, and many mutants have been studied.
Together these facts make C. elegans a favourable model organism
for the detailed study of development at the level of single cells,
using both anatomical and genetic techniques, and it was chosen as
such by Sydney Brenner (1974).
The life cycle consists of
an embryonic stage, inside the egg, which takes about 16 hours,
followed by four larval stages, named L1 to L4. The course of
development is extremely reproducible. The pattern of cell
divisions from the fertilised egg to the adult has been determined
completely (Sulston and Horvitz, 1979, Kimbe and Hirsh, 1979,
Sulston et al., 1983) and is essentially invariant.
Not
only are the pattern of cell division and the general body plan of
C. elegans simple and reproducible at a cellular level, but so is
its nervous system. The complete nervous system of the adult
hermaphrodite has been reconstructed by White et al. (1986) from
electron micrographs of serial thin sections. The neurons have
simple branching structures, and both the dispositions of cell
processes, and the connections they make, appear to be largely
invariant between animals. They can be assigned to 118 different
neuronal classes on the basis of morphology and synaptic
connectivity (the system of nomenclature is described in Chapter
2). An overview of the nervous system of an L1 larva is shown in
Figure 1.2. Its central processing region is a loop of neuropil
around the pharynx, called the nerve ring, containing around 175
nerve processes. Running from this is a set of longitudinal
process bundles that connect the ring to sensory receptors, the
body motor nervous system, and several small ganglia in the tail.
There are also circumferential commissures carrying processes from
one lonitudinal bundle to another. The most important of the
longitudinal bundles is the ventral nerve cord, which runs from the
retrovesicular ganglion (RVG) just behind the nerve ring to the
preanal ganglion (PAG) at the beginning of the tail, and containing
the motor neuron cell bodies for the body motor circuitry.
Nerve cells in C. elegans are small (less than 5 microns in
diameter) and it is not currently practical to impale them with
microelectrodes. However intracellular recording from selected
neurons has been possible in the larger nematode, Ascaris
lumbricoides. Attention has been focussed on the ventral cord
motor circuitry (reviewed in Stretton et al., 1985), and the
distribution of cell types seen there corresponds anatomically very
closely to that in C. elegans.
Figure 1.1
Transverse section of a 515 minute embryo (the C reconstruction of Chapter
3). The gut, muscle quadrants (M) and outer hypodermis (h) are all
labelled. There are two nerve processes in the ventral nerve cord (AVG and
DD3), and one motor neuron cell body (DB4).
A left handed commissure is growing out from the DB4 cell body towards the
dorsal hypodermis. In its growth cone can be seen a number of small
vesicles. Scale bar is 2 microns.
Figure 1.2
A general view of the L1 larva and its nervous system. All the neuronal
cell bodies and process tracts behind the retrovesicular ganglion on the
midline or the left side are shown. The main region of neuropil is the
nerve ring, which is a loop around the pharynx. The ventral cord runs back
from this and contains motor neuron cell bodies in addition to processes.
Those ventral cord motor neurons that do not send a commissure around the
left side of the body to the dorsal cord send one to the right side. There
are four small tail ganglia: the preanal ganglion, the dorsorectal
ganglion, and two lumbar ganglia, one on each side.
Previous studies on neural process guidance in C. elegans have been
restricted to examining the structure of the adult nervous system in both
wild type animals and mutants in which processes go astray. White (1983)
discusses some possible factors that may be important in neural guidance on
the basis of the adult electron microscope reconstructions. Chapter 9 of
this thesis also considers process placement in the nerve ring using data
from the adult reconstructions. Several techniques (mostly unpublished)
have been developed to visualise processes by light microscopy, and these
have been used to screen mutants that have possible neural defects, such as
uncoordinated mutants that do not move well. Hedgecock et al. (1985)
filled certain classes of sensory neurons with fluorescein by simple
immersion of animals in the dye. Mutants in five unc genes showed guidance
defects in these neurons, with processes either growing erratically in
abnormal locations, or stopping prematurely. Several mutants are also
known in which the outgrowth of the touch neurons is defective (Chalfie and
Suslton, 1983). Further studies have been undertaken using monoclonal
antibodies (S. McIntire, S. Siddiqui and J. Culotti, unpublished) and by
electron microscopereconstruction of mutants (J. White, unpublished).
The study of neural outgrowth undertaken here has concentrated on the
ventral cord, and to a lesser extent the ganglia at either end (RVG and
PAG). Figure 1.3 shows in schematic form all the neurons and nerve
processes behind the RVG in a newly hatched L1 larva. The ventral cord
contains the motor neurons that innervate body muscles as well as
interneuron processes that run to and from the nerve ring. There are two
groups of processes in the ventral cord, one on each side of the hypodermal
ridge. They are very asymetrical. The right hand cord contains 25 to 30
processes, including the motor neuron processes and many pairs of
interneurons which are bilaterally symmetric in the nerve ring, while the
left hand cord contains only 3 or 4 processes. The other main longitudinal
bundle is the dorsal cord, which contains motor neurons processes and just
one interneuron, RID.
The ventral and dorsal cords contain the motor circuitry controlling body
movement. There are three classes of motor neuron at the L1 stage, DA, DB and DD (five
more classes are added during postembryonic development). In addition to
having their cell body and a process in the ventral cord, all these motor
neurons send a commissure round the body of the animal to the dorsal cord,
where they have another process. Muscle arms from ventral muscles extend
to the ventral cord, while those from dorsal muscles extend to the dorsal
cord. Movement of the body is limited to the dorsal-ventral plane. The
head has more freedom of movment, owing ot more complex innervation of the
muscles in the head directly from the nerve ring, but motion of the whole
animal is caused by propogating dorsal-ventral waves along the body. DA and DB neurons
both have their neuromuscular output in the dorsal cord, and receive input
from (different) interneurons in the ventral cord. However they have
different polarities: both ventral and dorsal DA processes grow
forward, while DB processes grow
backward. DD motor neurons
receive input in the dorsal cord from the DA and DB neurons, by
ïntercepting" their neuromuscular junctions, and have output in the
ventral cord, which is thought to be inhibitory, ensuring relaxation of the
ventral musculature while the dorsal musculature is contracted.
Figure 1.3
All the nerve processes and cell bodies behind the RVG. This diagram is a
schematic cylindrical projection of the inner surface of the hypodermis and
nervous system, obtained by conceptually cutting along the dorsal midline
and unfolding flat. The dorsal cord is shown at the left hand edge,
anterior is at the top, and posterior at the bottom. The positions of the
four longitudinal muscle quadrants are shown by hatched regions. Nerve
processes in C. elegans branch only rarely and reproducibly and all the
branches in this region are shown. Processes entering the ventral, lateral
or dorsal cords from the front are indicated at the top. Those with
asterisk after the neuron's name only run part way back along the body.
Posterior interneuron processes running forward along the ventral cord are
indicated at the top. Those with an asterisk after the neuron's name only
run part way back along the body. Posterior interneuron processes running
forward along the ventral cord are indicated similarly at the front of the
preanal ganglion. All the anterior axons in the ventral cord without an
asterisk terminate in the preanal ganglion, except for that of AVG, which
is shown ascending into the dorsorectal ganglion. The PHA and PHB neurons
from the lumbar ganglia also have posteriorly directed processes that
terminate in the phasmid sensilla. Note the different directionalities of
outgrowth of the different ventral cord motor neuron classes.
In addition to those in the ventral and dorsal cords there are a few
neuronal cells and processes on the lateral hypodermal ridge and four small
ganglia at the back of the animal (figure 1.3). The lateral neurons ALML/ALMR
and PLML/PLMR
are touch receptor classes (Chalfie and Sulston, 1980), while CANL/CANR
and ALAL/ALAR
are associated with the excretory canals, which run through the lateral
ridges. In the front half of the animal there are four processes running
back under each muscle quadrant from the nerve ring. These sublateral
processes are possibly proprioceptive, involved in controlling head
movement, since the neurons they belong to are closely associated with the
head motor circuitry, SMBDL/SMBDR
and SMDDL/SMDDR
being motor neuron classes themselves. The preanal ganglion contains three
interneuron cell bodies, DD6,
DA8
and DA9.
The lumbar ganglia on the sides at the back contain the cell bodies of the
ALNL/ALNR
and PLML/PLMR
neurons, which have lateral processes, and of the phasmid chemoreceptors PHAL/PHAR
and PHBL/PHBR
and the ventral cord interneurons PVQL/PVQR,
PVCL/PVCR,
LUAL/LUAR
and PVR,
all of which send anterior processes down to the preanal ganglion and the
ventral cord via the lumbar commissures. Finally there are two neurons in
the dorsorectal ganglion on the top surface of the rectal epithelium behind
the anus, DVA
and DVC.
There are both practical and strategic reasons for choosing the ventral
cord as the target for study. First, although the final anatomy of the
nerve ring has been reconstructed, it is too complex a structure to be able
to easily study its development. Its final structure is, however, discussed
with respect to developmental considerations in the second part of this
thesis. Second, the method of observation used has been reconstructed from
electron micrographs, and it is relatively easy to reconstruct the ventral
cord region from transverse sections, since processes are mostly
lonitudinal, any commissures containing only a few processes. Third, and
perhaps most importantly, it is possible to at least some extent to examine
functionality defects in ventral cord structure, which allows the combining
of work on structure and function. A reasonable functional model of the
ventral cord motor circuitry has been proposed, both by analogy to the
results in Ascaris and as a result of ablation experiments in which
components of the circuitry were removed (Chalfie et al., 1985).
Movement is very easily observed, and a large number of uncoordinated
mutants have been ovtained that have various defects in movement (Brenner,
1974).
As mentioned previously, some of these mutants have been seen to have
defects in nerve process morphology (Hedgecock et al., 1985 S. McIntire, J.
White unpublished observations). Particular examples are that some or all
circumferential commissures go astray in unc-5,
unc-6
and unc-33
mutants, and the PHAL/PHAR
and PHBL/PHBR
processes get stuck at the bottom of the lumbar commissures in
unc-33 , unc-44 , unc-51 and unc-76 mutants.
These defects suggest that the affected genes may be involved in the
processes of neural outgrowth that have been studied here. Genes defined
in this way provide a possible link between the anatomical experiments and
observations described here and the molecular mechanisms involved. The
defects they induce are compared with the wild type development and the
effects of cell ablations in Chapter 5.