Max Planck Institute for Medical Research, Jahnstrasse 29, 69120 Heidelberg, Germany
(e-mail: hutter{at}mpimf-heidelberg.mpg.de)
Accepted 15 July 2003
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SUMMARY |
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Key words: Nervous system, Axon guidance, Laser ablation
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Introduction |
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Axon tracts in the C. elegans nervous system have been described
in great detail using electron microscopic reconstructions
(White et al., 1986;
White et al., 1976
) and a
single neuron (AVG) has been identified as the pioneer for the right ventral
cord axon tract (R. M. Durbin, PhD thesis, University of Cambridge, 1987). The
ventral cord in C. elegans consists of two axon bundles flanking the
ventral midline (Fig. 1). The
two axon bundles are highly asymmetrically despite an overall bilateral
symmetry of the nervous system. The left ventral cord axon tract consists of
only four axons, whereas the right axon tract contains about 50 axons in adult
animals. The number and arrangement of neuronal processes is highly invariant
from animal to animal. Elimination of the AVG pioneer by laser ablation
resulted in a disorganized ventral cord with the overall asymmetry still
preserved (R. M. Durbin, PhD thesis, University of Cambridge, 1987),
suggesting that this pioneer is essential for the correct navigation of later
outgrowing axons. The small sample size of the original experiment (the
ventral cord of one operated animal was analysed in detail with EM
reconstructions), however, precludes detailed conclusions on the importance of
this pioneer for the outgrowth of the different classes of follower axons.
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With the green fluorescent protein (GFP), its derivatives and other proteins with similar properties, a range of markers with different spectral properties is now available, which allows the simultaneous inspection of axon trajectories from different classes of neurons in a single animal. For this study a number of transgenic strains was generated, which express different combinations of GFP color variants in different subsets of neurons with axons in the ventral cord. With these markers, the dependencies of different axons in the ventral cord on identified pioneers and extracellular signals were analysed systematically on a single axon level. The results show that pioneers are not absolutely essential for correct outgrowth of follower axons. Axons of different classes of neurons navigate surprisingly independently and seem to use different combinations of extracellular cues as well as adhesion to certain early outgrowing axons in their navigation. The results give fundamental insights into the logic of axon outgrowth in the ventral cord of C. elegans. They provide a basis for the interpretation of phenotypes and suggest strategies for genetic screens to identify the missing guidance cues used by axons navigating in the ventral cord.
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Materials and methods |
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The GFP-coding part of the original constructs was replaced with one of the following variants of GFP (A. Fire vector kit) or DsRed (Clontech).
Nematode strains and mutant analysis
In addition to the multi-color GFP strains described above the following
GFP strains and mutants were used: NW1229, evIs111[F25B3.3::GFP];
EG1306, oxIs12[unc-47::GFP]; PY1058, oyIs14[sra-6::GFP];
OH2012, otEx1082 (GFP under the control of the promoter of an innexin-like
gene expressed in a subset of neurons, including AVG) (A. S. Wenick and O.
Hobert, unpublished); MT633, lin-11(n389) I; him-5(e1467) V; NW434,
unc-6(ev400) X; CB53, unc-5 (e53) IV; CB271,
unc-40(e271) I; CX5000, slt-1(eh15) X; CX3198,
sax-3(ky123) X; IM324, nid-1(ur41) V; NW987,
unc-129(ev554) IV; CZ337, vab-1(dx31) II; VH582,
mab-20(ev574) I; CB191, unc-30(e191) X. Mutants were
analysed after crossing the GFP markers into the corresponding strains.
Strains were grown at 20°C under standard conditions and analysed as
growing population. Typically, late larval stages and adults were scored for
axonal defects. DsRed matures rather slowly so that sufficient signal could
only be detected in adult animals. DsRed2 in contrast already gave strong
signals in newly hatched larvae. Axonal defects were scored using a Zeiss
Axioplan microscope with a 40x objective and narrow band pass filters to
separate the different color variants (CFP: D436/20, 455DCLP and D480/40; GFP:
HQ480/20, Q495LP and HQ510/20; YFP: HQ500/20, Q515LP and HQ535/30; DsRed:
HQ565/30, Q585LP and HQ620/60). Images were taken at a Leica SP2 confocal
microscope equipped with Ar and He lasers for excitation at 457 nm (CFP), 488
nm (GFP), 514 nm (YFP) and 543 nm (DsRed). For clean separation of the
channels it was sufficient in most cases to acquire data for the different
channels sequentially. Typical emission windows used were: 463-493 nm (CFP),
520-550 nm (YFP) and 580-640 nm (DsRed).
For counting cells in the rvg in evIs111 and lin-11(n389); evIs111 animals stacks of confocal images of the rvg were taken from a random set of animals displaying a ventral aspect. Only samples where individual cells could be counted unambiguously were used.
Laser ablation experiments
A Photonic Instruments Micro Point Laser System in combination with a Zeiss
Axioplan II microscope was used for laser ablation experiments. The laser
system consists of a nitrogen-pulsed dye laser with Coumarin (440 nm, 5 mM) as
dye. Ablations were carried out at the end of gastrulation at about 270
minutes of development, a time where cells can be identified easily by virtue
of their position in the embryo. Cells were irradiated with 100-200 pulses at
a frequency of 2 Hz. Before each session the amount of irradiation necessary
for immediate killing of the cell was determined experimentally. Irradiation
were carried out at about 80% of the immediate lethal dose. For AVG ablations,
ABprpapppa was irradiated in VH318. This also eliminates the RIR interneuron,
which sends its process into the nerve ring and has no connection to the
ventral nerve cord. Ablations were validated by checking for the absence of
the AVG cell body and the absence of the AVG axon in the tail spike with the
glr-1::YFP marker, which is expressed in AVG. Axon guidance defects
in the ventral cord, mainly erroneous outgrowth in the left axon tract or a
switch from the right to the left axon tract were scored after operated
animals were allowed to grow to adulthood. For RIF ablations, the following
cells were irradiated at about 270 minutes of development in VH414: RIFL and
SABVL:ABplppapaaa; RIFR and SABVR:ABprppapaaa; RIGL: ABplppappa; and RIGR:
ABprppappa. Ablation of ABplppappa also eliminated DD1 and ablation of
ABprppappa also eliminated DD2. Animals were allowed to grow to adulthood
before axon outgrowth defects were scored. Absence of RIG neurons was judged
with glr-1::DsRed, absence of RIF neurons was confirmed using
odr-2::CFP.
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Results |
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Dependencies between early and late outgrowing axons in the ventral
cord
Axons grow out sequentially in the ventral cord during embryonic
development. To analyse dependencies between early and late outgrowing axons,
navigational errors in various classes of neurons were correlated in mutants
affecting either the pioneer AVG or particular guidance cues using multi-color
GFP strains (Table 4). Axons of
two different classes of neurons only rarely crossed between axon tracts at
exactly the same point. This was true for all combinations of axons analysed
in all mutant backgrounds with only one exception. PVQL and PVPR axons were
almost always found to switch axon tracts together
(Table 4, row 1), suggesting a
strict dependency of PVQL on the earlier outgrowing PVPR axon
(Fig. 5D-F). However, in
individual unc-30 mutants PVQL axons were found to grow out normally
despite navigational errors of PVP axons, indicating that even PVQ axons are
able to navigate independently of PVP.
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Extracellular cues affect ventral cord asymmetry and directional
outgrowth of commissures
Pioneers as well as later outgrowing axons are known to depend on
extracellular cues for their navigation. Mutants in C. elegans
homologs of several extracellular axon guidance cues and their receptors have
been identified and shown to be involved in certain aspects of axon guidance.
I tested whether any of these mutants is involved in the generation of ventral
cord asymmetry or is a source of information for directional outgrowth of
motoneuron commissures. A small but significant fraction of animals with axons
extending inappropriately into the left ventral cord axon tract after exiting
the nerve ring was found in unc-6/netrin, nid-1/nidogen and
sax-3/Robo mutants, as well as in lin-11; unc-30 double
mutants (Table 2, column 1).
Axons found to enter the left axon tract usually stayed in this tract in
nid-1 mutants, leading to an almost symmetrical ventral cord
(Fig. 2E,F). This is in
contrast to sax-3 and unc-6 mutants, where these axons
frequently crossed into the right tract.
Some of the analysed mutants had defects in the directional outgrowth of motoneuron commissures. Penetrant defects in DA/DB commissures were found in sax-3 mutants, less penetrant defects also in unc-6/netrin, mab-20/semaII and vab-1/EphR mutants (Table 2, column 10).
Extracellular cues affect axon navigation within the ventral
cord
I tested whether any of known guidance cues/receptors is essential for
proper navigation of the ventral cord pioneer AVG. Surprisingly, mutants in
unc-6/netrin, slt-1/slit, sax-3/Robo,
mab-20/sema II, vab-1 (the only Ephrin receptor),
unc-129/TGFß and nid-1/nidogen showed no significant
defects in the navigation of the AVG axon
(Table 2, column 2), leaving
the source of information used by this pioneer obscure. In individual cases,
where the AVG axon erroneously crossed into the left axon tract, this had no
consequences for DD motoneuron axons, which grew out in the right axon tract
even at positions where AVG was in the left axon tract
(Fig. 5M-O).
Mutants in the above mentioned genes were also systematically tested for defects in the navigation of the other classes of axons present in the embryonic ventral cord (Table 2, columns 3-9). Mutations in slt-1 and vab-1 had no significant effects on axon outgrowth for any of the neuron classes with axons in the right axon tract. DD motoneuron axons, which grow out immediately after the AVG pioneer, were affected to a different extent in mab-20, unc-129, sax-3, nid-1 and unc-6 mutants. In a lin-11; unc-6 double mutant these defects were additive so that almost every animal was affected. In addition to inappropriate outgrowth in the left ventral cord tract and crossing between the left and right tracts, local defasciculations within the right ventral cord axon tract could be observed in unc-6 and lin-11; unc-6 double mutants. PVPL and PVQR axons were essentially unaffected by mutations in any of the genes tested. By contrast, the bilateral counterparts PVPR and PVQL, which pioneer the left axon tract, are dramatically misguided in sax-3, nid-1 and unc-6 mutants, and also somewhat affected in unc-129, vab-1 and mab-20 mutants. Glr-1::GFP expressing interneuron axons and DA/DB motoneuron axons which grow into the ventral cord rather late were only very moderately affected in sax-3, nid-1 and unc-6 mutants, and unaffected in all other mutants tested.
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Discussion |
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The idea that AVG has a role in guiding later outgrowing axons in the
ventral cord was first tested by Richard Durbin, who eliminated AVG by laser
ablation and studied ventral cord axons in one animal in great detail using
electron microscopic reconstructions (R. M. Durbin, PhD thesis, University of
Cambridge, 1987). He found that the right axon tract still contained the
majority of the axons. However, the axon bundle was disorganized and split
into distinct sub-bundles with axons wandering between bundles and
occasionally even crossing the midline to run in the left tract. He also
noticed that motoneuron axons were predominantly affected, whereas
interneurons were much less affected. This earlier work is extended here and
leads to a more refined picture concerning the importance of AVG as a pioneer.
A significant number of AVG-ablated animals showed axon outgrowth defects.
Affected were predominantly interneurons and D-type motoneurons and only
rarely DA or DB motoneurons. This indicates that some classes of neurons
depend more on the pioneer than others and implies that different classes of
neurons use different combinations of cues to navigate within the same axon
bundle. In more than half of the animals in which AVG was removed, no defects
could be detected in the outgrowth of the command interneurons of the
motorcircuit and of the major classes of motoneurons (DD, VD, DA, DB). This
suggests that the pioneer AVG is not absolutely essential for the proper
outgrowth of other axons in the right axon tract. Apparently, these axons can
use other sources of information, most likely extracellular cues, for their
navigation. The pioneer AVG is not uniquely endowed with pathfinding abilities
not shared by follower axons. It might be required as additional source of
information at times when a large number of axons grows out simultaneously to
ensure that every single axon navigates correctly. The observation that the
elimination of pioneers leads to a certain frequency of errors made by later
outgrowing axons is reminiscent of the situation found in Drosophila
or zebrafish, where similar results were obtained after elimination of
particular pioneers (Chitnis and Kuwada,
1991; Lin et al.,
1995
). Stalling or complete failure of outgrowth as described in
grasshopper after elimination of particular pioneers
(du Lac et al., 1986
;
Klose and Bentley, 1989
) was
never observed in the experiments presented here. This indicates that AVG is
not required for continuous growth of follower axons.
RIF axons pioneering the pathway between the ventral cord and the nerve
ring are obvious candidates for guiding axons exiting the nerve ring to the
right side of the ventral cord. The asymmetrical expression of the guidance
cue UNC-6/netrin in one of the RIF neurons and AVG led to the proposal that
RIFL and AVG generate a UNC-6/netrin labeled pathway on the right side of the
ventral midline (Wadsworth et al.,
1996). However, laser ablation of RIF neurons had no consequences
for the crossing of interneuron axons into the right axon tract. Even
elimination of all three pairs of neurons, which send their axons along the
RIFL/R trajectories (RIF, RIG, SABV) led to the same negative result. This
strongly suggests that these neurons either have no pioneering function for
axons exiting the nerve ring or that redundantly acting information systems
are present. More than one pioneer is also found for the longitudinal axon
tracts in Grasshopper and Drosophila, and in zebrafish motoneuron
axon tracts. In these cases, elimination of individual pioneers typically also
had no consequences for follower axons
(Eisen et al., 1989
;
Pike et al., 1992
;
Raper et al., 1983a
;
Raper et al., 1983b
;
Raper et al., 1984
).
lin-11 and unc-30 control the differentiation of
the pioneer neurons AVG and PVP
LIN-11 and UNC-30, two homeobox transcription factors, are known to affect
the differentiation of particular classes of neurons. LIN-11 has been shown to
affect differentiation and function of neurons of the thermosensory circuit
(Hobert et al., 1998), as well
as of olfactory and chemosensory neurons
(Sarafi-Reinach et al., 2001
).
UNC-30 is essential for the correct differentiation of D-type motoneurons
(Jin et al., 1994
). Both
transcription factors are also expressed in pioneer neurons of the ventral
cord, yet no defects have been associated with this expression so far.
lin-11 is expressed in the AVG neuron
(Hobert et al., 1998
) and both
lin-11 and unc-30 are expressed in the PVP neurons
(Hobert et al., 1998
;
Jin et al., 1994
).
lin-11 mutants fail to express AVG specific markers like
glr-1::GFP or odr-2::GFP, indicating that lin-11
interferes with the differentiation of the AVG neuron. lin-11 mutants
show axon outgrowth defects in ventral cord neurons, which do not express
lin-11, like command neurons of the motorcircuit and D-type
motoneurons. These defects match the defects seen after laser ablation of AVG
both qualitatively and quantitatively, suggesting that they are secondary
defects caused by a failure of AVG to provide its normal pioneer function. The
only difference between AVG-ablated animals and lin-11 mutants is the
severity of defects in glr-1::GFP expressing interneurons. The number
of axons in the left axon tract is higher in some lin-11 mutants when
compared with AVG-ablated animals. One explanation for this difference could
be the expression of lin-11 in other neurons, which might directly or
indirectly affect the outgrowth of these interneuron axons. lin-11 is
expressed in AVER/L, two of the glr-1 expressing interneurons
(Hobert et al., 1998
), and
might thus directly affect the ability of these neurons to navigate correctly.
lin-11 is also expressed in the PVP neurons, which grow out before
the glr-1 expressing interneurons. As lin-11 appears to
affect PVP differentiation (see below), this in turn could affect interneuron
axon outgrowth, provided that PVP also has some pioneering function in the
right axon tract.
Expression of the odr-2::GFP marker in PVP neurons is variable in lin-11 as well as unc-30 mutant animals, suggesting that these transcription factors control aspects of PVP differentiation. This idea is further supported by the nature of axon outgrowth defects found in these mutants. The PVPR neuron pioneers the left ventral cord tract and is known to be essential for the proper navigation of the PVQL axon. When PVPR is eliminated, the PVQL axon extends in the right ventral cord axon tract (R. M. Durbin, PhD thesis, University of Cambridge, 1987). Such defects are found in unc-30 mutants, indicating that unc-30 affects the pioneering function of PVPR. Interestingly such PVQL defects are not found in lin-11 mutants, suggesting that lin-11 and unc-30 control different aspects of PVP differentiation.
Left-right asymmetry in axon and commissure outgrowth
The overall asymmetry of the ventral cord never disappeared in any ablation
experiment, suggesting that neither the ventral cord pioneer AVG, nor the
neurons pioneering the path between the ventral cord and the nerve ring (RIF,
RIG, SABV) are important for generating this asymmetry. However, a failure of
axons to cross over into the right axon tract at the anterior end of the
ventral cord was found in a small but significant fraction of animals mutant
for lin-11. This contrasts with the observation that no such defects
were observed in AVG-ablated animals. The small sample size of AVG-ablated
animals might account for this difference. Alternatively, lin-11
function in neurons other than AVG might be responsible for the crossover
defects seen in lin-11 mutants. Axons extending inappropriately into
the left ventral cord tract were also observed in some animals mutant for
either unc-6/netrin, sax-3/Robo or nid-1/nidogen.
This confirms earlier observations in nid-1
(Kim and Wadsworth, 2000) and
implies that a combination of signals embedded in the basement membrane
provides the essential information for the generation of an asymmetrical
ventral cord.
The ventral cord consists of motoneuron axons originating from cell bodies located along the ventral midline and of interneuron axons entering the ventral cord mainly from the nerve ring in the head or from tail ganglia. Motoneuron axons invariably grow into the right ventral cord axon tract, indicating that motoneurons use positional information to distinguish left and right axon tracts. This information could either be intrinsic (polarization of the cell) or it could be extracellular information, which is read and interpreted by the neurons. The observation that the elimination of the pioneer neuron AVG leads to defects in the directional outgrowth of D-type motoneuron axons, suggests that cues provided by this pioneer are important sources of information. As a complete randomization of outgrowth was never observed in any experimental situation (laser ablation or mutant combination), other still undiscovered redundantly acting signals must exist. Navigation defects were much more prominent in D-type motoneurons when compared with DA and DB motoneurons, suggesting that different classes of motoneurons use AVG to a different extent for directed outgrowth of their axons. Directional outgrowth of DA/DB motoneuron commissures (on the left versus the right side) was affected in a large number of AVG-ablated animals, indicating that AVG provides essential left-right information for the navigation of commissures. Interestingly, the penetrance of these defects was highest for commissures normally growing on the left side, which suggests that the presence of AVG has a repulsive effect on the outgrowth of those commissures. As axons of these motoneurons were much less affected than commissures, this indicates that even different types of neuronal processes from the same neuron use different cues for their orientation.
The logic of axon navigation in the ventral cord of C.
elegans
Axons in the ventral cord of C. elegans are found at very
reproducible positions within the axon bundle. Axons belonging to the same
class of neuron form distinct sub-bundles within the right axon tract and even
sub-bundles are positioned precisely and reproducibly within the cord. Early
outgrowing axons are found dorsally in close contact with the basement
membrane, whereas later outgrowing ones end up in ventral positions far away
from the basement membrane. This well ordered structure implies a very precise
navigational system guiding axons not just towards the ventral cord but also
within the ventral cord. Precise positioning of axons within the ventral cord
is essential for the correct formation of synapses within the motor circuit,
as synapses are made within the ventral cord between neighboring processes
(White et al., 1986;
White et al., 1976
). Any
deviation from the normal placement of neuronal processes in the ventral cord
is likely to have consequences on the wiring of components of the motorcircuit
and the movement behaviour of the animal. Neuronal processes grow out
sequentially in the ventral cord (R. M. Durbin, PhD thesis, University of
Cambridge, 1987). Navigation of later outgrowing axons could therefore depend
on the presence of particular early outgrowing axons (pioneer-follower
relationship) and the order of outgrowth could already be a major determinant
of axon position. The data presented here argue against a strict
pioneer-follower relationship between any groups of axons. The only exception
are the PVQ axons, which strictly follow the earlier outgrowing PVP axons,
irrespective of whether these grow out correctly or not. Apart from this
example, later outgrowing axons typically do not simply follow earlier
outgrowing ones. The strict dependency of the PVQ axons on PVP axons might
indicate, that PVQ axons on their own are not able to navigate correctly.
However, in a few unc-30 mutants, where PVP axons were found in the
wrong axon tract, PVQ axons were normal, indicating that even they are able to
use other cues for their navigation.
When individual navigational errors were evaluated, no strong correlation was found between errors made by early outgrowing DD and later outgrowing DA/DB or interneuron axons. Each axon appears to commit errors individually and typically does not follow a misguided earlier outgrowing axon. Later outgrowing axons are able to ignore a misguided pioneer and to continue on their normal path, indicating that they constantly read and interpret several extracellular signals rather than simply following the trajectory of a pioneer. However, the presence or absence of early outgrowing axons is not completely irrelevant for later outgrowing ones. The percentage of animals showing glr-1::GFP-expressing interneuron axon defects is dramatically different depending on the presence or absence of the PVQL axon in the right axon bundle. The outgrowth of the PVQR axons is similarly affected by the presence or absence of either the AVG or the DD axons in the same axon tract. This suggests that early outgrowing axons are recognized and used as source of positional information, but not in a strict and absolute pioneer-follower relationship. This flexibility ensures that individual guidance errors of early outgrowing axons are not inevitably amplified by affecting follower axons.
Several guidance cues are known to affect neurons with axons in the ventral
cord of C. elegans (Colavita et
al., 1998; Hao et al.,
2001
; Kim and Wadsworth,
2000
; Roy et al.,
2000
; Wadsworth,
2002
; Zallen et al.,
1998
). Early outgrowing axons should depend more heavily on
extracellular cues than later outgrowing ones, which could use the presence of
earlier outgrowing axons as additional source of information. However, a
systematic analysis of the defects in the mutants affecting the different
extracellular cues or their receptors revealed that this is not necessarily
the case. Most surprisingly, the ventral cord pioneer AVG is not affected in
any of the mutants tested, leaving the primordial source of navigational
information for the ventral cord pioneer obscure. However, DD-motoneuron
axons, which grow out immediately after the AVG axon, are affected
significantly in several mutants, in particular in unc-6/Netrin,
sax-3/Robo, nid-1/nidogen and mab-20/semaII
mutants, suggesting that these axons depend heavily on extracellular cues and
use a variety of different cues in combination to navigate within the ventral
cord.
Among later outgrowing axons, the PVPL and PVQR axons are hardly affected in any of the analysed mutants. However, there is a strong correlation between AVG defects and corresponding PVQR/PVPL defects in nid-1 mutants, arguing for a role of AVG in guiding PVPL/PVQR axons. The fact, that essentially no PVPL/PVQR defects were found in lin-11 mutants suggests that other redundantly acting cues must be present. Most likely, these cues come from the DD axons, as there is also a strong correlation between defects in the DD axons and the PVPL/PVQR axons. DA and DB axons are only moderately affected in any of the single mutants tested. Significant defects, however, were found in lin-11; unc-30 and even more in lin-11; unc-6 double mutants, suggesting that unc-6/netrin in combination with early outgrowing axons provides guidance information for these axons. Nonetheless, even the most penetrant defects were well below 50%, indicating that major navigational cues for these axons are still unidentified. As DA/DB axons are found in ventral positions close to the epidermal cells, it might be worth considering these cells as potential sources for navigational cues. Finally, glr-1::GFP-expressing interneurons are moderately affected in unc-6/Netrin, sax-3/Robo and nid-1/Nidogen mutants. They are strongly affected in mutants interfering with the differentiation of earlier outgrowing axons, like lin-11 and unc-30. These interneuron axons might predominately navigate by using interactions with other axons and to a lesser extent rely on extracellular cues for their navigation. This makes sense in the light of the fact, that these interneuron axons are found in a central position within the right axon tract and not in close contact to the basement membrane where extracellular cues are likely to be located (Fig. 1D).
Taken together, the analysis presented here provides a detailed picture of the combination of cues used by individual axons to navigate towards the ventral cord and then further within the ventral cord of C. elegans (Fig. 6). The main conclusions are as follows.
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ACKNOWLEDGMENTS |
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