MRC Centre for Developmental Neurobiology, King's College London, Guy's Campus, 4th Floor New Hunt's House, London SE1 1UL, UK
* Authors for correspondence (e-mail: carol.irving{at}kcl.ac.uk and ivor.mason{at}kcl.ac.uk)
Accepted 30 August 2002
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SUMMARY |
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Key words: Trochlear nerve, Cranial motor nerve, Axon guidance, Fgf8, Isthmus, Organiser, Rhombomere, Chemotropic response
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INTRODUCTION |
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Here we investigate the formation of one relatively simple axon pathway:
that of the trochlear or IVth cranial nerve. We focus upon the initial
projection of trochlear axons as they extend from cell bodies in
ventro-anterior rhombomere one (r1) of the hindbrain and fasciculate, growing
along a dorsal trajectory that circumnavigates the isthmic organiser at the
midbrain-hindbrain boundary (MHB) to a dorsal exit point. At the latter
location axons become less tightly associated, before fasciculating once more
to project to the eye where they innervate the contralateral superior oblique
muscle (dorsal oblique in avian embryos)
(Colamarino and Tessier-Lavigne,
1995).
Within the brain (as opposed to the spinal cord), motor neuron organisation
is subservient to neuromeric organisation. Thus, the various classes of
cranial motor neurons branchiomotor, visceral motor and somatic motor
are organised within individual neuromeres or in adjacent neuromeric
pairs. The oculomotor (III) nucleus is located in the posterior midbrain, the
trochlear (IV) nucleus in anterior r1, while the trigeminal (V; r2 and r3),
facial (VI; r4 and r5), abducens (VII; r5 and r6) and glossopharyngeal (VIII;
r6 and r7) are organised in adjacent pairs of hindbrain segments. The midbrain
and each hindbrain segment have their own molecular `address' reflected by the
expression of a unique combination of transcription factors. Current evidence
suggests that the transcription factor hierarchy plays a major role in
determining the different properties of individual motor nuclei including
their axonal projections (Jacob et al.,
2001; Lumsden,
1990
; Lumsden and Krumlauf,
1996
; Lumsden and Keynes,
1989
).
Rhombomere 1, within which the trochlear motor nucleus develops, is
distinct from the remaining hindbrain segments since its pattern is
established through graded signals from the isthmic `organiser' at the
midbrain-hindbrain boundary (MHB), mediated at least in part through the
activity of Fgf8 (Irving and Mason,
2000; Meyers et al.,
1998
; Reifers et al.,
1998
) (reviewed by Rhinn and
Brand, 2001
; Wurst and
Bally-Cuif, 2001
). It is noteworthy that the dorsal projection of
trochlear motor axons to exit at the roof plate at the isthmus is unique among
motor neurons. Previous studies have shown that the dorsal projection of the
trochlear nerve is caused in part by chemorepulsive cues emanating from the
floor plate. Those motor nerves with dorsal trajectories (IV, V, VII, IX) are
repelled by factors secreted from the floor plate
(Colamarino and Tessier-Lavigne,
1995
; Guthrie and Pini,
1995
; Kennedy et al.,
1994
). Candidates for chemorepellent cues for trochlear neurons
are members of the netrin and semaphorin (Sema) families, since both netrin 1
and Sema3A repel growing trochlear axons in vitro
(Colamarino and Tessier-Lavigne,
1995
; Varela-Echavarria et
al., 1997
). netrin 1 is expressed by the floor plate, while Sema3A
is expressed by ventral tissues, suggesting that these molecules might govern
the dorsal projection of trochlear axons in vivo
(Kennedy et al., 1994
;
Puschel et al., 1995
;
Varela-Echavarria et al.,
1997
). In addition, Sema3F, which is expressed in both posterior
midbrain and anterior hindbrain in the mouse, also repels trochlear axons and
in mice lacking the Sema3F receptor, neuropilin 2, axons fail to exit the
neuroepithelium (Chen et al.,
2000
; Giger et al.,
2000
).
In this study we explore the relationship of the trochlear motor nucleus and its axonal projection within the neuroepithelium to the isthmic organiser. We show that trochlear axons project towards and extend within the organiser raising the possibility that the isthmus plays a role in guiding trochlear axons. We have examined the role of the isthmus and Fgf8 in trochlear axon navigation and provide direct evidence that Fgf8 acts as a chemoattractant, which guides trochlear axons into the isthmic region and subsequently maintains their axon pathway within it.
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MATERIALS AND METHODS |
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Rat tail collagen gels were prepared as described previously
(Guthrie and Lumsden, 1994).
MHB explants and dorsal isthmus, dorsal r1 or FGF-soaked beads were placed
into gels 100-500µm apart and cultured for 48 hours in media as described
previously (Colamarino and
Tessier-Lavigne, 1995
). Affi-gel blue beads were soaked in Fgf8b
(R&D Systems) or PBS (control beads) as described previously
(Irving and Mason, 2000
;
Shamim et al., 1999
) and
implanted into the collagen matrix. To inhibit Fgf signalling, either the
chemical inhibitor of FGF signalling, SU5402 (at 10 µM or 20 µM;
CalBiochem), or a neutralising FGF8 antiserum (R&D Systems) at a
concentration five times the stated neutralisation dose (ND50),
were included in both collagen matrix and cell culture media.
To score the extent of axonal turning towards potential sources of
chemotropic cues a simple grid system was used (see
Fig. 3B). An inverted T-bar
grid was oriented with its stem aligned along the original direction of
trochlear axon growth through the explant, perpendicular to the floor plate,
as we found that axons did not deviate towards a chemotropic cue until their
emergence into the gel, in agreement with previous studies
(Colamarino and Tessier-Lavigne,
1995). Axons extended within the collagen gels either singly or in
small fascicles and the numbers growing in each sector, either side of the
T-bar stem, were scored. Numerical data were analysed using Student's
t-test.
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Implantation of FGF beads in ovo
Heparin acrylic beads were soaked in Fgf8b (R&D Systems) or PBS
(control beads) and implanted into HH12 chick embryos as described previously
(Irving and Mason, 2000;
Shamim et al., 1999
). Embryos
were incubated for a further 72 hours until HH25.
Immunostaining and in situ hybridisation
Whole embryos were immunostained as described previously
(Irving and Mason, 2000) using
SC1 antibody (Hybridoma Bank; 1:5 for 5 days) and a horseradish
peroxidase-conjugated secondary antibody (Sigma; 1:200). Explants embedded in
collagen gel were immunostained using either F84.1 antibody
(Prince et al., 1992
;
Varela-Echavarria et al.,
1997
) (1:1000) or anti-160 KDa neurofilament antibody (Zymed;
1:10,000) for 3 days and a horseradish peroxidase-conjugated secondary
antibody (Sigma; 1:200). Whole-mount in situ hybridisation of embryos was
performed using probes reported previously
(Irving and Mason, 2000
).
Embryos were then post-fixed in 4% paraformaldehyde in PBS and immunostaining
was performed using anti-Isl1/2 antibody
(Thor et al., 1991
) as
described previously (Mason,
1999
).
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RESULTS |
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In accordance with previous studies, Isl+ motor neuron cell bodies were
located ventrally on either side of the floor plate, along the entire
rostrocaudal axis of posterior hindbrain segments (r2-7). By contrast, the
cell bodies of the trochlear nucleus were detected in only the most rostral
part of r1 and also within the Fgf8-positive isthmic tissue
(Fig. 1A). Small numbers of
trochlear motor neurons were first detected at Hamburger and Hamilton stage 17
(HH17; onset of limb bud outgrowth) both within and ventral to the
Fgf8 expression domain (Fig.
1B). Fgf8 transcripts form a characteristic stripe at the
isthmus in all vertebrate classes
(Christen and Slack, 1997;
Crossley and Martin, 1995
;
Crossley et al., 1996
;
Heikinheimo et al., 1994
;
Mahmood et al., 1995
;
Ohuchi et al., 1994
;
Reifers et al., 1998
;
Shamim et al., 1999
). However,
the presence of trochlear motor neurons ventral to the
Fgf8-expressing cells indicated that Fgf8 was not expressed
in isthmic cells closest to the floor plate
(Fig. 1B,C). By HH19 many more
trochlear motor neurons were detected lying both within the isthmus and
immediately posterior to it within anterior r1
(Fig. 1C). At this stage, cell
bodies were most closely-packed in the isthmic region. By HH25, cell bodies of
the trochlear nerve formed a cluster with a sharp anterior limit exactly
coincident with the anterior limit of Fgf8 expression
(Fig. 1D). Posteriorly, a few
Isl+ cell bodies were seen in mid to posterior r1 but the majority were
located within the anterior half of that rhombomere
(Fig. 1A,D). The asymmetric
location of trochlear neurons within r1 suggests that their specification may
be mediated at least in part by signals from the isthmus.
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Trochlear axons extend dorsally both towards and within isthmic
tissue
The finding that trochlear motor neuron cell bodies are located both within
and posterior to Fgf8-positive isthmic tissue prompted us to
investigate the relationship of the latter to trochlear axonal projections. We
examined trajectories and timing of trochlear axons growth by immunostaining
for SC1/DM-GRASP/BEN (hereafter called SC1). SC1 is an axonal surface
glycoprotein that is expressed on all hindbrain motor axons and floor plate
cells (Burns et al., 1991;
Guthrie and Lumsden, 1992
;
Pourquie et al., 1990
).
Unfortunately, the SC1 antigen was destroyed when we combined
immunohistochemistry with in situ hybridisation for Fgf8.
However the relationship of trochlear axon trajectories to the
Fgf8-positive tissue was derived by comparison with the
Isl/Fgf8 study.
When the formation of the trochlear projection within the CNS was complete, it was noted that whereas axons from anteriorly located cell bodies extended dorsally (i.e. within the Fgf8-positive tissue), those located posteriorly followed an anterodorsal route. The most anterior axons followed a straight trajectory dorsal to the roof plate, eventually forming a single large bundle. By contrast, more posterior axons appeared to fasciculate and defasciculate in smaller bundles as they extended anteriorly towards the isthmus. Within the isthmus these small bundles joined to form larger fascicles and eventually exited the brain at three or four points in the isthmic roof plate (Fig. 2A and data not shown).
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We investigated the spatiotemporal formation of the trochlear projection within the CNS. Anterior trochlear cell bodies first extended axons at HH18 (Fig. 2B); prior to this no SC1 staining was detected in r1 (data not shown). Initially axons were short, extending independently of one another and by HH19 the first pioneer axons had reached the roof plate (Fig. 2C). Anterior cell bodies extended axons in a direction perpendicular to the floor plate, thereby extending within Fgf8-expressing isthmic tissue. By contrast, posteriorly located neurons projected axons at a more acute angle relative to the floor plate, growing rostrally and eventually joining axons from more anterior cells within the isthmic region (Fig. 2D,E). At HH20, axons were becoming organised into fascicles as they extended dorsally. While some fasciculation and defasciculation was observed in ventral r1, axons became organised into several large bundles in the dorsal isthmus (Fig. 2D). By HH25, a large number of anterior axons had reached their exit point, while those extending from posterior cell bodies were much shorter, with growth cones still traversing ventral r1 (Fig. 2E,F). The most mature neurons might be expected to extend axons before those more recently born, suggesting that trochlear neurons might be born in an anteroposterior wave with the youngest cells located posteriorly. Double immunostaining for both SC1 and Isl1/2 confirmed that all SC1-positive axons in r1 were of trochlear origin (Fig. 2G-J) such that direct comparison could be made with the Fgf8/Isl1/2 study.
Isthmic tissue acts as a chemoattractant for trochlear axons in
vitro
The relationship of trochlear axonal projections, particularly those from
posterior cell bodies, towards the isthmic organiser suggested that the latter
might play a role in trochlear axon guidance within the CNS. In particular, it
raised the possibility that their route might be established by an attractive
cue(s) from the isthmus in addition to the established repulsion from the
floor plate. We therefore used collagen gel co-cultures
(Colamarino and Tessier-Lavigne,
1995; Varela-Echavarria et
al., 1997
) to test the possible influence of isthmic tissue upon
trochlear axons.
A region of ventral r1 and isthmus (mid-hindbrain region) was isolated from
embryonic rat brains and cultured for 48 hours at a distance from explants of
either dorsal isthmus tissue or posterior r1 tissue
(Fig. 3A). Normally,
F84.1-immunoreactive trochlear axons extend perpendicular within MHB explants
and defasciculate to some extent upon entering the collagen gel, however
generally they do not deviate greatly from their original trajectories
(Fig. 3C)
(Colamarino and Tessier-Lavigne,
1995). To measure deviation towards potential sources of
chemotropic cues, an inverted T-bar grid was oriented according to the
trajectory of the projection within the explant and numbers of individual
axons/fascicles were counted in the sector containing a source and the
adjacent sector (Fig. 3B).
Explants were not scored if the potential source was located on or near the
midline of the grid.
When a piece of dorsal isthmus tissue was placed at a distance from such an
MHB explant, axons followed an altered trajectory; turning and growing towards
the isthmus tissue (Fig. 3D,E;
n=20/29; Table 1). By
contrast, posterior dorsal r1 tissue did not cause trochlear axons to deviate
towards it (Fig. 3F;
n=7/8; Table 1). In
the latter experiments we had anticipated a possible repulsive effect, as
Sema3F, which has been shown to repel trochlear axons, is expressed by
posterior r1 tissue in the mouse embryo
(Chen et al., 2000). Taken
together, or data indicate that isthmic tissue, but not posterior r1, contains
a diffusible molecule that can influence the direction of growth of trochlear
axons at a distance.
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Fgf8 is a chemoattractant for trochlear axons in vitro
Our previous studies have shown that Fgf8 secreted by the isthmus patterns
r1 and that Fgf8 protein diffuses across the entirety of that segment to
position the r1/r2 boundary (Irving and
Mason, 2000). Furthermore, studies by several groups have
suggested that Fgf8 forms a gradient extending from the isthmus both
anteriorly into the midbrain and posteriorly into r1 (for reviews, see
Rhinn and Brand, 2001
;
Wurst and Bally-Cuif, 2001
).
Thus, Fgf8 is a candidate for the isthmic guidance cue for trochlear
axons.
To test whether Fgf8 can act directly to influence the directed growth of trochlear axons we performed co-cultures of MHB explants with a source of Fgf8 provided by a bead loaded with recombinant Fgf8b protein (Fig. 4A). Axonal deflection was scored as described above (Fig. 3B). Trochlear axons reproducibly turned towards the FGF bead (Fig.4B; n=17/25; Table 1) and similar results were obtained using Fgf4-soaked beads (data not shown). By contrast, axons rarely turned towards a bead that had been pre-incubated in PBS alone (Fig.4C; n=5/38; Table 1) and statistical analysis showed that this effect was not significant when compared to the effects of Fgf8. This response to Fgf8 was not a general feature of all MHB neuronal populations (Fig. 4D). Thus, Fgf8 protein is sufficient to mimic isthmic tissue as a guidance cue for growing trochlear axons in vitro.
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It was interesting that in some instances trochlear axons turned towards either isthmic tissue or an Fgf8 bead while still within the explant. This might have been due to either a direct chemotropic influence of ectopic Fgf8 that had entered the periphery of the explant or to its indirect action in inducing an unknown chemoattractant. However, in other cases ectopic Fgf8 promoted turning of axons after they had exited the explant (Fig. 4E) suggesting that Fgf8 can itself act directly to guide trochlear axons, although additional indirect effects within explant tissue cannot be excluded.
Ectopic Fgf8 redirects trochlear axons in vivo
To investigate whether Fgf8 could influence trochlear axon growth in vivo,
we implanted Fgf8-coated beads into chick embryo hindbrains. Beads were
inserted unilaterally into dorsal, posterior r1, prior to the onset of
trochlear axon outgrowth (Fig.
5A), and embryos were incubated for 72 hours until approximately
HH25. The effects of this posterior, "competing" source of Fgf8
were examined by staining with anti-SC1 antibody. Embryos receiving implants
of PBS-soaked beads served as controls.
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Following insertion of a PBS bead into r1, normal motor axonal trajectories were generally observed (Fig. 5B; n=44/50; Table 2). By contrast, embryos that had received Fgf8-coated beads, frequently showed obvious abnormalities in the trochlear projection within r1 (n=43/91). Changes in trochlear axon pathfinding could be grouped into 4 classes. The most frequently encountered phenotype (type 1; Fig. 5C; n=31/91) was that axons of posteriorly-located cells did not have an anterodorsal trajectory but instead projected dorsally. Moreover, they failed to coalesce into the 3 or 4 large fascicles that normally exit at the isthmus but instead exited dorsal r1 as a series of small parallel-projecting fascicles. Thus, trochlear axons emerged from dorsal r1 over a much broader domain than in control embryos. In addition, in some cases a subset of caudal axons stalled within posterior r1 and did not reach the dorsal neuroepithelium. Thus, in the most frequent phenotype encountered, posteriorly located trochlear axons appeared to have lost their ability to navigate towards the isthmus.
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The second phenotype (type 2; n=4/91) revealed a dramatic turning of the entire anterior pioneer axon fascicle to project posteriorly towards the ectopic source of Fgf8 (Fig. 5D). Some axons initially extended along a trajectory perpendicular to the floor plate, before making a sharp turn towards the bead. Remaining axons did not turn in this manner but instead generally grew perpendicular to the floor plate (i.e. with a type 1 phenotype). In other instances (type 3 phenotype), implantation of an FGF bead resulted in a complete splitting of the trochlear nerve into 2 main axon groups. The anterior axons followed a normal trajectory to exit the neural tube in the dorsal isthmus region, while posterior axons formed a series of loose fascicles growing caudally and dorsally directly towards the bead (Fig. 5E, F; n=2/91).
Implantation of Fgf8 beads into the hindbrain is sufficient to induce gene
expression characteristic of the midbrain-hindbrain region, suggesting that
the FGF protein was either acting as an ectopic organiser or inducing one. In
addition, ectopic motor neurons (Isl+) were present within posterior r1, which
is usually devoid of motor neurons (Irving
and Mason, 2000). In the present study, we found that a subset of
embryos developed a morphology reminiscent of an ectopic isthmus at the level
of the bead implant. In these cases, SC1-positive axons were observed
projecting from ectopic, ventrally located motor neurons in that region (type
4 phenotype). We believe that these are most likely to represent ectopic
trochlear axons, since trigeminal axons in r2 and r3 stain only weakly for SC1
(Fig. 5G)
(Chedotal et al., 1995
). These
ectopic axons extended from cell bodies in the ventral r1/r2 boundary region
and grew dorsally towards the ectopic source of Fgf8
(Fig. 5G; n=7/91).
Taken together, these data suggested that ectopic Fgf8 can redirect trochlear axons along ectopic pathways. Specifically, it was the anterior component of their pathfinding that was affected, while dorsoventral extension, which is probably largely a product of repulsive cues from the floor plate, seemed unaffected. In addition, in some instances an ectopic morphological isthmic structure appeared to have been generated and was associated with ectopic motor neurons with axonal SC1 staining characteristic of trochlear rather than trigeminal neurons.
Fgf8 is required for guidance of trochlear motor axons
Our in vitro and in vivo studies strongly suggested a role for Fgf8 in
navigation of trochlear motor axons towards and within the isthmus during the
establishment of their projection within the CNS. To test the idea that Fgf8
was required for trochlear axon projections, we performed a series of
inhibition studies using both a pharmacological inhibitor of FGF receptor
(FGFR) activation and a neutralising antiserum raised against Fgf8. These
studies were undertaken using rat MHB explants in collagen gels and explants
included dorsal tissue, since we wished to assay the effects of the inhibitory
reagents on axon growth across the entire trochlear axon pathway within the
isthmic region (Fig. 6A).
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We examined the effect of inhibition of FGFR activity using SU5402, which
specifically inhibits signalling through all FGF receptors
(Mohammadi et al., 1997). In
control explants, trochlear axons extended through the explant as a closely
associated bundle of fascicles emerging in the dorsal region of the explant
(Fig. 6B) and reproducing the
projection pattern observed in vivo
(Colamarino and Tessier-Lavigne,
1995
). By contrast, inhibition of FGFR activity resulted in fewer
axons and fascicles, but those that were present failed to become organised
into a single closely organised projection within the explant. Rather,
individual axons and fascicles followed diverse pathways through the explant,
although a general dorsal direction was maintained
(Fig. 6C,D). In only a subset
of cases trochlear axons emerged from the explant into the collagen gel (10/20
for SU5402 at 10 µM; 3/12 for SU5402 at 20 µM; 14/25 for SB402451) but
in these instances they exited over a much wider region of the explant border
than in controls.
These data suggested a role for FGFR activation in establishment and
maintenance of the normal trochlear projection, although the severity of the
effects may be indicative of other functions for FGF signalling. Moreover,
there is a body of evidence indicating that FGFRs can be activated not only by
FGF ligands but also by certain members of the CAM and cadherin families of
cell adhesion molecules. We therefore used the same explant assay but in
combination with a neutralising antiserum raised against Fgf8
(Hunter et al., 2001;
Irving and Mason, 2000
) to
demonstrate a requirement for the latter in the formation of the trochlear
projection (Fig. 6E-G).
Explants treated with this antiserum did not show the reduction in axon
fascicle number or length observed with SU5402, however the projection of
axons within the explant was highly abnormal. In some cases, axons initially
began to form a tight bundle projecting dorsally but, in more dorsal regions
of explants, extensive defasciculation occurred with axons following rostral
and caudal trajectories (Fig.
6E). In other cases, axons appeared misrouted from the time of
their initial projections within the ventral-most tissue, with some axons
never entering the main axon bundle (Fig.
6F,G). Instead, they followed random abnormal projections within
the explant with many projecting posteriorly before exiting over a broad
region of the explant. By contrast, control explants cultured in the presence
of an antibody that specifically blocks Fgf4 activity
(Shamim et al., 1999
) did not
exhibit any of the above defects (data not shown). These data indicate a
requirement for Fgf8 in trochlear axon guidance, both in establishment and
maintenance of the projection within the isthmic region.
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DISCUSSION |
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By studying the expression of Isl proteins, some of the earliest molecular
markers for differentiated motor neurons
(Thor et al., 1991), we have
shown that trochlear motor neurons develop within both the
Fgf-8-positive isthmic tissue and anterior r1. Unexpectedly, we found
that Fgf-8 transcripts did not extend as far ventral as the floor
plate and that trochlear motor neurons also developed within this
Fgf-8-negative region.
It has been shown that Fgf8 acts in concert with sonic hedgehog to regulate
the induction of dopaminergic neurons in the posterior midbrain and
serotonergic neurons in anterior hindbrain; the differential competence of
these two regions being dependent upon further unidentified factors
(Ye et al., 1998). It
therefore seems likely that Fgf8 might play a role in the induction
of trochlear neurons and also of the oculomotor nucleus located in the
posterior midbrain. Indeed preliminary data suggests that Fgf8 is able to
induce ectopic Isl-positive motor neurons in posterior r1 and that the axonal
projections of these cells is characteristic of the trochlear nucleus
(Fig. 5G).
The most anterior trochlear motor neurons (i.e. those within the isthmus) extend axons before those located within r1, suggesting that they are more mature and are probably born first. Thus it is unlikely that cells are born within the isthmus and then migrate posteriorly, but rather there is an anterior-posterior wave of induction of trochlear motor neuron differentiation. Indeed, Isl-positive cells become progressively more sparse as distance from the isthmus increases (Fig. 1) consistent with their induction being regulated by a gradient of signal from the isthmus.
Extension of trochlear axons towards and within the isthmus
Consistent with several earlier studies we found that trochlear axons
extended circumferentially in a series of fascicles along a characteristic
trajectory to the dorsal midline. This projection is unique among motor
neurons and is conserved among all vertebrate classes
(Chedotal et al., 1995;
Colamarino and Tessier-Lavigne,
1995
; Fritzsch and Northcutt,
1993
; Fritzsch and Sonntag,
1988
; Matesz,
1990
; Sinclair,
1958
; Szekely and Matesz,
1993
). We investigated the relationship of the trochlear
projection to the isthmic organiser cells. We found that while axons from
anterior cell bodies took a dorsal trajectory i.e. extending within the
Fgf-8-positive territory, axons from more posterior motor neurons
followed a dorso-anterior path towards the isthmus. Upon arrival within the
isthmus, they fasciculated with axons from the anterior cells and projected
dorsally to their exit points. Thus, initial axon projection was established
within the isthmus by axon pioneers from the most anterior cells, with more
posterior cells extending processes only later.
Guidance of trochlear axons: roles of the isthmus and Fgf8
The extension of trochlear axons towards and within the isthmic organiser
region suggested that the latter tissue might be a source of guidance cues for
their growth cones. This was examined in collagen gel co-cultures, previously
used by others to examine chemotropic influences on trochlear axons
(Colamarino and Tessier-Lavigne,
1995; Varela-Echavarria et
al., 1997
). We found that axons extended towards and grew within
isthmic tissue, whereas they were neither attracted towards nor repelled by
tissue taken from the dorsal part of posterior r1.
These data raised the question of what the isthmic chemoattractant cue
might be, and Fgf8 was an obvious candidate. Many studies have shown that FGFs
stimulate axon extension in vitro, both from primary neurons and from cell
lines with neuronal characteristics (for a review, see
Eckenstein, 1994;
Mason, 1994
). However, there
is little data concerning their ability to guide the formation of axonal
pathways in vivo, with perhaps the best studies being those on the formation
of the retinotectal projection in the frog (for a review, see
Dingwell et al., 2000
). In
this system, initial axonogenesis seems to be dependent upon FGFR activation
but via an N-cadherin ligand (Lom et al.,
1998
). By contrast, signalling regulated by an FGF ligand is
required for axon growth (McFarlane et
al., 1995
) and, significantly, for turning towards and entry into
the optic tectum (McFarlane et al.,
1996
). As yet, it is unclear whether the role of FGF signalling is
to promote turning of the growth cone towards the tectum by changing its
response to environmental cues, or whether FGF is acting as a chemoattractant.
However, there is evidence that FGFs have chemotropic potential in other
systems e.g. in migration of neural crest cells and limb myogenic cells in
vitro (Murphy et al., 1994
;
Sieber-Blum and Zhang, 1997
;
Webb et al., 1997
) and in
development of the Drosophila tracheal system
(Affolter and Shilo, 2000
).
Our study suggests that Fgf8 is a chemoattractant for trochlear neurons both in vitro and in vivo. Ectopic Fgf8, delivered from beads attracts trochlear axons in vitro, and redirects their growth towards a bead in vivo. In the most severe cases in vivo, the trochlear nerve became split into two with axons extending both anteriorly to the isthmus and posteriorly towards the Fgf8 bead. In addition, the most anterior pioneer axon bundle occasionally turned and projected posteriorly towards the ectopic Fgf8 source. It is not clear why only the most anterior fascicle behaved in this manner, although it may reflect rapid depletion of the Fgf8 protein or the growth of r1, which is considerable at the developmental stages used and might move the bead distant from the site of implantation. In either case, later-extending axons might be expected to be unaffected by ectopic protein.
It remains possible that changes in trajectory of the trochlear motor nerve observed following bead implants in ovo or inhibition studies in vitro may also reflect additional effects of Fgf8-regulated tropic signals. In addition, deflection of trochlear axons towards either isthmic tissue or a source of Fgf8 while within mhb explants in vitro might be due to either direct chemoattractant effects of Fgf8 or its indirect effects in inducing an unidentified chemotropic cue. However, trochlear axons were also found to reorient towards Fgf8 beads after they had left the explant and were extending within the collagen gel. The simplest interpretation of the latter data is that Fgf8 can itself provide a direct chemotropic influence, although although it remains possible that additional, unidentified guidance cues may be regulated by it within MHB tissue following both in vivo and in vitro manipulations.
We further showed that inhibition of FGFR activity disrupts the formation
of the trochlear projection within explants in vivo. Most significantly, a
specific anti-Fgf8 antiserum causes mis-routing of axons within MHB explants
in a manner consistent with a role for Fgf8 in guiding trochlear axons towards
the isthmus and maintaining their growth within it. Moreover, a recent study
has suggested that higher Fgf8 concentrations are present dorsally in the
isthmus (Carl and Wittbrodt,
1999), raising the possibility that Fgf8 might also contribute to
dorsal guidance of trochlear axons.
Within the isthmic region, trochlear axons axons come together to form
three or four main fascicles that circumnavigate the isthmus. It was notable
that there was considerable axon defasciculation as a result of the
application of both a pharmacological FGFR inhibitor and an anti-Fgf8
neutralising antiserum, suggesting that Fgf8 might also play a role in
inducing or maintaining fasciculation. Indeed, inhibition of FGFR activity
promotes defasciculation in other systems
(Brittis et al., 1996).
However, emergent trochlear axons defasciculate in collagen gel cultures and
Fgf8 protein did not noticeably reduce this behaviour.
Multiple chemotropic cues establish the trochlear projection within
the CNS
Our observations showed that growing trochlear axons initially extended
away from the floor plate in a near-perpendicular direction, presumably
reflecting their response to chemorepellents from that tissue. Trochlear axons
are repelled by both floor plate tissue and netrin 1 in vitro
(Colamarino and Tessier-Lavigne,
1995; Varela-Echavarria et
al., 1997
), although in netrin-deficient mice the trochlear
trajectory is largely normal (Serafini et
al., 1996
). This presumably reflects the presence of other
chemorepellents in the floor plate, such as semaphorins. Sema3A can act as a
chemorepellent for trochlear axons in vitro, although its spatio-temporal
location within the hindbrain may exclude it from fulfilling this role in vivo
(Varela-Echavarria et al.,
1997
). Sema3F has also been demonstrated as a direct
chemorepellent for trochlear axons in vitro, and is expressed in both the
anterior midbrain and posterior r1 these may reflect domains of
repulsion that channel trochlear axons on their course around the isthmus. In
support of this, mice lacking Neuropilin 2, the preferred Sema3F receptor,
show normal positioning of trochlear neuron cell bodies but exhibit a dramatic
loss of trochlear axons projecting into the periphery. Instead, axons follow
random projections within the CNS (Chen et
al., 2000
; Giger et al.,
2000
). Furthermore, Sema3F is also expressed in tissues
surrounding the nervous system, and it has been proposed that, following
dorsal decussation and exit from CNS, the trochlear nerve may be guided to the
eye by the same molecule acting as a repulsive cue
(Giger et al., 2000
). However,
it should be noted that we found no evidence of a diffusible chemorepellant
produced by rat posterior r1 tissue.
Our study developed from the observation that the most anterior trochlear axons followed a simple dorsal trajectory through the isthmic organiser, whereas those located more posteriorly in r1, grew antero-dorsally until they reached the isthmus. We propose that the trochlear projection reflects the sum of repulsive cues (including netrin) from the floor plate, and possibly Sema3F repulsion from posterior r1, and an attractive cue from Fgf8 at the isthmic organiser (Fig. 7).
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ACKNOWLEDGMENTS |
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