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First published online September 12, 2006
doi: 10.1242/10.1242/dev.02564
1 SORST, Japan Science and Technology, Japan.
2 Graduate School of Frontier Biosciences, Osaka University, Yamadaoka 1-3,
Suita, Osaka 565-0871, Japan.
3 MRC Centre for Developmental Neurobiology, King's College London, Guy's
Campus, London SE1 1UL, UK.
* Authors for correspondence (e-mail: yan.zhu{at}fbs.osaka-u.ac.jp; murakami{at}fbs.osaka-u.ac.jp)
Accepted 3 August 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Commissural axons, Ephrin A, Longitudinal polarity, Hindbrain, Chick
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Murakami and Shirasaki,
1997; Stoeckli and Landmesser,
1998; Salinas,
2003; Guthrie,
2004). By contrast, little is known about the mechanisms that
control the pathfinding of commissural axons after midline crossing, in
particular, those that regulate their rostrocaudal polarity along the
longitudinal axis.
Two recent studies have shown that Wnt4 and sonic hedgehog (Shh) control
the rostrocaudal polarity of post-crossing spinal commissural axons in rodent
and chick, respectively, by forming rostrocaudally orientated gradients in and
around the ventral midline (Lyuksyutova et
al., 2003; Bourikas et al.,
2005). Wnt4 and Shh gradients might account for the longitudinal
polarity of most spinal commissural axons, which turn rostrally in close
contact to the contralateral surface of the floor plate. However, the
trajectories of post-crossing commissural axons are less stereotypical than
previously thought. In chick and rodent spinal cord, some post-crossing
commissural axons have also been shown to grow caudally or bifurcate
rostrocaudally, and some turn longitudinally at a distance away from the floor
plate within the ventral spinal cord
(Oppenheim et al., 1988;
Yaginuma and Oppenheim, 1991;
Erskine et al., 1998;
Imondi and Kaprielian, 2001;
Kadison and Kaprielian, 2004).
This diversity is even more pronounced in the developing hindbrain, a wider
structure along the dorsoventral axis than the spinal cord. Hindbrain
commissural axons not only turn within the ventral aspect of the neural tube,
but also at intermediate and dorsal positions, into either rostral or caudal
directions (Glover and Petursdottir,
1991; Clarke and Lumsden,
1993; Glover,
1993; Shirasaki et al.,
1995; Shirasaki and Murakami,
2001). We envisage that multiple molecular mechanisms, possibly
involving components with distinct spatial distributions, account for the
diversity of post-crossing commissural projections in the hindbrain. Thus, the
hindbrain represents a complex model that is well suited for uncovering novel
molecular mechanisms that regulate the guidance of post-crossing commissural
axons.
In the present study, we have focused on commissural axons that turn
longitudinally at a distance from the ventral midline. The axons, which we
identified as coming from the caudal group of the contralateral second order
vestibular neurons (cC-VC) (Diaz and
Puelles, 2003), turn rostrally at a dorsal position in the
hindbrain. Here, we show that caudal hindbrain possesses a graded
non-permissive/repulsive activity for growing cC-VC axons. This activity is
mediated at least in part by ephrin A. We present multiple lines of evidence
that demonstrate ephrin A/EphA signalling plays a crucial role in controlling
the rostrally directed turning of post-crossing cC-VC axons.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Animal studies were carried out in accordance
with the guidelines of the Animal Studies Committee of Osaka University.
Organotypic culture and grafting
Organotypic culture of chick hindbrains was carried out as previously
described (Chédotal et al.,
1997; Zhu et al.,
2003).
In transplantation experiments, grafts from donor and host hindbrains were prepared in ice-cold Gey's Balanced Salt Solutions (GBSS, Sigma) with flame sharpened tungsten needles. The grafts were soaked in GBSS containing 30 µg/ml 3,3'-dioctadecyloxacarbocyanine perchlorate (DiO, Molecular Probe, Invitrogen) for 5 minutes. Both the graft and the host were transferred onto millicell culture inserts (CM, Millipore) and the graft was positioned using a fire-polished pulled glass pipette. The borders of the grafts were discerned by DiO signals and bright-field illumination.
Anterograde- and retrograde-labelling
Small crystals of
1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine
perchlorate (DiI) or
1,1'-dioctadecyl-3,3,3',3'-tetramethylindo-dicarbocyanine,
4-chlorobenzenesulfonate salt (DiD) (Molecular Probes, Invitrogen) were
inserted by a glass micropipette with a broken tip, on cultured hindbrains or
open-book hindbrains fixed in 4% paraformaldehyde (PFA)/0.1 M
phosphate-buffered saline (PBS, pH 7.4). The position of the root of
vestibuloacoustic ganglion (gVIII) was marked by Carmine (Wako), and DiI or
DiD crystals were inserted at a specific distance from the Carmine mark,
guided by a scaled eyepiece graticule. For fixed hindbrains, samples were
stored in 4% PFA for 4 weeks at room temperature. Hindbrains were
organotypically cultured for 1 day in vitro (div) to 2 div after dye
insertion, before fixation. All samples were mounted in Mowiol
(Calbiochem).
PI-PLC, hFc, EphA3-Fc and ephrin A2 functional blocking antibody treatment in vitro
Phosphatidylinositol-specific phospholipase C (PI-PLC, Molecular Probes,
Invitrogen) at a final concentration of 200 mU/ml was added to the culture
medium at the onset of culture, with a fresh supplement of PI-PLC after 1 div.
Human Fc fragment of IgG (Jackson ImmunoResearch) or recombinant mouse
EphA3-Fc Chimera (R&D Systems) were added at a final concentration of 1500
ng/ml and 2000 ng/ml, respectively, to the culture medium at the onset of
culture. A function-blocking antibody against chick ephrin A2 (B3) (a kind
gift from Dr H. Tanaka) (Yamada et al.,
2001) was used at a final concentration of 20-30 µg/ml.
Immunohistochemistry and antibodies
Immunohistochemistry on flat whole-mount hindbrains was performed as
described (Tashiro et al.,
2000), except that 0.04% NiSO4 was added to
diaminobenzidine tetrahydrochloride (DAB; 0.1% in Tris-buffered saline)
solution. Samples were cleared in 90% glycerol for imaging.
Fluorescence immunohistochemistry was performed on 20 µm frozen sections, as described above, with a few modifications: (1) treatment of 0.3% H2O2 was omitted; (2) Alexa594-conjugated streptavidin (Molecular Probe, 1:1000) was used; (3) Triton-X100 was used at 0.2%; and (4) slides were mounted in Mowiol (Calbiochem) with 2.5% 1,4-diazabicyclo-[2,2,2]octane (DABCO, Sigma).
Primary antibodies used were a monoclonal antibody against a chick
neurofilament associated protein (3A10) (from the Developmental Studies
Hybridoma Bank, The University of Iowa) and a monoclonal antibody against
chick ephrin A2 (kind gift from Dr H. Tanaka)
(Yamada et al., 2001).
Secondary antibody used was biotinylated horse anti-mouse IgG (H+L) (1:200,
Vector Laboratories).
Affinity probe in situ binding
Chick ephrin A2-alkaline phosphatase (AP) and chick EphA3-AP were kind
gifts from Dr H. Tanaka. Affinity probe in situ binding on whole-mount chick
hindbrains or frozen sections was carried out largely as previously described
(Cheng et al., 1995) with some
modifications: the blocking buffer was composed of Hanks' Balanced Salt
Solution (HBSS):PBS at 1:1 ratio, with 10% sheep serum. For AP in situ on
frozen sections, 30 µm sections were air dried for 2 hours and fixed in
99.5% ethanol for 30 seconds, followed by 5 washes in PBS. The rest of the
procedure follows the protocol for the whole-mount AP in situ binding.
RNA in situ hybridisation and probes
In situ hybridisation on whole-mount chick hindbrains was performed as
previously described (Henrique et al.,
1995). The following plasmid templates were used to generate
digoxigenin-labelled anti-sense or sense riboprobes. Plasmids containing chick
ephrin A2, ephrin A5 and ephrin A6 were kind gifts from Dr
H. Tanaka (Iwamasa et al.,
1999), Dr U. Drescher (Drescher
et al., 1995) and Dr N. Wada (University of California, Irvine),
respectively. Partial cDNA fragments of chick EphA3, EphA4, EphA5 and
EphA7 were generated by RT-PCR from total RNA prepared from E6 chick
hindbrains. Primers for RT-PCR correspond to nucleotide positions on cDNA:
831-1620 of EphA3; 410-1219 of EphA4; 826-1641 of EphA5; and
865-1507 of EphA7. The cDNA fragments were cloned into pGEM-T Easy
Vector (Promega) for subsequent riboprobe preparation.
In ovo electroporation
In ovo electroporation was carried out essentially as described previously
(Nakamura and Funahashi,
2001). Briefly, stage 20-21 eggs were windowed and extra-embryonic
membranes were removed from the hindbrain area. Plasmid solution (1 µl)
coloured with Fast Green (w/v 0.05%) was injected into the IVth ventricle. Two
silver wire (0.3 mm diameter) electrodes as anode and cathode were placed in
parallel on either side of the hindbrain and five square pulses of 15 V, 50 ms
duration were applied with an Electro Square Porator (ECM830, BTX). The
embryos were cooled immediately with a few drops of cold PBS. Eggs were sealed
for further incubation until E6.
Two avian retroviral constructs used for electroporation are
RCASB(P)-EphA3
C (a kind gift from Dr J. G. Flanagan and Dr M. Nakamoto)
(Nishida et al., 2002;
Feldheim et al., 2004) and
RCASB(P) mock vector [EphA3C insert was deleted from
RCASB(P)-EphA3C by ClaI digestion and the vector re-ligated].
Full-length chick ephrin A2-coding sequences were amplified by RT-PCR and
cloned into an expression vector pCAGGS
(Niwa et al., 1991). Each of
the above three constructs (1 mg/ml in PBS) was co-electroporated with an EGFP
expression vector pCAGGS-EGFP (Hatanaka
and Murakami, 2002) at 2:1 ratio.
Image recording
Fluorescence and bright field images were captured with a charge-coupled
digital (CCD) camera (Axiocam, Zeiss) linked to an upright microscope (BX-60,
Olympus). In some cases, fluorescence images were obtained by a laser-scanning
confocal microscope (MRC 1024ES, BIORAD).
Data quantification and statistics
Quantitative measurement on fluorescence images was performed using
MetaMorph (Version 6.1, Universal Imaging Corporation). Mann-Whitney U-test
was subsequently performed on the processed data.
In the cases of caudal turning error of cC-VC axons after addition of PI-PLC and EphA3-Fc in vitro, it was unfeasible to perform quantitative measures as described above because of the retrogradely labelled cells right below the turning point. These samples were arbitrarily divided into two categories: samples with all axons turning rostrally and samples with axons turning caudally. The datasets fit binomial distribution; thus, Fisher's test was used for statistical analysis.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Developmental course of a cerebellar-projecting lateral commissural tract in chick hindbrain
The developmental course of the lateral commissural tract was first
revealed by DiI anterograde-labelling on fixed hindbrains in open book
configurations at various stages. Small crystals of DiI were inserted into the
dorsal hindbrain 700-1200 µm caudal to the root of vestibuloacoustic
ganglion (gVIII) (schematics in Fig.
1A). At stage 24, DiI labelled a tract that crossed the midline
and grew rostrally within the lateral aspect of the contralateral hindbrain
(Fig. 1B). At stage 26, the
tract extended further rostrally and reached the base of the cerebellar plate
(Fig. 1C). By stage 27, some
axons defasciculated from the longitudinal tract and turned into the
cerebellar primordium (Fig.
1D). The development of this lateral commissural tract could be
recapitulated by DiI anterograde-labelling on organotypically cultured
hindbrains (Fig. 1E-H),
although the labelled tract in cultures appeared to be less fasciculated than
that in vivo. The possibility of following the growth of this tract in vitro
allowed us to investigate the tissues and molecules that influence the
pathfinding of this tract in organotypically cultured hindbrains (see later
sections).
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) (see
Fig. S1 in the supplementary material).
Caudal hindbrain possesses a graded nonpermissive/repulsive activity for rostrally growing cC-VC axons
To investigate the mechanisms that control the rostral turning of cC-VC
axons, we focused on the guidance potential of hindbrain tissue posterior to
the turning point of cC-VC axons. The tissues in this location were grafted to
intersect the rostral path of the cC-VC tract, in organotypically cultured
stage 25-26 hindbrains. Behaviour of cC-VC axons was then observed following
anterograde-labelling of the tract as described in
Fig. 1. When the graft was
taken from about 1.2 mm caudal to gVIII, a position close to but caudal to the
turning point of the cC-VC axons, many axons stalled or turned away before
entering the graft, yet a small percentage of axons continued to grow into and
through the graft (Fig. 2A,D,
n=12). A graft from a further caudal position (about 1.8 mm caudal to
gVIII) caused a more marked effect; almost all cC-VC axons stalled or turned
away without entering the graft (Fig.
2B,D, n=12). By contrast, when a piece of tissue was cut
from the rostral path of cC-VC and put back to its original position, most
labelled cC-VC axons grew through the graft
(Fig. 2C,D, n=10).
These results suggest that caudal hindbrain posterior to the turning point of
cC-VC axons is inhibitory (nonpermissive/repulsive) to cC-VC axons.
Furthermore, the degree of this inhibition is high caudally and low
rostrally.
PI-PLC and EphA3-Fc treatment alleviate the caudal inhibition
Inhibitory axon guidance cues in the CNS have been attributed to a limited
number of receptor-ligand families, namely Eph-ephrin,
Neuropillin/Plexin-Semaphorin, Robo-Slit and Unc5H-Netrin families
(Yu and Bargmann, 2001;
Dickson, 2002). Among these,
both the ephrin A and Semaphorin VII families are GPI-anchored molecules. The
GPI anchor can be enzymatically cleaved by PI-PLC, which in turn result in the
removal of these membrane-attached ligands. We first tested whether PI-PLC
treatment can alleviate the inhibitory activity in the caudal hindbrain, using
the grafting scheme depicted in Fig.
2B. Without adding PI-PLC, almost all axons failed to enter the
graft as shown in Fig. 2B
(Fig. 3A1, n=12).
Addition of PI-PLC to the culture medium alleviated the caudal inhibition, as
indicated by invasion of many cC-VC axons into the graft
(Fig. 3A2, n=20). This
suggests GPI-anchored molecule(s) is involved in mediating the caudal
inhibition.
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;
Wilkinson, 2000;
Wilkinson, 2001). To test the
potential involvement of ephrin As in the caudal inhibition, we used the
soluble recombinant mouse EphA3-Fc to block specifically ephrin A binding to
its receptors (Yates et al.,
2001; Dufour et al.,
2003). When purified human Fc (hFc) region of IgG was added to the
medium, almost all cC-VC axons failed to enter the graft
(Fig. 3B1, n=7). By
contrast, many cC-VC axons grew through the graft when EphA3-Fc (without
clustering) was added to the culture medium
(Fig. 3B2, n=7),
although the effect of EphA3-Fc was slightly weaker than PI-PLC. Inhibition
imposed by the more rostral graft as in
Fig. 2A could also be
alleviated by EphA3-Fc (data not shown). The caudal inhibition could also be
reduced when a function-blocking antibody against chick ephrin A2 (B3)
(Yamada et al., 2001) was
added to culture medium, although the effect is weaker than PI-PLC and
EphA3-Fc (see Fig. S2 in the supplementary material). Taken together, these
results suggest that ephrin A is involved in mediating the inhibitory effect
of the caudal hindbrain.
Distribution of ephrin A ligand and EphA receptor in stage 25 chick hindbrain
The involvement of ephrin A in mediating the inhibitory activity of the
caudal hindbrain, presumably through the EphA receptor, predicted the
expression of these molecules in the caudal hindbrain. To test this, we first
investigated the distribution of ephrin A on stage 25 hindbrains, using
EphA3-AP affinity probe in situ binding, and found a strong ephrin A activity
in the caudal hindbrain (Fig.
4A, n=3). Ephrin A activity was also observed in the
rostral cerebellum, consistent with previous reports
(Karam et al., 2000;
Nishida et al., 2002). In the
caudal hindbrain, ephrin A was enriched in the lateral half of the
neuroepithelium and its expression appeared to be graded - low in rostral and
high in caudal. The rostral extent of the gradient reached 
700 µm
caudal to the gVIII root. Thus, both the position of ephrin A active domain
and its graded nature in the caudal hindbrain correlate with the
characteristics of the inhibition revealed by the grafting experiment
(Fig. 2).
We next examined EphA receptor activity by ephrin A2-AP in situ binding. In the lateral half of the hindbrain, EphA distribution appeared to be roughly complementary to that of the ephrin A activity (Fig. 4B, n=9). It was distributed in a lateral column extending rostrally into the caudal cerebellum, and caudally into r8 with a tapering end. The caudolateral EphA-positive domain in r6, r7 and possibly rostral r8 was well correlated with the position of retrogradely labelled cC-VC neurons at the equivalent stage (Fig. 4B, compare with Fig. S1A,B in the supplementary material).
To confirm that EphA receptors were indeed expressed in cC-VC neurons, we first retrogradely labelled cC-VC neurons on organotypically cultured stage 25 hindbrains (as in Fig. S1A in the supplementary material). Such hindbrains were cryosectioned and those sections containing retrogradely labelled neurons were imaged immediately (Fig. 4C, top panel), followed by ephrin A2-AP in situ binding, to reveal EphA activity (Fig. 4C, middle panel). These two images were then overlaid to compare the localisation of DiI signal with AP signal on the same section (Fig. 4C, bottom panel). DiI-positive cC-VC neurons were located in the EphA-positive area around the lateral mantle zone (Fig. 4C, arrowhead). Furthermore, cC-VC commissure was positive for EphA signal (Fig. 4C, white and black arrows). Thus, EphA is expressed in the cC-VC axons in a manner that it can interact with the ephrin A in the caudal hindbrain.
|
;
Drescher et al., 1995). In the
caudal hindbrain, ephrin A2 was distributed posterior to the otic vesicle, in
a rostral-low/caudal-high gradient (Fig.
5C). On transverse sections, ephrin A2 immunoreactivity was
detected in the lateral half of the hindbrain with its signal stronger in
caudal sections (Fig. 5D). From
these data, we conclude that ephrin A2, EphA5 and EphA3 are the main
components that account for the ephrin A/EphA activities detected by the
AP-fusion proteins in the developing cC-VC system.
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Perturbing ephrin A/EphA signalling lead to inappropriate caudal turning of some cC-VC axons
Our findings that ephrin A contributes to the laterally located inhibitory
activity for cC-VC axons in the caudal hindbrain raise the possibility that
ephrin A is involved in the rostral turning polarity of cC-VC axons. We tested
out this possibility by perturbing ephrin A/EphA signalling both in vitro and
in ovo.
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C) (Nishida et al.,
2002; Feldheim et al.,
2004). Overexpression of exogenous EphA3C in the developing
chick cerebellum causes a dramatic reduction of endogenous ephrin A ligand
activity in this tissue, possibly owing to the masking of the endogenous
ephrin A by the ectopic EphA3C
(Nishida et al., 2002). We
therefore introduced RCASB-EphA3C by in ovo electroporation
unilaterally into the caudal hindbrains of stage 20-21 chick embryos. EGFP was
co-electroporated to discern the electroporated domain. We first confirmed by
EphA3-AP in situ binding that a marked reduction of ephrin A activity was
caused by ectopic expression of EphA3C (n=8/8)
(Fig. 8J), but not by
electroporation of RCASB mock vector (n=4/4) (data not shown). We
then analysed cC-VC axon trajectories of the electroporated hindbrains at
stage 27-28 by DiI anterograde labelling. Samples with electroporated domains
spanning the cC-VC turning point were selected for further analysis. When an
RCASB mock vector was electroporated, cC-VC axons turned rostrally in the
majority of samples (n=41/55)
(Fig. 8A-D). However, when
RCASB-EphA3C was electroporated, caudal-turning axons were detected in
the majority of samples (n=33/49)
(Fig. 8E-I). DiI-labelling at
stage 27-28 appeared to result in fewer or no retrogradely labelled cells,
allowing us to perform quantitative analysis by measuring the fluorescence
intensity of caudal- and rostral-turning axons using Metamorph. The degree of
inappropriate caudal turning was represented by the ratio of caudal versus
rostral turning axons and is summarised in
Fig. 8K. The samples with
EphA3C showed both higher incidence and higher percentage of
inappropriate turning axons, with the caudal turning axons being on average
27% of those that turned rostrally.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The function of ephrin A gradient in the caudal hindbrain
The developing cC-VC axons extend circumferentially at the border of the
ventricular and mantle zone. Upon reaching the contralateral lateral
hindbrain, cC-VC axons course towards the pial surface and execute rostral
turning into longitudinal axis (Fig.
4C; see Fig. S1B in the supplementary material). The rostral
extent of an ephrin A gradient, demonstrated by EphA3-AP in situ binding and
ephrin A2 immunohistochemistry, reaches the region within which the cC-VC
axons turn rostrally. This raises the possibility that ephrin A controls the
rostral turning polarity of cC-VC axons by its gradient, rather than forming a
caudal inhibitory barrier. Two observations support this possibility: (1)
cC-VC axons were only partially inhibited when a piece of caudal hindbrain
located posterior and close to their turning point was transplanted to
intersect the axonal path (Fig.
2A); and (2) in ovo introduction of RCASB-EphA3C to a
region caudal to but not overlapping the cC-VC axon turning point did not
disrupt the rostral turning of cC-VC axons (data not shown). Therefore, we
envisage that cC-VC axons, upon entering their presumptive turning zone,
encounter a rostral-low/caudal-high ephrin A gradient across the rostrocaudal
span of their growth cones, which is in turn translated into a preference for
turning rostrally. The importance of an ephrin A gradient has been
demonstrated in the establishment of topographic projection of several CNS
circuits, including the projection of retinal ganglionic axons to various
cortical and subcortical targets, the hippocamposeptal system and the
thalamocortical projection (Vanderhaeghen
et al., 2000; Yue et al.,
2002; McLaughlin et al.,
2003; Garel and Rubenstein,
2004). The role of ephrin A in topographic projection is generally
thought to rely on its ability to cause growth cone collapse and retraction of
EphA-expressing axons in a concentration-dependent manner. Yet whether ephrin
A can guide axons by inducing growth cone turning has remained unclear. Here,
we showed in vivo that ectopically expressed ephrin A2 could induce turning as
well as stalling of cC-VC axons. Our finding gains support from recent in
vitro studies that showed that, in addition to growth cone collapse, ephrin A5
bound to beads or in the form of a gradient could induce the turning of
Xenopus and chick retinal axons
(Weinl et al., 2003;
Weinl et al., 2005). At
present, it remains unknown which condition preferentially induces turning
versus collapse in our system. Nevertheless, these data render support to our
model that EphA-expressing axons could be induced to change their growth
polarity upon growing into a gradient of ephrin A.
It is conceivable that the caudal ephrin A gradient also controls the
rostrocaudal polarity of other axon tracts that grow longitudinally through
the lateral hindbrain, especially those destined for dorsal structures, such
as the cerebellum. One candidate is the inferior olivary axons that extend
from the caudal hindbrain towards the cerebellum through the lateral hindbrain
(Zhu et al., 2003). The high
level of ephrin A in the caudal extreme hindbrain could also function as an
inhibitory barrier that obliges the commissural axons emanating from the
caudal end of the hindbrain to turn at an intermediate position (Y.Z. and
F.M., unpublished). A similar role has been proposed for ephrin-B, in the
control of commissural axons in the chick and mouse spinal cord
(Imondi and Kaprielian, 2001).
Finally, the trajectory of gVIII descending afferent, which grows within the
ephrin A gradient and stops at the caudal extreme of the hindbrain, raises the
possibility that ephrin A gradient might control the topography of the gVIII
afferents to the secondary vestibular neurons, as well as defining the caudal
limit of its growth.
Rostrocaudal turning polarity of post-crossing commissural axons
Post-crossing commissural axons make rostrocaudal turning errors when their
pre-contact with the floor plate was perturbed genetically, surgically or
pharmacologically (Stoeckli and
Landmesser, 1995; Matise et
al., 1999; Zou et al.,
2000; Shirasaki and Murakami,
2001). However, the molecular cues that directly instruct the
rostrocaudal turning of post-crossing commissural axons have remained elusive
until recently when Wnt4 and Shh gradients adjacent to the floor plate were
found to direct the rostral turning of spinal commissural axons in mice and
chick, respectively (Lyuksyutova et al.,
2003; Bourikas et al.,
2005). In both cases, the spinal commissural axons turn rostrally
immediately after crossing the floor plate. However, the developing hindbrain
as a relay station between the spinal cord and the higher brain structures,
hosts a higher diversity of commissural tracts, many of which turn
longitudinally at a distance further from the floor plate, within the lateral
aspect of the hindbrain. We postulated that molecular cues distributed in the
lateral hindbrain exist to control the longitudinal turning polarity of these
lateral axon tracts. Our results have demonstrated a rostral-low/caudal-high
gradient of ephrin A in the caudolateral hindbrain and that ephrin A/EphA
signalling is important to ensure the rostral turning polarity of a lateral
hindbrain commissural tract.
Several observations point to the possibility that additional factors might
function in concert with ephrin A/EphA in the control of the rostral turning
of cC-VC. First, the effect of EphA3-Fc appears to be weaker than PI-PLC
treatment in alleviating the caudal inhibition
(Fig. 3). Second, cC-VC axons
might be a heterogeneous population in their sensitivity to ephrin A, as
evidenced by the small percentage of cC-VC axons entering the ectopic ephrin
A2 domain (Fig. 6). Yet all
cC-VC axons turn rostrally, implying mechanisms other than ephrin A might
control the rostral turning of these ephrin A insensitive cC-VC axons. Third,
the average percentage of axons turning caudally upon disruption of ephrin
A/EphA signalling was only around 27% of the rostral-turning axons. Shh is not
a very likely candidate to control the rostrocaudal turning of laterally
located commissural axons, as its activity is largely confined to the ventral
region of the developing neural tube
(Marti et al., 1995;
Briscoe and Ericson, 2001).
Wnt4 and Wnt3a have been previously shown to express in a
caudal-high/rostral-low manner in the lateral hindbrains of developing chick
(Hollyday et al., 1995). If
they were to control the rostral turning of lateral commissural axons, they
would have to function as chemorepellents, rather than having the
chemoattractive role demonstrated for Wnt4 in the rostral turning of spinal
commissural axons. Indeed, a recent study has shown that the caudally directed
growth of corticospinal axons is controlled by the repulsive gradients of Wnt1
and Wnt5a, acting via the Ryk receptors
(Liu et al., 2005). Thus, Wnt
proteins might act either as repellents or attractants, depending on whether
they act through Ryk or Frizzled receptors, respectively. Furthermore, Sema3C
also appears to be distributed high in the caudal hindbrain at st27
(Chilton and Guthrie, 2003).
The role of these molecules in controlling rostrocaudal turning of commissural
axons awaits further investigation.
Conclusion
Taking advantage of the relatively large size of the developing hindbrain,
we used transplantation on organotypically cultured hindbrains to dissect out
the guidance properties of caudal lateral hindbrain at a gross level, with
respect to a developing lateral hindbrain commissural tract, cC-VC. This led
us to identify an ephrin A gradient in the caudal lateral hindbrain that plays
a role in the control of rostrocaudal turning polarity of cC-VC. The diverse
trajectories of hindbrain commissural axons may require a combination of
different guidance cues that intricately pave the hindbrain to set the routes
for these tracts. In addition, the high diversity could be achieved through
each axon tract displaying different responsiveness to the same guidance
cue(s).
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/133/19/3837/DC1
|
ACKNOWLEDGMENTS |
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C construct. We also thank Drs
H. Kobayashi and K. Nishida for critical reading of the manuscript. This work
was supported by SORST, Japan Science and Technology Corporation, Inoue
Foundation, a Grant-in-Aid from MEXT and Development travel fellowship. |
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