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First published online 30 May 2007
doi: 10.1242/dev.001958
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1 Laboratory for Neurobiology of Synapse, RIKEN Brain Science Institute, 2-1
Hirosawa, Wako-shi, Saitama 351-0198, Japan.
2 Core Research for Evolutional Science and Technology, Japan Science and
Technology Agency, Osaka 560-0082, Japan.
3 Department of Molecular and Cellular Biology, Harvard University, 16 Divinity
Avenue, Cambridge, MA 02138, USA.
* Author for correspondence (e-mail: miyasaka{at}brain.riken.jp)
Accepted 12 April 2007
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Stromal cell-derived factor-1 (SDF-1), GPCR, Cell migration, Axon guidance, Pioneer axons, Zebrafish
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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; Guan and Rao,
2003), suggesting that these two developmental processes use
common guidance mechanisms, at least in part.
The olfactory system is an excellent model for studying both cell migration
and axon guidance. Olfactory sensory neurons (OSNs) develop within the
olfactory placode, a thickened ectoderm that later gives rise to the olfactory
epithelium (OE). Subsequently, OSNs extend axons toward the developing
olfactory bulb (OB) with exquisite precision. In zebrafish, a previous fate
map study has revealed that a large cellular field located along the lateral
edge of the anterior neural plate converges through cell movements to form the
olfactory placode (Whitlock and
Westerfield, 2000). However, the factors responsible for directing
the assembly of the olfactory placode have not yet been identified.
Chemokines are small, secreted proteins that were originally identified as
molecules regulating leukocyte trafficking in the immune system. Recently, it
has become clear that chemokines and their receptors play prominent roles, not
only in the immune response, but also in various cellular events during
development of the nervous system (Tran
and Miller, 2003). In particular, Cxcl12 (also known as SDF-1,
stromal cell-derived factor-1) influences the guidance of both migrating
neurons (Bagri et al., 2002;
Zhu et al., 2002;
Stumm et al., 2003;
Belmadani et al., 2005;
Knaut et al., 2005;
Borrell and Marin, 2006) and
growing axons (Xiang et al.,
2002; Chalasani et al.,
2003; Li et al.,
2005; Lieberam et al.,
2005) through its receptor Cxcr4. As the olfactory placode is one
of the regions where Cxcr4 is expressed during embryogenesis
(Chong et al., 2001;
Tissir et al., 2004;
Schwarting et al., 2006), it
is conceivable that Cxcl12/Cxcr4 signaling may play a role in olfactory
development. Here, we provide genetic evidence demonstrating that Cxcl12/Cxcr4
signaling mediates the assembly of olfactory placodal precursors into a
compact cluster to form the olfactory placode in zebrafish. Furthermore, the
loss of Cxcl12/Cxcr4 signaling results in impaired pathfinding of pioneer
olfactory axons and failure of OSN axons to project to the OB. Our results
indicate that chemokine signaling plays a dual role in controlling cell
positioning and subsequent axon pathfinding during early development of the
primary olfactory system in zebrafish.
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MATERIALS AND METHODS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). Embryos were staged according to hours postfertilization
(hpf) at 28.5°C and morphological criteria
(Kimmel et al., 1995).
Collected embryos were maintained in 1/3 Ringer's solution (39 mM NaCl, 0.97
mM KCl, 1.8 mM CaCl2, 1.7 mM HEPES, pH 7.2) supplemented with 100
units/ml penicillin and 100 µg/ml streptomycin. In some cases, 0.002%
phenylthiourea (PTU) was added after 12 hpf to prevent pigmentation. We
confirmed that PTU treatment at a dose of 0.002% does not affect olfactory
placode assembly and OSN axon projection, although the treated embryos show a
slight delay of hatching, as observed in previous work
(Elsalini and Rohr, 2003).
Fish lines
A transgenic line, Tg(OMP2k:gap-YFP)rw032a
(abbreviated as omp:yfp), in which membrane-tethered YFP is expressed under
the control of the olfactory marker protein (OMP) promoter, were used to
visualize olfactory neurons, including unipolar pioneer neurons and ciliated
OSNs (Miyasaka et al., 2005;
Sato et al., 2005).
odysseus (ody) homozygous adults were crossed with
heterozygous adults to obtain 
50% ody/ody embryos and
50% ody/+ embryos for synchronized development.
ody/ody embryos were identified by the impaired migration of
germ cells (Doitsidou et al.,
2002; Knaut et al.,
2003) and neuromasts (David et
al., 2002), as assessed by immunofluorescence staining for Vasa (a
marker for germ cells) and PCAM (a marker for neuromasts and OSNs; also known
as Ncam3 - ZFIN), respectively. In some cases, ody mutant embryos
were genotyped by PCR amplification of DNA fragments containing the
ody mutant locus, followed by direct sequencing of the PCR products.
Primers used are as follows: ody-f, 5'-TGGAGTTTGGCTTCCAGCGAC-3';
ody-r, 5'-CAGCATAGTCAAAGCGTCCAC-3'. A transgenic line, hsp:cxcl12a
(previously called heatshock-SDF-1a)
(Knaut et al., 2005) was used
for ubiquitous heat-induced misexpression of Cxcl12a.
Whole-mount in situ hybridization
cDNA fragments for probes against cxcl12a, cxcl12b, cxcr4a, cxcr4b,
cxcr7a and cxcr7b were amplified by PCR from cDNA libraries of
zebrafish embryos or adult heads. Zebrafish cxcr7 orthologs to the
human and mouse Cxcr7 gene were identified by TBLASTN searches using
the sixth draft zebrafish genome assembly (Zv6: Sanger Center). The sequences
used as probes against cxcr7a and cxcr7b were deposited in
GenBank with the accession numbers EF467374 and EF467375, respectively.
Digoxigenin (DIG)-labeled cRNA probes were generated by in vitro transcription
from their cDNA clones. Whole-mount in situ hybridization was performed as
previously described (Miyasaka et al.,
2005).
Immunohistochemistry
Whole-mount immunohistochemistry was carried out as previously described
(Miyasaka et al., 2005), with
the following modifications. Embryos were fixed overnight at 4°C in 4%
paraformaldehyde in PBS and permeabilized in acetone for 7 minutes at
-20°C.
Antibodies used are as follows: rabbit polyclonal anti-GFP antibody
(1:1000, a kind gift from Dr N. Tamamaki, Kumamoto University, Kumamoto,
Japan); rat monoclonal anti-GFP antibody (1:1000, Nacalai Tesque); mouse
monoclonal zns-2 antibody (1:20, supernatant, Developmental Studies Hybridoma
Bank); rabbit polyclonal anti-PCAM antibody (rabbit IgG; 0.4 µg/ml)
(Mizuno et al., 2001;
Miyasaka et al., 2005); rabbit
polyclonal anti-Vasa antibody (1:2000)
(Knaut et al., 2000); rabbit
polyclonal anti-DsRed antibody (1:300, Clontech); Cy3-conjugated donkey
anti-mouse IgG antibody (1:300, Jackson ImmunoResearch); Cy3-conjugated goat
anti-rabbit IgG antibody (1:300, Jackson ImmunoResearch); Alexa 488-conjugated
goat anti-rabbit IgG antibody (1:300, Molecular Probes); Alexa 488-conjugated
goat anti-rat IgG antibody (1:300, Molecular Probes); peroxidase-conjugated
goat anti-rabbit IgG Fab antibody fragment [Histofine Simple Stain Max PO(R),
Nichirei Bioscience].
Morpholino injection
Antisense morpholino oligonucleotides (MOs: Gene Tools, LLC) were dissolved
at a concentration of 4 ng/nl in 1x Danieau buffer [58 mM NaCl, 0.7 mM
KCl, 0.4 mM MgSO4, 0.6 mM Ca(NO3)2, 5 mM
HEPES, pH 7.6] containing 0.1% Phenol Red, and injected into one-cell stage
embryos carrying the omp:yfp transgene at a volume of 1 nl with an
air pressure microinjector (FemtoJet, Eppendorf). The volume of MO solution to
be injected was calibrated by measuring the diameter of a droplet at the tip
of glass needle in the air. MO sequences are as follows: cxcr4b MO,
5'-AAA TGA TGC TAT CGT AAA ATT CCA T-3'
(Doitsidou et al., 2002);
cxcl12a MO, 5'-ACT TTG AGA TCC ATG TTT GCA GTG-3'
(Li et al., 2005);
cxcl12b MO, 5'-CGC TAC TAC TTT GCT ATC CAT GCC A-3'
(Knaut et al., 2003).
Cxcl12a misexpression
For ubiquitous misexpression, hsp:cxcl12a/+ fish were crossed with
omp:yfp/+ fish. Embryos obtained from such a cross were heat shocked
at either 12 hpf or 19 hpf for 1 hour in a 37°C water bath, raised at
28.5°C, and imaged at 26-29 hpf and 3.5 days postfertilization (dpf) using
a confocal laser-scanning microscope (Olympus FV500). To identify hsp:cxcl12a
transgenic embryos, genomic DNAs were extracted from the embryos after image
acquisition, and genotyped by PCR using the following primers: 5'-CAT
GTG GAC TGC CTA TGT TCA TC-3'; 5'-ATC AGA GCG ACT ACT ACG ATC
AC-3'.
For mosaic misexpression, omp:yfp/+ embryos were injected at the
one- to two-cell stage with a 50 µg/ml solution of phsp:mDsRed or
phsp:cxcl12a-mDsRed DNA, and heat shocked at 12 hpf and again at 19 hpf. The
second heat-shock treatment was given to facilitate identification of cells
expressing mDsRed or Cxcl12a-mDsRed. The embryos were fixed at 24 hpf and
analyzed by immunohistochemistry using anti-GFP and anti-DsRed antibodies. The
DNA constructs were prepared as follows. To generate phsp:mDsRed,
EGFP cDNA of the pHSP70/4-EGFP
(Halloran et al., 2000) was
removed by AgeI and NotI digestion and replaced with
DsRed-Monomer cDNA from the pDsRed-Monomer (Clontech). To generate
phsp:cxcl12a-mDsRed, the stop codon of cxcl12a was removed by PCR and
the cxcl12a gene was inserted between the SalI and
AgeI sites of the phsp:mDsRed so that the DsRed-Monomer was
in frame with the last codon of the cxcl12a gene.
Time-lapse imaging
Embryos younger than 2 dpf were anesthetized in 0.016% tricaine and mounted
in 1.5% low-melting point agarose. Embryos older than 2.5 dpf were
anesthetized, inserted into a silicone tube (1 mm inner diameter, 2.5 mm outer
diameter, 5 mm length) to allow the embryos to stand, and mounted anterior
side down on a glass-bottomed petri dish with a small amount of 1/3 Ringer's
solution. The embryos were imaged at each time point using a confocal
microscope. The embryos were removed from the agarose or silicon tube and kept
at 28.5°C between the time points for observations.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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), suggesting that this receptor might play a role in the
development of the olfactory system. To corroborate this possibility, we
analyzed the spatiotemporal expression of cxcr4b in the head region
in more detail. At 12 hpf, cxcr4b was expressed in a row of cells
along the anterolateral edge of the neural plate
(Fig. 1A) that corresponds to
the presumptive olfactory placodal field
(Whitlock and Westerfield,
2000). During the next few hours, cxcr4b-expressing cells
increased in number and arranged at the edge of the developing anterior neural
tube, forming bilateral bands along the anteroposterior axis
(Fig. 1C,E). By 20 hpf, most of
the cxcr4b-expressing cells had assembled into the olfactory placode
(Fig. 1G,I). From 24 to 30 hpf,
when the initial olfactory axons were extending toward the developing OB,
cxcr4b expression in the olfactory placode was still evident
(Fig. 1K,M,O), but cells
adjacent to the apical surface, where mature OSNs reside, were devoid of
cxcr4b expression (Fig.
1O). From 36 hpf onward, cxcr4b expression in the
olfactory placode was greatly diminished, and only a few cells expressing
cxcr4b remained in the basal part of the OE at 48 hpf
(Fig. 1Q,S). In the zebrafish genome, there are two genes (cxcl12a and cxcl12b) that encode potential ligands for Cxcr4b. Analyzing the expression patterns of the two Cxcr4b ligands, we found that only cxcl12a (right panels in Fig. 1), but not cxcl12b (see Fig. S1D-F in the supplementary material), was present in close proximity to the cxcr4b-expressing olfactory placode. The developing anterior neural tube was located between the two cxcr4b-expressing stripes and started to express cxcl12a by 14 hpf (Fig. 1D). Over the next ten hours, the cxcl12a expression domain gradually became restricted to the anterior tip of the telencephalon (Fig. 1F,H,J,L) and the two cxcr4b-expressing stripes closely followed this refining cxcl12a expression and, by convergence movements, formed the two olfactory placodes (Fig. 1E,G,I,K). At 24 to 36 hpf, in addition to the telencephalic expression, cxcl12a was expressed by cells residing at the basal margin of the olfactory placode, on the border between the placode and the telencephalon where olfactory axons cross to enter the telencephalon (Fig. 1L,P,R). At 48 hpf, the expression of cxcl12a in the telencephalon was less intense but still visible, while the expression in the margin of the OE was barely detectable (Fig. 1T). Thus, the expression of cxcl12a in close proximity to cxcr4b-expressing cells that will form the olfactory placodes is consistent with the idea that chemokine signaling might play a role in olfactory placode assembly and OSN axon pathfinding.
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ody/ody or ody/+ embryos were examined for
cxcr4b expression during the period of olfactory placode formation.
At 12 and 14 hpf, the arrangement of cells expressing cxcr4b in
ody/ody embryos was indistinguishable from that in
ody/+ embryos (Fig.
2A,B,F,G). At 16 hpf, some cxcr4b-expressing cells in
ody/ody embryos were not arranged along the lateral edge of
the developing anterior neural tube and dispersed laterally or anteromedially
(Fig. 2H). At 18 hpf, a
substantial number of cxcr4b-expressing cells were mispositioned
ventromedially (Fig. 2I). At 20
hpf, when the olfactory placode had formed, the ventromedially displaced cells
were still detectable at ectopic positions, although reduced in number
(Fig. 2J). Total number of
cxcr4b-expressing cells in the anterior head region of
ody/ody embryos did not differ from that of ody/+
embryos during the period of olfactory placode assembly
[ody/ody versus ody/+, 51±4 versus
51±2 cells/side (16 hpf), 85±2 versus 83±2 (18 hpf),
98±5 versus 103±3 (20 hpf), n=6 for each,
mean±s.e.m.], suggesting that the loss of Cxcr4b-mediated signaling
causes mispositioning of olfactory placodal precursors without affecting their
proliferation.
To examine whether the mispositioned cells in ody/ody
embryos develop into olfactory neurons, we used the omp:yfp transgenic line in
which membrane-tethered EYFP starts to be expressed at 20 hpf in a
subpopulation of olfactory neurons, including unipolar pioneer neurons and
ciliated OSNs (Miyasaka et al.,
2005; Sato et al.,
2005). In ody/ody embryos carrying the
omp:yfp transgene, YFP-positive cells were less tightly organized
than those in wild-type embryos and often dispersed ventromedially from the
normal position of the olfactory placode at 24 hpf
(Fig. 3A-C). Similarly,
reducing the expression level of Cxcr4b by MO-mediated knockdown frequently
led to a mispositioning of YFP-positive cells at 24 hpf
(Fig. 3D), mimicking the
phenotype observed in ody mutants. The fact that mispositioned cells
express YFP under the control of the omp promoter suggests that the
specification of olfactory neurons is independent of Cxcr4b signaling. To
trace the subsequent fate of these ectopically located olfactory neurons, we
analyzed ody mutants until 3.5 dpf. The ectopically positioned
YFP-positive olfactory neurons observed at 1 dpf were rapidly reduced in
number during the second day of development, and they had completely
disappeared by 3.5 dpf (Fig.
9E-H).
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Due to phenotypic variability in embryos with compromised cxcr4b-mediated signaling (Fig. 3), we quantitated to what degree olfactory neurons are mispositioned at 24 hpf by measuring the distances from the midline to the medialmost and lateralmost cells (medial and lateral limits), and the distances from the ventral edge of the head to the ventralmost and dorsalmost cells (ventral and dorsal limits) (Fig. 4). In wild-type embryos, virtually no olfactory placode failed to assemble, although two out of 30 placodes showed a ventromedial displacement of a single olfactory neuron (Fig. 4A,G). In ody/+ embryos (Fig. 4B,H) and cxcl12b morphants (Fig. 4F,L), essentially no olfactory placode displayed mispositioning of olfactory neurons. By contrast, a large proportion of placodes in ody/ody embryos (Fig. 4C,I), cxcr4b morphants (Fig. 4D,J) and cxcl12a morphants (Fig. 4E,K) exhibited ventromedially displaced olfactory neurons; the averages of ventral limit (ody/ody, 47±4 µm, n=34; cxcr4b MO, 55±4 µm, n=30; cxcl12a MO, 40±5 µm, n=20; mean±s.e.m.) and medial limit (ody/ody, 40±3 µm, n=34; cxcr4b MO, 40±3 µm, n=30; cxcl12a MO, 29±4 µm, n=20) were significantly shifted ventrally and medially, respectively, when compared with wild-type placodes (ventral limit, 68±3 µm; medial limit, 51±2 µm; n=30). In ody/ody embryos, the average of lateral limit was slightly shifted laterally when compared with wild type (ody/ody, 97±2 µm, n=34; wild type, 90±1 µm, n=30), but neither cxcr4b nor cxcl12a morphants showed such a shift. Thus, ventromedial displacement of olfactory neurons is a common phenotype when reducing or removing Cxcl12a/Cxcr4b signaling. These results indicate that Cxcl12a signaling through Cxcr4b is required for assembly of the olfactory neuron precursors into a compact cluster.
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; Burns et al.,
2006). We searched the zebrafish genome database and identified
two genes (cxcr7a and cxcr7b) equally homologous to the
mammalian cxcr7. In situ hybridization analyses revealed that neither
cxcr7a nor cxcr7b was expressed in the developing olfactory
placode (see Fig. S1M-R in the supplementary material). These results indicate
that Cxcr4b is the only known Cxcl12a receptor expressed in the olfactory
neuron precursors, suggesting that olfactory neuron precursors can sense
Cxcl12a only through Cxcr4b during placode assembly.
Misexpression of Cxcl12a perturbs the olfactory placode assembly
The Cxcl12a expression domain at the anterior neural tube becomes
restricted to the anterior tip of the telencephalon simultaneously with the
assembly of the olfactory placode (Fig.
1), suggesting that Cxcl12a might act as a directional cue to
guide migration of olfactory placodal precursors. To test this possibility, we
first sought to perturb the local source of Cxcl12a by raising the Cxcl12a
levels throughout the embryo. We heat shocked hsp:cxcl12a;omp:yfp
double-transgenic embryos before (12 hpf) and after (19 hpf) olfactory placode
assembly, and analyzed the positioning of YFP-positive olfactory neurons at
26-29 hpf. Embryos in which Cxcl12a was heat induced at 12 hpf (six of 11
embryos) showed impaired assembly of olfactory neurons
(Fig. 5B), which resembled the
Cxcl12a/Cxcr4b morphant and ody mutant phenotypes
(Fig. 3). Heat induction of
Cxcl12a at 19 hpf (n=12) did not affect the placode assembly
(Fig. 5C). Similarly, heat
shocking omp:yfp single transgenic embryos did not cause any defects in
olfactory neuron assembly (Fig.
5A). These results suggest that a spatially and temporally
localized Cxcl12a source is crucial for correct positioning of olfactory
neurons.
Next, to determine whether Cxcl12a attracts migrating olfactory neuron precursors, we analyzed the effects of localized Cxcl12a misexpression. We injected omp:yfp transgenic embryos with a DNA construct containing a monomeric DsRed (mDsRed)-tagged Cxcl12a under the control of hsp70 promoter, creating ectopic sources of Cxcl12a after heat shock. In embryos that showed mosaic Cxcl12a-mDsRed expression in the anterior head, we frequently observed abnormal appearance of YFP-labeled olfactory neurons ventral to the correct position (n=14 of 16 embryos). In such cases, some olfactory neurons were found in close proximity to the ventral ectopic Cxcl12a sources (Fig. 6B-D). By contrast, we never found dorsally displaced olfactory neurons, despite the presence of ectopic sources in the dorsal region (open arrowheads in Fig. 6B). In control embryos with mosaic mDsRed expression in the anterior head (n=15), olfactory placode assembly was never perturbed (Fig. 6A). These results indicate that ectopically expressed Cxcl12a in the ventral region can compete with the endogenous Cxcl12a and recruit olfactory neurons ventrally.
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). Using the
PCAM antibody, a marker that labels axons from both ciliated and microvillous
OSNs (Miyasaka et al., 2005),
we examined axon trajectories of OSNs in ody mutants at 3 dpf, when
prototypic glomerular targeting by OSN axons has been established. In
ody mutants, we frequently observed an impaired OSN axon projection
(Fig. 7). Due to phenotypic
variability, we divided ody mutants into three classes: 28% of the
ody mutants (n=18 embryos) showed bilateral OSN axon
projection defects (class 3); 28% showed projection defects only on one side
(class 2); and 44% showed normal axon projection to the OB (class 1). In
ody mutants with projection defects, OSN axons failed to exit the OE
or occasionally stalled immediately after exiting the OE, accumulating near
the OE-telencephalon border (Fig.
7C,D). Intriguingly, embryos with projection defects on one side
showed no detectable proto-glomerular innervation defects on the contralateral
side (Fig. 7C). OSN axon
projection defects were also seen when a subpopulation of OSN axons were
labeled by membrane-tethered YFP in ody mutants carrying the
omp:yfp transgene (Fig.
9H). Reducing the activity of Cxcr4b by MO injections yielded
similar defects in OSN axon projection, although the incidence of the defects
was lower when compared with that of ody mutants (4 and 41% of OE,
respectively; Table 1). Such a
discrepancy in phenotypic incidence between mutants and morphants was not
observed in the defects of the olfactory placode assembly (see
Fig. 4). cxcl12a
morphants also exhibited similar defects in OSN axon projection at a
comparable incidence with cxcr4b morphants (3 and 4% of OE,
respectively; Table 1), whereas
cxcl12b morphants never displayed OSN axon projection defects
(n=134 OE). The lower incidence of defects in
cxcl12a/cxcr4b morphants than ody mutants that lack
Cxcr4b function is probably explained by an incomplete removal of
Cxcl12a/Cxcr4b signaling after MO injections. These results indicate that
Cxcl12a/Cxcr4b signaling is important to allow OSN axons to exit the OE and to
extend toward the OB, and suggest that the OSN axon projection would be
ensured by less activity of Cxcl12a/Cxcr4b than that required for the
olfactory placode assembly.
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), suggesting that the axons of unipolar neurons may provide a
track for OSN axons to follow. Therefore, it is possible that Cxcr4b-mediated
signaling helps axons of unipolar neurons to pioneer to the presumptive OB. To
explore this possibility, we investigated the trajectory of pioneer axons of
1.5 dpf ody mutant embryos expressing YFP in both unipolar neurons
and ciliated OSNs. Axons of unipolar neurons were identified by co-labeling
with zns-2 antibody (Whitlock and
Westerfield, 1998). We found that most of the YFP-positive axons
emerging from the olfactory placode at 1.5 dpf were also immunoreactive for
zns-2, and that projection of double-labeled pioneer axons were frequently
impaired in ody mutant embryos
(Fig. 8). The defects in
pioneer axon projection from ody mutant olfactory placodes did not
occur in an all-or-none manner, differently from PCAM-positive OSN axon
projection at 3 dpf (see Fig.
7). In 19% of the ody mutant placodes, all pioneer axons
failed to exit the placode and accumulated near the placode-telencephalon
border (n=16 placodes; Fig.
8A-C); in 19% some pioneer axons reached the presumptive OB and
the remaining axons failed to exit the placode
(Fig. 8D-F), and in 62% most
pioneer axons showed a normal projection to the presumptive OB
(Fig. 8G-I). ody/+
(see Fig. 9B) and wild-type
(Fig. 8J-L) embryos never
exhibited such pioneer axon projection defects. To assess whether the variable projection patterns of pioneer axons from unipolar neurons are associated with projection patterns of following OSN axons, we carried out time-lapse observation of the YFP-expressing axons from both unipolar neurons and ciliated OSNs until 3.5 dpf. We found that about 44% of the ody mutant olfactory placodes (n=16) extended a smaller number of pioneer axons toward the presumptive OB (arrow in Fig. 9F) than did ody/+ and wild-type olfactory placodes at 1.5 dpf. Whenever we observed the pioneer axons clearly not reaching the presumptive OB at 1.5 dpf, we later found that the following axons of ciliated OSNs from the same placode also failed to grow out (Fig. 9G,H; left side) and eventually accumulated at the border between the OE and the telencephalon by 3.5 dpf (arrowheads in Fig. 9H). By contrast, if a substantial number of the pioneer axons reached the presumptive OB by 1.5 dpf, we never detected impaired axon projection of ciliated OSNs at 3.5 dpf in ody mutant embryos (Fig. 9F-H; right side). In ody/+ (Fig. 9A-D) and wild-type (data not shown) embryos, the pioneer axons always successfully navigated to the presumptive OB, and ciliated OSNs also established correct projection to the OB. These results suggest that Cxcr4b-mediated signaling is important for pathfinding of zns-2-positive pioneer axons from unipolar neurons to exit the olfactory placode and that impaired pathfinding of the pioneer axons might be the reason why OSN axons subsequently fail to project to the OB in the absence of Cxcr4b function.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Role of Cxcl12a/Cxcr4b signaling in olfactory placode assembly
Olfactory neuron precursors initially lie in the olfactory placodal field,
the region stretching along the lateral edge of the anterior neural plate
(Whitlock and Westerfield,
2000; Whitlock,
2004). As development progresses, they converge to form the
olfactory placode. The region expressing cxcr4b in the anterior head
corresponds to the olfactory placodal field. Previous studies have
demonstrated that Cxcr4b-mediated signaling guides migration of various types
of cells in different ways in zebrafish, including primordial germ cells
(Doitsidou et al., 2002;
Knaut et al., 2003), posterior
lateral line primordia (David et al.,
2002) and trigeminal sensory neurons
(Knaut et al., 2005). The
cxcr4b-expressing germ cells and posterior lateral line primordia
move along their migratory route, maintaining direct contacts with the
cxcl12a expression domains. By contrast, most
cxcr4b-expressing trigeminal sensory neurons are initially located at
a distance from the cxcl12a expression domains, and then move toward
the Cxcl12a sources. These observations suggest that Cxcl12/Cxcr4 signaling
may act in either short or long range, depending on the situations of
individual cell types. In the case of olfactory neuron precursors, they are
closely adjacent to the cxcl12a expression domain until the olfactory
placode is formed, suggesting a short-range action of Cxcl12a in this
process.
In the absence of Cxcr4b-mediated signaling, olfactory neuron precursors
dispersed anteriorly, resulting in ventrally displaced olfactory neurons.
Hence, chemokine signaling is required for correct positioning of olfactory
neurons. Moreover, global and localized misexpression of Cxcl12a perturbed
olfactory placode assembly, suggesting that Cxcl12a/Cxcr4b signaling is not
merely permissive, but acts as a guidance cue to position the olfactory
neurons. This notion is consistent with the observation that spatial
restriction of the cxcl12a expression domain occurs synchronously
with convergence of the olfactory placodal field. There is a significant
anterior tissue movement during early segmental stages
(Karlstrom and Kane, 1996),
and this movement should result in ventral cell streaming at the most anterior
part of the developing head. The appearance of ventrally displaced olfactory
neurons in the absence of Cxcr4b signaling raises the possibility that Cxcl12a
provides olfactory neuron precursors with a retention signal to withstand the
anterior/ventral movement of neighboring cells. Our observation that ectopic
Cxcl12a sources in the ventral region, but not in the dorsal region, can
recruit olfactory neurons to positions close to the ectopic Cxcl12a sources
could also be explained by the competition between the endogenous Cxcl12a
source and the anterior/ventral streaming of cells expressing exogenous
Cxcl12a. Such a mechanism, by which Cxcl12 might act to retain
cxcr4b-expressing cells in the correct position, has been implicated
in trigeminal sensory ganglion assembly
(Knaut et al., 2005). In this
case, cxcl12 expression domains are located posteriorly adjacent to
the final position of ganglia, opposite to the direction of morphogenetic
movements. By contrast, the cxcl12a expression domain in the anterior
head was positioned medially to the array of olfactory neuron precursors,
although a similar mispositioning phenotype was observed in both olfactory
neurons and trigeminal sensory neurons upon disruption of Cxcr4b signaling.
Direct in vivo observations on dynamic behaviors of olfactory neuron
precursors and neighboring cells in wild-type and ody mutant embryos
may clarify the mode of Cxcl12a action in more detail.
Role of Cxcl12a/Cxcr4b signaling in olfactory axon pathfinding
In ody mutants, 50% of the embryos exhibited defects in OSN
axon projection to the OB. The simplest interpretation is that Cxcl12a may act
as a chemoattractant for olfactory axons, because cxcl12a is
expressed in the placode-telencephalon border where the axons cross to enter
the telencephalon and also in the anterior tip of the telencephalon toward
which the axons grow. However, Cxcl12 does not appear to exhibit obvious
attractive activity when assayed on several classes of vertebrate neurons in
vitro (Chalasani et al., 2003).
Secondly, axon guidance defects might be an indirect consequence of loss of
Cxcr4b signaling. In the absence of Cxcr4b signaling, olfactory neurons are
mispositioned and outgrowing axons are challenged with a new
micro-environment, and failure to interpret this new environment correctly may
cause axons to navigate to new targets. However, such an indirect effect of
loss of Cxcr4b signaling on axon guidance is unlikely, because ubiquitous
misexpression of Cxcl12a at 12 hpf, which induces mispositioning of olfactory
neurons, did not cause the ody-like OSN axon pathfinding defects
(Fig. 5B,E). As Cxcl12 has been
shown to reduce the growth cone responsiveness to chemorepellents in vitro
(Chalasani et al., 2003) and in
vivo (Chalasani et al., 2007),
the third possibility is that exposure of olfactory axons to Cxcl12a at the
placode-telencephalon border might reduce the growth cone responsiveness to
chemorepellent factors that exist around the path of olfactory axon
navigation, allowing them to exit the olfactory placode and to grow toward the
OB. This permissive model is consistent with our observation that forced
ubiquitous expression of Cxcl12a at either 12 or 19 hpf induces no significant
defect in OSN axon projection to the OB
(Fig. 5). A previous study
(Yoshida et al., 2002) has
demonstrated that alteration of protein kinase A (PKA) activity in olfactory
axons is important for pathfinding and has proposed that the interaction of
growing axons with the placode-telencephalon border may downregulate PKA
signaling, modulating the response of growth cone to guidance cues. Cxcl12a
hence appears to be a promising candidate to fulfill this function via Cxcr4b,
which signals through Gi proteins, thereby decreasing the intracellular cyclic
AMP level (Tran and Miller,
2003).
The defects in OSN axon projection in ody mutants are variable and
appear to occur in an all-or-none manner. Once OSN axons can reach the OB, the
pattern of proto-glomerular targeting by OSNs in ody mutants is
indistinguishable from that in wild type. This all-or-none phenotype could be
explained by the notion that pioneer axons provide a scaffold essential for
subsequently projecting OSN axons
(Whitlock and Westerfield,
1998; Miyasaka et al.,
2005). Indeed, the pathfinding by pioneer axons was frequently
impaired in ody mutants. In some cases, a portion of zns-2-positive
pioneer axons failed to exit the olfactory placode, but the remaining ones
from the same placode could reach the OB at 1.5 dpf. Such defects with an
intermediate severity are rarely observed in OSN axon projection at 3.5 dpf.
These findings imply that there might be a threshold in the number of
OB-reaching pioneer axons that is required for establishment of a sound
projection of OSN axons. Our observation that mature OSNs did not express
cxcr4b also suggests that Cxcr4b-mediated signaling influences the
early growing axons from either unipolar neurons or newly differentiated
immature OSNs.
Cxcl12/Cxcr4 signaling has recently been shown to regulate retinal axon
projection in zebrafish (Li et al.,
2005) and motor axon projection in mouse
(Lieberam et al., 2005). In
cxcr4b mutant zebrafish, some ganglion cell axons fail to exit the
eye but extend in aberrant directions within the retina. Similarly, in the
absence of Cxcr4 signaling in mice, some axons of a set of motoneurons, termed
vMNs, aberrantly exit through the dorsal exit point of the spinal cord instead
of correctly choosing the ventral exit point. In both cases, the initial phase
of axon pathfinding is perturbed similarly to the case for olfactory axons
deprived of Cxcr4b function. These findings suggest that Cxcl12/Cxcr4
signaling might be used as a general molecular tool to create a favorable
environment and to shape an initial trajectory for various types of early
growing axons in vertebrates.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/13/2459/DC1
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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