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First published online 16 February 2005
doi: 10.1242/dev.01698
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1 Laboratory for Neurobiology of Synapse, RIKEN Brain Science Institute, 2-1
Hirosawa, Wako-shi, Saitama 351-0198, Japan
2 Laboratory for Developmental Gene Regulation, RIKEN Brain Science Institute,
2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan
3 Department of Neurobiology and Anatomy, University of Utah Medical Center,
Salt Lake City, UT 84132, USA
4 Core Research for Evolutional Science and Technology (CREST), Japan Science
and Technology Corporation (JST), 3-4-5 Nihonbashi, Chuo-ku, Tokyo 103-0027,
Japan
Author for correspondence (e-mail:
yoshihara{at}brain.riken.go.jp)
Accepted 14 January 2005
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SUMMARY |
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TOP
SUMMARY
Introduction
Materials and methods
Results
Discussion
REFERENCES |
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Key words: Axon guidance, Pioneer neurons, Glomerulus, Transgenic zebrafish
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Introduction |
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TOP
SUMMARY
Introduction
Materials and methods
Results
Discussion
REFERENCES |
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1,000 genes in rodents and 100 genes in
fish (Mombaerts, 1999
). In
mice, OSNs expressing a given OR are widely distributed within the olfactory
epithelium (OE), yet they converge their axons onto a few specific glomeruli
in the olfactory bulb (OB), creating an odor map
(Ressler et al., 1994;
Vassar et al., 1994;
Mombaerts et al., 1996).
This feat is accomplished by sophisticated processes of axon guidance and
synapse formation during development, which can be divided into at least three
steps. First, nascent olfactory axons exit the OE, coalesce to form fascicles,
and grow toward the OB primordium at the rostral tip of the telencephalon.
Second, upon reaching the OB primordium, the olfactory axons defasciculate
tangentially and sort out into smaller subsets toward restricted domains of
the OB. Third, the olfactory axons make synaptic connections in target
glomeruli with the dendrites of OB projection neurons and interneurons. What
molecules are responsible for the establishment of the topographic odor map?
Genetic deletions or substitutions of specific OR genes in mice have suggested
that the ORs themselves play an instructive role in glomerular targeting
(Mombaerts et al., 1996;
Wang et al., 1998;
Feinstein and Mombaerts, 2004;
Feinstein et al., 2004).
Several cell recognition molecules have been implicated as guidance ligands
and receptors for OSN axons that function at a series of choice points along
the navigation course from the OE to the target glomeruli
(St John et al., 2002). For
instance, semaphorin 3A and ephrin-As have been shown to be involved in axon
sorting within the olfactory nerve layer and in axon termination onto precise
glomerular positions, respectively
(Schwarting et al., 2000;
Taniguchi et al., 2003;
Cutforth et al., 2003).
However, it remains largely unknown how the early growing olfactory axons are
precisely guided to the OB primordium. Roundabouts (Robos) and Slits,
chemorepulsive receptors and ligands, appear to be good candidates to achieve
this function in the light of their spatiotemporal expression patterns in the
developing olfactory system (Yuan et al.,
1999; Lee et al.,
2001; Marillat et al.,
2002).
Robos are evolutionarily conserved transmembrane glycoproteins belonging to
the immunoglobulin (Ig) superfamily (Kidd
et al., 1998; Sundaresan et
al., 1998; Zallen et al.,
1998). Robo was originally identified from studies of
Drosophila mutants in which axons misroute at the midline in the
ventral nerve cord (Seeger et al.,
1993). Drosophila Robo protein prevents commissural axons from
inappropriately recrossing the midline by sensing the repulsive ligand Slit
secreted from the midline glia (Kidd et
al., 1998; Kidd et al.,
1999). In vertebrates, the roles of Robo/Slit repulsive signaling
have been implicated in axon pathfinding of various types of neurons
(Bagri et al., 2002;
Nguyen-Ba-Charvet et al.,
2002; Plump et al.,
2002; Knöll et al.,
2003; Long et al.,
2004). We have previously shown that the zebrafish mutant
astray (ast) exhibits deviation of retinal axons from their
normal route, and that ast is defective in the gene encoding Robo2
(Fricke et al., 2001;
Hutson and Chien, 2002).
Here, we demonstrate, by using ast mutants and Slit2-overexpressing zebrafish, that Robo/Slit signaling is required for proper navigation of the early growing olfactory axons toward the OB primordium. Furthermore, we propose that the establishment of a sound glomerular map in the adult OB requires the precise formation of the initial axon scaffold, which is mediated by Robo2 at early developmental stages.
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Materials and methods |
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TOP
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 at pH 7.2) supplemented with 100
units/ml penicillin and 100 µg/ml streptomycin. In some cases, 0.002%
phenylthiourea was added after 12 hpf to prevent pigmentation.
Generation of transgenic zebrafish
A bacterial artificial chromosome (BAC) clone containing the zebrafish
olfactory marker protein (OMP) gene was isolated from Down-to-the-Well BAC
pools (Genome Systems). Roughly 6-kb and 2-kb fragments upstream of the OMP
translation start site were subcloned into pEGFP-1 (Clontech) to generate
pOMP6k:GFP and pOMP2k:GFP, respectively. To facilitate
axonal localization of reporter protein, the EGFP cDNA of
pOMP6k:GFP and pOMP2k:GFP was replaced with
EYFP-Mem cDNA (Clontech), which encodes a fusion protein consisting
of the N-terminal 20 amino acids of GAP-43 and EYFP. The resulting plasmids,
pOMP6k:gap-YFP and pOMP2k:gap-YFP, were purified,
linearized, and diluted to 50 ng/µl in distilled water containing 0.1%
Phenol Red. The DNA solution was injected into the blastomere of one-cell
stage embryos. Embryos with fluorescence were raised to sexual maturity and
founder fish were identified by the expression of YFP-fluorescence in their
progeny. Four transgenic lines were obtained from injections with
pOMP6k:gap-YFP and pOMP2k:gap-YFP (two lines for each
construct). Two lines, termed Tg(OMP6k:gap-YFP)rw031a
and Tg(OMP2k:gap-YFP)rw032a, in which strong YFP
fluorescence was observed, were used in this study.
Fish lines
astray mutants (astti272z) were kept as
homozygotes because they are partially adult viable and fertile
(Fricke et al., 2001).
ast homozygotes (ast/ast) were crossed with
heterozygous Tg(OMP6k:gap-YFP)rw031a transgenic fish
(abbreviated as omp:yfp/+), and then
ast/+;omp:yfp/+ fish were crossed with ast
homozygotes to obtain ast/ast;omp:yfp/+ embryos. To
identify ast/ast;omp:yfp/+ fish, genomic DNA was
extracted from embryos, or fin-clips of adults, and typed by PCR amplification
of a DNA fragment containing the astti272z allele using
the primers 5'-GAA TGA CTC CTC GTC GCT CT-3' and 5'-TAT GGT
GGT AGG GCT AAG GAC-3', followed by direct sequencing of the PCR
products. A transgenic line, Tg(hsp70:Slit2-GFP)rw015d (previously
called HS2E-4S) (Yeo et al.,
2001; Yeo et al.,
2004), in which the Slit2-GFP fusion protein can be heat-induced,
was used to overexpress Slit2.
Whole-mount in situ hybridization
Digoxigenin (DIG)-labeled cRNA probes for robo2
(Lee et al., 2001),
slit1a, slit1b (Hutson et al.,
2003), slit2 and slit3
(Yeo et al., 2001) were used.
Whole-mount in situ hybridization was performed as previously described
(Hauptmann and Gerster, 1994),
with the following modifications. DIG-labeled probes synthesized by in vitro
transcription were purified with Micro Bio-Spin 30 columns (BioRad). Embryos
were hybridized with probes overnight at 55°C in hybridization solution
(2.5 mM EDTA, 300 mM NaCl, 50% formamide, 1 mg/ml yeast RNA,
1xDenhardt's solution, 5% dextran sulfate, 20 mM Tris-HCl at pH 8.0).
After hybridization, embryos were treated with RNase A (20 µg/ml) for 30
minutes at 37°C.
DiI-staining
DiI-staining of OSNs was carried out according to Dynes and Ngai
(Dynes and Ngai, 1998).
Immunohistochemistry
Whole-mount immunohistochemistry was carried out as previously described
(Macdonald, 1999), with the
following modifications. Embryos older than 2 days postfertilization (dpf)
were fixed in 2% trichloroacetic acid in phosphate-buffered saline (PBS), and
permeabilized in acetone for 7 minutes at -20°C. PBS containing 1%
dimethylsulfoxide and 0.1% Tween-20 (PBDT) was used as washing solution.
For simultaneous in situ detection of robo2 transcripts and YFP antigens, immunostaining with diaminobenzidine was done first, as described above, except that MAB (100 mM maleic acid, 150 mM NaCl), containing 2% blocking reagent (Roche Diagnostics) and 0.5% heparin, was used for blocking and incubation solutions.
For immunostaining of adult OB sections, 12- to 14-month-old female fish
(4 cm body length) were anesthetized with 0.016% tricaine, and
telencephalic hemispheres including OBs were dissected out. Tissues were fixed
overnight at 4°C in 4% paraformaldehyde in PBS, equilibrated in 30%
sucrose, frozen in O.C.T. Compound, sectioned on a cryostat (20 µm
thickness), and mounted onto silane-coated glass slides. The sections were
incubated sequentially with 5% NGS in PBS containing 0.1% Triton X-100,
primary antibodies, and fluorescent dye-conjugated secondary antibodies.
Antibodies used were as follows: rabbit polyclonal anti-GFP antibody (1:1000, a kind gift from Dr N. Tamamaki); mouse monoclonal anti-calretinin antibody (1:1000, Swant); mouse monoclonal anti-SV2 antibody [1:20, supernatant, Developmental Studies Hybridoma Bank (DSHB) at University of Iowa]; mouse monoclonal zns-2 antibody (1:200, supernatant, DSHB); rabbit polyclonal anti-PCAM antibody (rabbit IgG; 0.4 µg/ml); Alexa488-conjugated goat anti-rabbit IgG antibody (1:300, Molecular Probes); Cy3-conjugated goat anti-mouse IgG (1:300, Jackson ImmunoResearch); peroxidase-conjugated secondary antibody (Histofine Simple Stain Max PO, Nichirei, Tokyo, Japan). Anti-PCAM antiserum was produced by Sawaday Technology (Tokyo, Japan). A synthetic C-terminal peptide (20 amino acid residues) of zebrafish PCAM was conjugated with keyhole limpet hemocyanin, and a rabbit was immunized with the conjugate. The generated antiserum was purified by immunoaffinity chromatography with peptide-coupled resin.
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Results |
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TOP
SUMMARY
Introduction
Materials and methods
Results
Discussion
REFERENCES |
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).
robo2 expression in the olfactory placode could be detected at 20 hpf
(Fig. 1B). From 24 to 36 hpf,
when the early olfactory axons emerging from the olfactory placode grew toward
the OB primordium, robo2 mRNA was strongly expressed in the olfactory
placode in addition to in the brain (Fig.
1C-E). At 48 hpf, when the pioneering olfactory axons had arrived
at the OB primordium and started to form discrete axonal condensations
(proto-glomeruli), robo2 expression in the olfactory placode was
greatly diminished and detectable only in a few cells located near the nasal
pit, although its expression remained high in the brain
(Fig. 1F). We were unable to
detect evident robo2 expression in adult OE by section in situ
hybridization (data not shown).
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). Because OSNs have dendrites whose cilia are exposed to the
environment, we labeled OSNs by dipping the embryos into a solution containing
DiI at 3.5 dpf (Dynes and Ngai,
1998). Following at least a 1-hour incubation to allow diffusion
of DiI into axons, the trajectories of OSN axons were viewed from anterior and
dorsal directions by confocal laser scanning microscopy. In wild type, OSN
axons exited the OE through a restricted region on its medial side, forming a
tightly fasciculated bundle (Fig.
2B). They extended dorsally soon after exiting the OE and then
defasciculated tangentially on the surface of the OB
(Fig. 2B,C). In ast
homozygotes, many OSN axons reached the OB, but several axonal fibers
misrouted posteriorly and penetrated into the diencephalon without reaching
the OB (Fig. 2D-G). The
posteriorly misrouting axons occasionally crossed the midline to the
contralateral side (arrows in Fig.
2F,G). To assess phenotypic penetrance and strength, we counted
the number of embryos having a given pathfinding error
(Table 1). All ast
homozygous embryos exhibited posterior pathfinding errors with some
differences in maximal reach value, and 50% of ast homozygotes
had midline-crossing errors; such trajectories were never observed in
wild-type or ast heterozygous embryos
(Table 1).
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;
Yoshida et al., 2002). We then
crossed ast to one of the transgenic lines,
Tg(OMP6k:gap-YFP)rw031a (abbreviated as omp:yfp), and
carried out time-lapse observation of dynamic OSN axon projections
(Fig. 3). The zebrafish
olfactory placode contains a transient population of neurons, which are
morphologically and spatially distinct from OSNs: they are dendrite-less
unipolar neurons whose cell bodies are situated in the ventromedial portion of
the olfactory placode (Whitlock and
Westerfield, 1998). At 1 dpf, YFP fluorescence was detected in
10 unipolar neurons (closed arrowheads in
Fig. 3A,F), and in several
early developing OSNs with dendrites extending toward the presumptive
olfactory pit (open arrowheads in Fig.
3A,F). Emergence of the axons from the olfactory placode was
significantly retarded in ast homozygotes when compared with
heterozygotes at 1 dpf (Fig.
3A,F). In ast homozygotes, many axons grew dorsally
toward the OB primordium, but several axons misrouted ventromedially soon
after exiting the olfactory placode at 1.5 dpf (short arrows in
Fig. 3G). At 2 dpf, the axons
that had misrouted at the exiting point formed fascicles and became prominent,
with an increase in number of YFP-labeled OSNs (short arrows in
Fig. 3H). By 3 dpf, the
aberrant axonal fascicles had often penetrated the diencephalon, occasionally
crossing the midline (Fig.
3I,J). At 3 dpf, the gross projections to OB were reduced in
homozygotes when compared with heterozygotes
(Fig. 3D,I). Notably,
YFP-labeled dorsolateral glomeruli (thick arrows in
Fig. 3D) disappeared in
homozygotes (Fig. 3I). These
observations demonstrate that the posteriorly projecting and midline-crossing
errors arise in ast mutants at the time when the first olfactory
axons leave the olfactory placode.
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; Whitlock and
Westerfield, 2000). Thus, it is likely that the misrouting axons
in ast embryos at the time when the first olfactory axons leave the
olfactory placode are derived from the unipolar neurons. To clarify whether
the unipolar neurons express robo2, we performed double in situ
detection for robo2 transcripts and YFP antigens in the omp:yfp
transgenic line at 30 hpf. As described above, the unipolar neurons were
readily distinguishable from OSNs by their unique location and morphology.
Hybridization signals for robo2 transcripts were uniformly
distributed within the olfactory placode
(Fig. 4A,B), and seen on the
cell bodies of the unipolar neurons (arrowheads in
Fig. 4B), as well as in the
early developing OSNs (arrows in Fig.
4B).
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)
recognizes the axons of unipolar neurons
(Whitlock and Westerfield,
1998). We therefore performed zns-2-labeling of 36-hpf
ast embryos to assess whether or not the unipolar neurons make
pathfinding errors in the absence of Robo2 function. In wild type, the
zns-2-positive axons formed a tight fascicle and extended dorsally along the
surface of the telencephalon (Fig.
4C), whereas in ast mutants, the zns-2-positive axons
defasciculated immediately after growing out of the olfactory placode and some
of them misrouted ventromedially toward the midline
(Fig. 4D). These trajectories
resembled those of the early growing YFP-labeled axons observed in
ast/ast;omp:yfp/+ embryos
(Fig. 3G). Because the
dendrite-less unipolar neurons are unlabeled by external application of DiI,
the misrouting axons labeled by DiI in ast embryos should have
originated from OSNs (Fig.
2D-G). Taken together, these results indicate that both unipolar
neurons and OSNs make pathfinding errors in ast embryos.
Spatial expression patterns of four members of Slit genes
To examine whether the ast phenotype correlates with the
expression of Slit genes, the Robo2 ligands, we conducted a detailed spatial
expression analysis by whole-mount in situ hybridization
(Fig. 5). All four members of
the zebrafish Slit family, slit1a, slit1b, slit2 and slit3
(Yeo et al., 2001;
Hutson et al., 2003) were
expressed close to the olfactory axon trajectory during the initial phase of
olfactory axon pathfinding. At 30 hpf, slit1a, slit1b and
slit2 were expressed in bilateral clusters of cells located near the
boundary between the telencephalon and diencephalon (arrowheads in
Fig. 5C,F,I). slit1a
and slit1b were also expressed in bilateral clusters in the
telencephalon (arrows in Fig.
5B,C,E,F), adjacent to the region where the pioneering olfactory
axon termini were extending (Fig.
5N,O). slit2 and slit3 were expressed along the
midline in the ventral forebrain (thick arrows in
Fig. 5H,K). These regions of
Slit expression were located posteriorly or ventromedially adjacent to the
olfactory axon pathway (Fig.
5P), consistent with a function of Slits as chemorepellents for
the Robo2-expressing olfactory axons. Such spatial expression patterns of Slit
genes were maintained from 24 to 36 hpf (data not shown). These results
suggest that the ast phenotype may be attributed to a defect in
Robo2/Slit signaling that normally prevents olfactory axons from entering into
inappropriate areas.
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). We
therefore investigated whether localized Slit sources are critical for OSN
axon pathfinding, by inducing ubiquitous misexpression of Slit. We used a
transgenic line, Tg(hsp70:Slit2-GFP)rw015d (abbreviated as
hsp:slit2-gfp), in which a Slit2-GFP fusion protein could be induced
ubiquitously following an increase in ambient temperature (to 39°C for 50
minutes) (Yeo et al., 2001;
Yeo et al., 2004).
Heterozygous hsp:slit2-gfp embryos were heat-induced at 19 hpf and re-induced
at 31 hpf to maintain the Slit2 expression level during the period of initial
olfactory axon outgrowth. At 3.5 dpf, OSN axons in the heat-induced
hsp:slit2-gfp transgenic embryos were labeled by external application of DiI
and the trajectories were analyzed by confocal microscopy. Intriguingly, the
resulting phenotype of Slit2-overexpressing embryos resembled the Robo2
loss-of-function phenotype: some axonal fibers misrouted posteriorly and
penetrated into the diencephalon, occasionally crossing the midline
(Fig. 6). The phenotypic
penetrance and strength were also similar to ast homozygous mutants
(Table 1). It is likely that
when Slit2 is ubiquitously expressed, the olfactory axons are impaired in
their ability to respond to the local repulsive cues of endogenous Slit
proteins. These results suggest that olfactory axons can indeed respond to
Slit, and that the precise patterns of Slit expression are critical for
navigation of OSN axons to the OB.
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), labeled
all OSN axon shafts and termini, with no staining of cell bodies
(Fig. 7A). In wild type,
olfactory axons were tightly fasciculated to form a single bundle until they
entered the OB (brackets in Fig.
7A,C). By contrast, we found in ast embryos that
olfactory axons were defasciculated before reaching the OB (brackets in
Fig. 7D,F) and some small axon
bundles entered the OB from improper entry sites (arrows in
Fig. 7D,F). PCAM
immunoreactivity of axon termini was somewhat weaker in ast mutants
than in wild type (Fig.
7A,D,G,J). The proto-glomeruli immunostained for SV2, a synaptic
vesicle protein, in ast embryos were irregular in shape and were less
clearly defined than those in wild type
(Fig. 7H,I,K,L).
Anti-calretinin antibody labeled cell bodies of a small subpopulation of OSNs
and their target proto-glomeluli (Fig.
7B). The majority of calretinin-positive axons projected laterally
to form two discrete proto-glomeruli in wild type (thick arrows in
Fig. 7B,C), whereas in
ast mutants, only one irregularly shaped proto-glomerulus was seen at
the lateralmost position of the OB (thick arrows in
Fig. 7E,F). This aberrant
calretinin-positive proto-glomerulus in ast mutants was somewhat
larger in size than the normal calretinin-positive proto-glomerulus seen in
wild type, implying the loss of proper segregation of these proto-glomeruli in
ast mutants. These results demonstrate that Robo2 function is
necessary for maintaining the integrity of the olfactory nerve and
proto-glomerular organization.
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;
Yoshida et al., 2002). As the
projection patterns of YFP-labeled axons were essentially the same in the four
independent lines generated (data not shown), the restriction of expression to
a subpopulation of OSNs is probably not due to a positional effect of the
transgene integration sites. To elucidate whether the loss of Robo2 function
affects the topographic glomerular map of the OB in adult fish, we examined
the spatial arrangement of glomeruli innervated by YFP-labeled axons in
ast/ast;omp:yfp/+ adults. OBs of ast
adults [dorsal-to-ventral (D-V) thickness, 460±10 µm;
anterior-to-posterior (A-P) length, 458±9 µm; medial-to-lateral
(M-L) width, 458±4 µm; n=5, mean±s.e.m.] were
slightly smaller in size than those of wild-type adults (D-V, 508±6
µm; A-P, 492±8 µm; M-L, 442±6 µm; n=5;
Fig. 8A-D). Whole-mount
observation of OBs revealed that in ast mutants, dorsally projecting
YFP-labeled fibers were reduced in number, whereas the intensity of YFP
fluorescence in the ventral portion of the OB was increased
(Fig. 8A,C). Notably, the
posterolaterally located YFP-labeled glomeruli observed in wild type
(arrowheads in Fig. 8A) were
not observed in ast adults (Fig.
8C), whereas ectopic glomerulus-like structures were found in the
posteroventral portion of the OB (arrows in
Fig. 8C). A similar projection
pattern was observed in all individual ast adults (n=6). We
also found that only one ast adult exhibited a posterior
misprojection outside of the OB (Fig.
8E,F), although all ast embryos showed posterior
pathfinding errors at 3.5 dpf (Table
1). This implies the retraction or elimination of the posteriorly
misrouted axons during maturation, so that the ectopic glomerulus-like
structures seen in the posteroventral portion of OB (arrows in
Fig. 8C) might be formed by the
retracted axons. To analyze the spatial arrangement of glomeruli in more
detail, we carried out immunohistochemical staining of OB sagittal sections
(Fig. 8B,D). The sections were
counterstained with an anti-SV2 antibody to distinguish the glomerular layer
from the olfactory nerve layer. In ast mutants, YFP-labeled axons
ectopically innervated anteroventral and posteroventral glomeruli (arrows in
Fig. 8D). In addition, aberrant
axonal fascicles penetrated deep into the cellular layer of the OB (arrowheads
in Fig. 8D) and eventually
formed glomeruli in the posterodorsal region. Thus, glomerular organization of
the OB is perturbed in ast adults despite the fact that
robo2 expression in the OE is restricted to embryos at stages between
20 and 36 hpf (Fig. 1). These
results suggest an importance of early olfactory axon guidance in determining
a topographic glomerular map in the adult OB.
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Discussion |
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TOP
SUMMARY
Introduction
Materials and methods
Results
Discussion
REFERENCES |
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Robo2 steers early olfactory axons toward the OB primordium
Whitlock and Westerfield reported that the initial connections between the
olfactory placode and the OB primordium were provided by the axons of a
transient population of unipolar neurons, which they called `pioneer' neurons
(Whitlock and Westerfield,
1998). robo2 is transiently expressed in the olfactory
placode and its temporal expression parallels the axon outgrowth of unipolar
neurons (Fig. 1). Moreover, the
unipolar neurons express robo2 and make pathfinding errors without
Robo2 function (Fig. 4). These
results suggest that Robo2-mediated signaling directly regulates the axon
pathfinding of the unipolar neurons.
OSN axons also make pathfinding errors in ast embryos. Similar to
the unipolar neurons, Robo2 may directly regulate the axon pathfinding of
early developing OSNs, because these neurons also express robo2
(Fig. 4). Alternatively, the
axons of OSNs may make pathfinding errors non-autonomously by following the
misrouted axons of the presumptive `pioneer' unipolar neurons in ast
mutants. It has been reported that ablation of the unipolar neurons results in
misrouting of the following OSN axons
(Whitlock and Westerfield,
1998), suggesting that the unipolar neurons provide an essential
scaffold for subsequently projecting OSN axons. In the absence of the unipolar
neurons, the misrouted OSN axons typically extend posteriorly toward the
diencephalon (Whitlock and Westerfield,
1998), resembling the trajectory in ast embryos observed
in this study (Fig. 2). Further
experiments, such as inhibition of Robo2 function selectively in the unipolar
neurons, will clarify whether or not the OSN axons make pathfinding errors
cell-autonomously.
Slit proteins act as surround repulsive cues for olfactory axons
The summed region of Slit expression and the trajectory of
robo2-expressing early olfactory axons are situated in a
complementary manner, consistent with a function of Slit proteins as
chemorepulsive ligands for Robo2. The spatial expression patterns of Slit
genes could explain how the pathfinding errors observed in ast are
normally prevented. slit1a, slit1b and slit2 expressed near
the telencephalon/diencephalon boundary (arrowheads in
Fig. 5C,F,I) could function as
a barrier to prevent olfactory axons from entering into the diencephalon.
slit2 and slit3 along the midline in the ventral forebrain
(thick arrows in Fig. 5H,K)
could act to promote dorsally directed outgrowth and to prevent
midline-crossing. Telencephalic slit1a and slit1b (arrows in
Fig. 5B,C,E,F) could play a
role in limiting the extension of olfactory axons on the surface of the OB.
Thus, the spatial expression patterns of Slit genes support a `surround
repulsion' model, in which chemorepulsive cues produced by surrounding tissues
channel axons into a specific route
(Keynes et al., 1997).
We demonstrate that ubiquitous misexpression of Slit2 causes OSN axon
pathfinding errors resembling the ast phenotype. It is surprising
that the OSN axons do not simply retract or stall. However, a similar
situation was observed following Slit2 overexpression with Mauthner axons
(Yeo et al., 2004) and retinal
axons (L.D.H. and C.-B.C., unpublished). Mauthner axons aberrantly re-cross
the midline and retinal axons display an ast-like phenotype.
Moreover, when Slit is pan-neurally expressed throughout the CNS of
Drosophila, commissural axons exhibit an abnormality resembling the
loss-of-function phenotype of Robo (Kidd
et al., 1999). In all cases, ubiquitous Slit overexpression does
not prevent axon outgrowth, but causes axon misrouting. Such phenotypes of
Slit overexpression could be explained by a hypothetical `gradient reading'
model in which axonal growth cones would change their direction by reading a
concentration gradient of a guidance cue
(Walter et al., 1990). In this
model, the repulsive cue does not influence the axonal outgrowth activity of
neurons. Therefore, even if the gradient of Slit were lost by forced
ubiquitous expression of Slit2, the OSN axons would not stall, and would grow
well on a uniform field of the repulsive cue. However, an alternative
possibility cannot be excluded: the OSN growth cones habituate and lose
sensitivity to Slit by the continuous exposure of a high concentration of
exogenous Slit2. In either case, the phenotype of Slit2 overexpression
strongly suggests that OSN axons are responsive to Slit secreted from local
sources and that the precise patterns of Slit expression are crucial for OSN
axon pathfinding.
Although Robo2/Slit signaling is important for the proper navigation of the
nascent olfactory axons toward the OB primordium, other guidance mechanisms
must be involved in this process, as evidenced by the fact that many olfactory
axons can reach the OB primordium in ast embryos
(Fig. 3). In rodents, cell
adhesion molecules, such as L1 and NCAM, have been implicated in the initial
assembly of olfactory pathway (Gong and
Shipley, 1996; Whitesides and
LaMantia, 1996). In zebrafish, L1
(Tongiorgi et al., 1995), NCAM
and its related molecule PCAM (N.M. and Y.Y., unpublished) are also expressed
in the olfactory placode at the time of initial axon outgrowth, and are thus
candidate molecules functioning in concert with Robo2. Netrins act as
attractive cues via transmembrane receptors of the DCC subgroup of Ig
superfamily (Chisholm and Tessier-Lavigne,
1999). In rat, Netrin 1 expression is associated with DCC-positive
olfactory axons only during the period of initial olfactory axon outgrowth
(Astic et al., 2002),
suggesting that Netrin 1 may play a role in promoting outgrowth of the nascent
olfactory axons toward the OB primordium. Thus, it is likely that the
combinatorial actions of attractive and repulsive cues mediate the proper
navigation of the early growing olfactory axons.
Robo2 is required for maintaining integrity of the olfactory nerve and proto-glomerular organization
Analysis of the olfactory nerve in 72-hpf ast embryos revealed
that Robo2 is required to maintain olfactory axons in a tightly fasciculated
state until they reach the developing OB. A feasible mechanism is that
secreted Slit proteins near the olfactory nerve act to maintain axons within
the main bundle through surround repulsion. Alternatively, a Slit-independent
mechanism may be involved in fasciculation of the olfactory nerve, because the
fasciculation defect in Slit2-overexpressing embryos is somewhat less severe
than that in ast mutants (data not shown). Homophilic and
heterophilic interactions of cell adhesion molecules are thought to be
important for axonal fasciculation. A recent in vitro study has shown that
human Robo1 and Robo2 exhibit homophilic binding activity, as do the Ig
superfamily adhesion molecules (Hivert et
al., 2002). Thus, Robo2 may regulate the adhesive property of
olfactory axons via a homophilic binding mechanism in vivo.
A bilateral symmetric and stereotyped arrangement of proto-glomeruli in the
developing OB becomes evident between 48 and 72 hpf, after robo2
expression in the olfactory placode has been downregulated. The
proto-glomerular organization is impaired in ast embryos at 72 hpf,
as revealed by staining with antibodies against SV2, PCAM and calretinin
(Fig. 7). The precise mechanism
that could explain such defects in ast embryos is unclear. One
possibility is that a tightly fasciculated state of the olfactory nerve before
reaching the developing OB could be crucial for the subsequent formation of
proto-glomeruli. Aberrant defasciculation of the olfactory nerve in
ast embryos results in the entrance of some fibers into OB from
improper positions (arrows in Fig.
7D-F). These axons could encounter an inappropriate environment of
putative guidance cues within the developing OB, leading to abnormal axonal
sorting and impaired proto-glomerular formation. Alternatively, Robo2
expressed in the developing OB (Fig.
1) may contribute to dendritic morphogenesis of OB neurons and
formation of proto-glomeruli, because Robo/Slit signaling has been shown to
regulate dendritic development of cortical pyramidal neurons in rodents
(Whitford et al., 2002).
Selective removal of Robo2 function in the peripheral olfactory neurons or the
OB neurons will be necessary to distinguish these possibilities.
Impaired glomerular map in adult ast OB
ast adults exhibit impaired spatial arrangement of glomeruli in
the OB (Fig. 8). Robo/Slit
signaling has recently been implicated in topographic axonal projections in
the Drosophila olfactory system
(Jhaveri et al., 2004) and in
the mouse vomeronasal system (Knöll
et al., 2003; Cloutier et al.,
2004). In Drosophila, different populations of OSNs
express distinct combinations of Robos, and perturbation of Robo levels by
loss of function or ectopic expression causes aberrant positioning of OSN axon
termini in the antennal lobe. In mouse, Robo2 is expressed by the axons of
vomeronasal sensory neurons located in the basal zone of the vomeronasal organ
(VNO), whereas Slit1 and Slit3 are expressed in the accessory olfactory bulb
(AOB), with a higher concentration in the anterior region where the axons from
basal VNO do not project (Knöll et
al., 2003). slit1-deficient mice exhibit ectopic
innervation of anterior AOB by the axons from basal VNO
(Cloutier et al., 2004). In
both cases (fly and mouse), Robos are expressed in peripheral sensory neurons
throughout the period when the topographic axonal projection is
established.
By contrast, the expression of zebrafish robo2 in peripheral olfactory neurons is restricted between 20 and 36 hpf (Fig. 1). Thus, Robo2 function in OSNs should be limited to the initial phase of olfactory axon pathfinding. We found that ast adults exhibit abnormal innervation of ventral glomeruli by YFP-labeled axons, concomitant with the reduction of dorsally projecting fibers, despite the continual renewal of OSNs throughout life. This finding raises a possibility that later developing OSNs project their axons to target glomeruli using the pre-existing fibers as a scaffold. Removal of Robo2 function causes ventromedial and posterior pathfinding errors of early growing olfactory axons (Figs 2, 3), probably due to the loss of sensitivity to Slit2/Slit3 and Slit1a/Slit1b expressed ventromedially and posteriorly adjacent to the OB primordium, respectively (Fig. 5). Later developing OSN axons would follow the trajectory of early misrouting axons, resulting in the ectopic innervation of ventral glomeruli and thus the maintenance of an aberrant topographic map in adult. However, it also seems possible that later OSN axons do not rely on any pre-existing fibers for pathfinding, but rather depend on their targets, as the formation of proto-glomeruli is impaired in ast embryos (Fig. 7). Further experiments will be required to verify these possibilities.
In conclusion, Robo2/Slit signaling guides early growing olfactory axons toward the OB primordium and is required for constructing a precise glomerular map in the adult OB.
|
ACKNOWLEDGMENTS |
|---|
|
Footnotes |
|---|
Present address: Department of Biology, Williams College, Williamstown, MA
01267, USA
|
REFERENCES |
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TOP
SUMMARY
Introduction
Materials and methods
Results
Discussion
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