Department of Molecular Biology, Umeå University, Umeå, S-901 87, Sweden
* Author for correspondence (e-mail: staffan.bohm{at}molbiol.umu.se)
Accepted 26 November 2002
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SUMMARY |
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Key words: RNCAM, OCAM, NCAM2, apCAM, Fasciclin 2, Neural cell adhesion molecule, Splice variants, Olfactory, Sensory map, Gene expression, Odorant receptors, Axon guidance, Mouse, Glycosylphosphatidylinositol, Fasciculation
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INTRODUCTION |
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To study the importance of RNCAM isoforms in olfactory map formation, we
have used the olfactory marker protein (OMP) gene promoter to generate
transgenic mice with ectopic (in Z1) and elevated (in Z2-4) expression of
either GpiRNCAM or TmRNCAM in OSNs. Our identification of NADPH diaphorase
(NADPHd) activity as a novel marker for Z1 axons and the use a genetically
modified mouse (P2-IRES-taulacZ)
(Mombaerts et al., 1996) have
allowed us to conclude that OMP-RNCAM transgenic mice have an intact regional
division of axon projections while showing errors in OR-specific axon
segregation. The topographic distribution of glomeruli with axons having more
than one OR identity in mice overexpressing either of the two RNCAM isoforms
alone or in combination, suggest that OSNs use a dual, isoform-specific,
regulatory function of RNCAM when precisely selecting target glomeruli.
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MATERIALS AND METHODS |
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Generation of the anti-RNCAM antibody
A pBAD (Invitrogen, Carlsbad, CA) vector was used to generate a
Myc/His-tagged TmRNCAM protein that corresponded to amino acid residues
442-685 in the extracellular domain. Bacterial expression (strain Top10,
Invitrogen, Carlsbad, CA) was induced with 1% Arabinose (Sigma-Aldrich,
Sweden) at 37°C for 1 hour. Bacteria was sonicated and tagged peptide was
recovered on Ni-NTA agarose (Qiagen, Germany). Rabbits were immunized with 500
µg of purified protein followed by several boosting injections with 100
µg of protein (Agrisera, Sweden). Antiserum was purified using a column of
Top 10 bacterial extract coupled to CNBr Sepharose (Amersham Pharmacia
Biotech, Sweden) and subsequently affinity purified by binding and elution
from a column of tagged RNCAM protein coupled by cyanogen bromide (BioRad,
Sweden). Specificity of purified anti-RNCAM antibody was confirmed by western
blot analyses using homogenates from olfactory bulb and brain.
Immunocytochemistry
Tissue from 2-week-old mice was fixed in 4% paraformaldehyde/PBS for 30-90
minutes at room temperature and decalcified in 0.5 M EDTA at 4°C for 3-10
days. Tissue was cryoprotected by 30% sucrose at 4°C for 24 hours,
embedded in OCT compound (Sakura Finetek, Torrance, CA) and cryosectioned (16
µm). Retrieval of RNCAM antigen was achieved by microwaving at 650 W for 10
minutes in 10 mM citrate buffer (pH 6.0). Sections were incubated for 1 hour
at room temperature in blocking solution (3% normal goat serum, 0.1 M
phosphate buffer, 2 mM MgCl2, 5 mM EGTA, 0.02% Nonidet P-40, and
0.001% sodium deoxycholate) followed by an overnight incubation at 4°C in
blocking solution containing affinity-purified anti-RNCAM antibody (1:100).
Sections were subsequently washed with PBS and a positive immunoreaction was
visualized by using biotinylated goat anti-rabbit IgG (1:250) (Vector
laboratories, Burlingame, CA) and Cy3-coupled strepavidin (1:2000) (Jackson
Immuno Research laboratories, West Grove, PE) for 1.5 hours and 30 minutes,
respectively. Sections were counter stained with Hoechst 33258 (Sigma-Aldrich,
Sweden) and mounted with Dako fluorescent mounting media (Dako, Sweden).
Microphotographs were taken using fluorescent optics on a Zeiss Axioskop
microscope (Zeiss, Sweden)
In situ hybridization
Pretreatment of sections was carried out according to Breitschopf et al.
(Breitschopf et al., 1992).
Cryosections (10 µm) were hybridized to 35S-labelled RNCAM cRNA
probes; hybridization and washing conditions were as described previously
(Sassoon et al., 1988
). The
probe that recognized both isoforms of RNCAM corresponded to a 1.4 kb fragment
coding for the extracellular RNCAM domain. Isoform-specific probes
corresponded to published sequences of GpiRNCAM (bp 2204-2900, Accession
Number, AF016619) and TmRNCAM (bp 312-842, Accession Number, AF016620),
respectively. Slides were dehydrated and processed for autoradiography using
NTB-emulsion (Kodak, Sweden), and exposed for 5-14 days at 4°C.
Histological staining of sections was performed with the nuclear dye Hoechst,
and analysis was carried out using both fluorescent and dark-field optics on a
Zeiss Axioskop microscope.
NADPH diaphorase and ß-galactosidase histochemistry
NADPH diaphorase histochemistry was carried out essentially as previously
described (Dellacorte et al.,
1995). Cryosections (16-40 µm) of OE and OB were mounted on
slides, washed twice with 100 mM Tris HCl (pH 8.0), incubated for 60 minutes
at 37°C in 1 mM ß-NADPH, 0.3% Triton X-100, 1.5 mM L-Arginine, 0.8 mM
NBT 100 mM Tris-HCl (pH 8.0), rinsed in PBS and counterstained with Hoechst.
ß-Galactosidase histochemistry was carried out on serial coronal OB
cryosections (40 µm). Slides were rinsed three times for 20 minutes in 0.1
M phosphate buffer, 2 mM MgCl2, 5 mM EGTA, 0.02% Nonidet P-40, and
0.001% sodium deoxycholate. The staining reaction was carried out in the same
buffer, supplemented with 1 mg/ml X-gal, 5 mM K3
Fe(CN)6, and 5 mM K4 Fe(CN)6, overnight (16
hours) at 37°C. Sections were subsequently rinsed three times in PBS,
counterstained with Hoechst and mounted with Dako farmount mounting media
(Dako, Sweden). Student's t-test (two tailed) was used to determine
statistical significance of all morphological data sets.
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RESULTS |
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Immature OSNs are located close to the basal cell layer of OE. A probe
specific for TmRNCAM hybridized to OSNs in OE layers containing both immature
and mature OSNs, whereas a GpiRNCAM-specific probe hybridized predominantly to
immature OSNs (Fig. 1I,J). This
result indicated that RNCAM isoforms showed a different laminar distribution
in OE because of post-transcriptional regulation of mRNA steady-state levels.
Surprisingly, the same distribution of RNCAM isoforms was evident in
transgenic mice (Fig. 1F,H).
Thus, exogenous and endogenous RNCAM transcripts showed the same relative
distribution in OE, regardless of the fact that the gene regulatory sequences
determining transcription were different, i.e. OMP or RNCAM promoters,
respectively. As cDNA of spliced transcripts was used to construct the
transgenes, the result indicated that the observed difference in distribution
of RNCAM mRNA isoforms was regulated by differential mRNA stability and not
differential splicing. Because both transgenes contained an identical 5'
UTR, the results further suggested that the determinant regulating the
differential mRNA stability was located within the 3' UTRs that
differed. The turnover of an mRNA can be regulated by cis-acting elements
located in the 3' UTR such as the AU-rich elements (AREs) that are
binding sites for proteins regulating mRNA turnover
(Ross, 1995). Interestingly,
two AREs (AUUUA and AUUUUA) were present within the 3' UTR unique to the
GpiRNCAM construct (Fig. 1A).
It has been shown that the expression of Gap43 (Basp2
Mouse Genome Informatics) mRNA is regulated post-transcriptionally via
AREs and that the protein is synthesized in high amount during neurite
outgrowth (Beckel-Mitchener et al.,
2002
). In this respect, it is interesting to note that we found
GpiRNCAM mRNA to be expressed in the same cell layer of OE as Gap43
mRNA (Verhaagen et al., 1989
).
These results suggested that the GpiRNCAM isoform might function in axons of
immature OSNs, while TmRNCAM in addition might have a function in more
differentiated OSNs.
In tissue sections of the OB, the GpiRNCAM-specific probe generated no
hybridization signal above background, while the TmRNCAM-specific probe
generated a hybridization pattern indistinguishable from that reported for a
probe recognizing both isoforms (Alenius
and Bohm, 1997; Yoshihara et
al., 1997
). Thus, TmRNCAM appears to be evenly distributed along
the dorsoventral and mediolateral extensions of granular, mitral and
periglomerular cell layers.
Differential distribution of Gpi and Tm RNCAM protein
We generated a polyclonal antiserum against the common extracellular
domain. To analyze for cellular distribution of RNCAM isoforms, we took
advantage of the fact that Z1 OSNs lack endogenous RNCAM. As expected, control
mice showed no RNCAM immunoreactivity in Z1 glomeruli
(Fig. 2A). For GpiRNCAM
transgenic mice, intense immunoreactivity was observed in the nerve layer,
whereas glomeruli showed less staining
(Fig. 2B). Thus, when GpiRNCAM
was expressed at levels higher than normal, this isoform preferentially
localized to axons in the nerve layer. This result indicated that the
immunoreactivity in Z2-4 glomeruli of control mice corresponded to TmRNCAM and
not GpiRNCAM (Fig. 2G). The
finding that transgenic TmRNCAM protein showed equally strong immunoreactivity
in nerve and glomerular cell layers supported this notion
(Fig. 2C). The differential
distribution of RNCAM isoforms may relate to the fact that Gpi-anchored
proteins, in contrast to many transmembrane proteins, are clustered in
sphingolipidsterol rich microdomains or rafts that influence subcellular
localization (Simons and Ikonen,
1997). An alternative interpretation is that the levels of
expression per se determine the axonal distribution of RNCAM isoforms. The
finding that endogenous expression levels of TmRnCAM was higher than that of
GpiRNCAM in control mice support such a notion
(Fig. 1E,G). However,
regardless of the mechanism causing the differential distribution observed,
the results suggested that both isoform were present on axons in the nerve
layer, whereas TmRNCAM was the predominant isoform on axon terminals within
glomeruli.
Unaltered Z1 topography in Gpi and Tm RNCAM transgenic mice
Our analyses addressing whether gain of RNCAM function alters the broad
zonal organization of projections were hampered by the fact that no marker,
except RNCAM, had been identified that distinguish this division. Previous
studies have reported that NADPH diaphorase (NADPHd) activity is restricted to
the dorsomedial OE and OB (Davis,
1991; Dellacorte et al.,
1995
). In situ hybridization analysis of RNCAM and NADPHd
histochemistry on serial sections of OE revealed that NADPHd activity was
complementary to RNCAM expression and thus restricted to Z1
(Fig. 2D,E). Importantly, no
RNCAM/NADPHd double-positive glomerulus was found
(Fig. 2F,G). Besides the
intriguing topographical restriction of NADPHd-activity, this represented a
useful marker for the majority of Z1 axons and their terminals in the OB.
Fig. 3 shows comparisons of
NADPHd activity in whole-mount preparations, as well as serial OB sections of
transgenic mice and littermate controls. These analyses revealed that control
mice (Fig. 3C,F) and transgenic
mice (Fig. 3A,B,D,E) had equal
surface areas and spreading of the NADPHd-positive region along dorsoventral,
mediolateral and rostrocaudal axes of the OB. Moreover, no NADPHd-positive
glomeruli or stray axons were present in the region of the bulb that normally
is innervated by RNCAM-positive axons. This indicated that topographic
expression of RNCAM does not determine the division of the glomerular field
into two major regions that receive input from OSNs located in Z1 and
Z2-4.
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A fraction of P2 axons targets incorrect glomeruli
As RNCAM-mediated adhesion theoretically can interfere with a number of
different axon guidance decisions, we analyzed the projections from one
defined OSN subpopulation (P2) in detail. The P2 OR gene is expressed by
RNCAM-positive OSNs located in Z2. Axons of the P2-positive OSN subpopulation
can be visualized with the aid of the P2-IRES-tau-lacZ mouse line,
which was generated by gene replacement in embryonic stem cells described by
Mombaerts et al. (Mombaerts et al.,
1996). This mouse line has a P2 allele that is modified to express
a bicistronic transcript generating normal P2 OR protein, as well as an easily
visualized reporter protein (the microtubule-associated protein tau fused to
ß-galactosidase). Histochemical staining for ß-galactosidase
activity allows for direct visualization of P2 axons. We analyzed mice
carrying one copy of the P2-IRES-tau-lacZ allele together with one
RNCAM transgene allele, either OMP-TmRNCAM or OMP-GpiRNCAM. P2 axons were
visualized in coronal sections of nasal tissue and OB of 2-week-old mice.
Transgenic and control mice had equal total numbers of morphologically similar
P2 glomeruli (Fig. 4A,B;
Fig. 5A). Interestingly,
examination of transgenic mice revealed increased numbers of a type of
glomeruli that was only partially innervated by P2 axons
(Fig. 4C,D). It has been
reported that intermingling of axons corresponding to two closely related ORs
occurs during early postnatal development, while misrouted axons are rarely
detected in adult animals (Conzelmann et
al., 2001
; Mombaerts et al.,
1996
; Potter et al.,
2001
; Royal and Key,
1999
). Royal et al. have however, reported a sporadic occurrence
of semi-innervated P2 glomeruli in which P2 axons appear to terminate in a
discrete subregion of the glomerulus, either with a diffuse or dense
distribution (Royal and Key,
1999
). The increased number of such semi-innervated P2 glomeruli
in transgenic mice indicated that gain of RNCAM function augmented the
frequency at which such axonal targeting errors occur. We estimated the number
of this type of glomeruli to be on average 0.6 per OB in 2-week-old control
mice and 1.5-2 per OB in RNCAM transgenic littermates
(Fig. 5B).
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Axons of P2 OSNs typically innervate one glomerulus (or a few juxtaposed
glomeruli) on the medial and lateral sides of each bulb. Thus, each wild-type
P2-IRES-tau-lacZ mouse has at least four fully innervated glomeruli
(Mombaerts et al., 1996). In
our experimental system, we detected on average five fully innervated
glomeruli per control mouse. It has been speculated that the variation in the
exact number of glomeruli might be a reflection of genetic background
differences and/or variations in the odorous environment of animal facilities
(Potter et al., 2001
). It is
conceivable that the occurrence of semi-innervated P2 glomeruli in control
mice will vary between laboratories for the same reasons. To control for
genetic background and odor environment, we performed all quantitative
analyses on transgenic and control littermates. As an additional control, we
analyzed on OMP promoter-driven transgenic mouse line that overexpressed a
cell-surface protein (neuropilin 2) unrelated to RNCAM (data not shown).
Transgenic OMP-neuropilin 2 mice and littermate controls were found to have
the same number of semi-innervated P2 glomeruli 0.7±0.2 per OB (SEM,
n=10) and 0.6±0.2 per OB (SEM, n=8), respectively.
These results showed that the increased number of targeting errors was an
effect specific to RNCAM overexpression.
OR genes are subject to the phenomenon of monoallelic expression
(Chess et al., 1994).
Appearance of semi-innervated P2 glomeruli in mice homozygous or heterozygous
for the targeted P2 allele was similar
(Fig. 4D). This excluded the
possibility that the unstained fraction of axons in the semi-innervated P2
glomeruli exclusively corresponded to OSNs that express P2 from the untargeted
P2 allele because of allelic inactivation. Moreover, the OE in RNCAM
transgenic mice had a normal structure and normal expression of NADPHd
activity, endogenous RNCAM (Fig.
1E,F,G,H), neuropilin 2 and OR genes specific for Z1 (K21), Z2
(P2, K20), Z3 (L45) and Z4 (A16) (data not shown). These results taken
together with the predominant localization of RNCAM protein on axons indicate
that the increased number of incorrectly targeted P2 glomeruli was a
consequence of aberrant axon navigation within the OB, rather than
disorganization of OSNs within the OE itself.
Isoform-specific effect on topography of axon navigational
errors
Interestingly, we detected a three- to fourfold increase of incorrectly
targeted glomeruli in both TmRNCAM and GpiRNCAM transgenic mice
(Fig. 5B). A close examination
of the distribution of semi-innervated P2 glomeruli revealed that they were
confined to different domains of the OB, depending on which isoform was
overexpressed. In control mice, sporadic semi-innervated P2 glomeruli were
located within domains of the glomerular field that could be represented by
circles with a diameter of 200 µm being centered 100 µm caudoventral to
the major P2 glomeruli (i.e. in the vicinity of the major P2 glomeruli)
(Fig. 6A). The increase of
semi-innervated P2 glomeruli detected in TmRNCAM transgenic mice occurred
within the same domains (Fig.
6B; Fig. 7).
Although semi-innervated P2 glomeruli in control mice were slightly biased to
the lateral side, the reverse was true in TmRNCAM transgenic mice
(Fig. 6A,B;
Fig. 7). Notably, a different
pattern was evident in GpiRNCAM transgenic mice. In these mice,
semi-innervated P2 glomeruli were predominantly located within a domain with a
diameter of approximately 200 µm, centered 340 µm caudoventral to the
major lateral P2 glomeruli (Fig.
6C; Fig. 7). The
result indicated that elevated levels of TmRNCAM enhance a type of axon
targeting error that occurs normally close to the major P2 glomeruli, while
elevated levels of GpiRNCAM result in a different type of aberrant axon
navigation and targeting (se below). Moreover, TmRNCAM predominantly affected
axons projecting to the medial map, while GpiRNCAM selectively affected P2
axons to target incorrect glomeruli within a domain distant to the main
lateral P2 glomeruli.
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The location of an OR-specific glomerulus is almost constant between
individuals but some variations do occur
(Schaefer et al., 2001;
Strotmann et al., 2000
). This
local permutation in the glomerular array precluded a precise determination of
whether P2 axons co-converged with axons of OSNs expressing the very same OR
gene in different mice. Importantly however, semi-innervated P2 glomeruli in
GpiRNCAM transgenics showed a similar location in different mice, i.e.
330±16 µm (n=22) relative to the major lateral P2
glomeruli. Using the same way of measuring revealed a distance of
705±18 µm (n=22) between major medial and lateral P2
glomeruli. These results then indicated that the positions of invariant P2
glomeruli and semi-innervated P2 glomeruli showed a similar degree of local
permutation. The GpiRNCAM-specific phenotype thus appeared to be a consequence
of co-convergence of P2 axons with axons corresponding to a limited set of OR
identities.
Axons forming P2 semi-innervated glomeruli in GpiRNCAM mice `bypass'
major lateral glomeruli
The trajectories of axons forming semi-innervated P2 glomeruli in GpiRNCAM
mice was followed from the glomeruli towards OE by analyzing serial sections
of the OB (Fig. 4E). Some P2
axons segregated from the lateral division of P2 axons close to the major
lateral P2 glomeruli. These misguided P2 axons thus bypass their target, turn
posteriorly, course in an ventral direction and innervate a glomerulus with an
OR specificity that is different from P2
(Fig. 6C). Examination of P2
axons forming semi-innervated glomeruli in TmRNCAM transgenic and control mice
revealed a different type of trajectory. Instead of bypassing the correct
target glomeruli, P2 axons terminated within incorrect glomeruli on the way
towards the correct target glomeruli (Fig.
6B; Fig. 8). An
estimation of the location of semi-innervated P2 glomeruli by comparative
NADPHd histochemistry on serial sections revealed that incorrectly targeted
glomeruli were not in a region of the OB that corresponded to Z1
(Fig. 8). This result indicated
that axons of a defined Z2 OSN subpopulation do not co-converge with axons of
Z1 OSNs that ectopically express RNCAM. Thus, the result supported the
conclusion that the Z1/Z2-4 division of the glomerular field is formed
independently of spatial RNCAM expression.
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Suppression of GpiRNCAM-specific targeting errors in Gpi/Tm double
transgenic mice
An increased number of semi-innervated P2 glomeruli were detected in
transgenic mice overexpressing either of the two RNCAM splice variants. This
suggested that the isoforms fulfill similar functions. The results also
suggested that TmRNCAM and GpiRNCAM have distinct functions as convergence
errors were located to different domains of the OB, depending on isoform
overexpressed. Interestingly, mating TmRNCAM and GpiRNCAM mice generated
offspring with a phenotype similar to that of TmRNCAM transgenic mice. Thus,
TmRNCAM/GpiRNCAM double transgenic mice showed an increased number of
semi-innervated P2 glomeruli within a domain in the vicinity of major P2
glomeruli (Fig. 6D). Moreover,
the increase was biased toward the medial P2 glomerulus, similar to the
situation in transgenic mice overexpressing TmRNCAM only
(Fig. 7). This result indicated
that TmRNCAM inhibits formation of GpiRNCAM-specific targeting errors distal
to the lateral P2 glomerulus. TmRNCAM, on the other hand, exhibits a
GpiRNCAM-independent function that results in targeting errors proximal to
major P2 glomeruli.
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DISCUSSION |
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If RNCAM does not determine formation of a broad regional division of
projections, it is possible that the topographic distribution of RNCAM
expression has evolved to play a role in axon convergence and/or structural
plasticity for neurons in Z2-4. In fact, previous studies of OSN-expressing
tagged ORs corresponding to a RNCAM-negative Z1 OR (M72) and a RNCAM-positive
Z2 OR (P2) have provided evidence for heterogeneity in glomerular formation
during development and sensitivity to targeted deletion of an olfactory cyclic
nucleotide-gated channel subunit (Potter
et al., 2001; Zheng et al.,
2000
). To determine if RNCAM function influences axon convergence,
we have used the P2-IRES-tau-lacZ allele to probe for navigational
errors of P2 axons. One finding is that increased expression of RNCAM in OSNs
results in an increased number of glomeruli that are innervated by axons that
represent more than one OR identity. This indicates that normal level of RNCAM
is important for the formation of highly refined axon projections of OSNs, to
which RNCAM expression is normally confined.
Alternative splicing generates two RNCAM isoforms that share an identical
extracellular domain that mediates homophilic cell adhesion
(Yoshihara et al., 1997).
Different functions of RNCAM isoforms can be envisioned in light of the fact
that TmRNCAM has a cytoplasmic domain that potentially could mediate signaling
and/or coupling to the cytoskeleton (Walsh
and Doherty, 1997
). Like other Gpianchored proteins, GpiRNCAM may
also affect intracellular signaling and not only represent adhesiveness as
such (Davy et al., 1999
).
Interestingly, we find that overexpressing TmRNCAM increases the number of
targeting errors of P2 axons within domains in the immediate vicinity of their
original glomerular targets, while overexpression of GpiRNCAM results in
semi-innervated P2 glomeruli located distant from the lateral P2 glomerulus.
Moreover, TmRNCAM suppresses the GpiRNCAM-specific phenotype, while the
reverse is not true for the TmRNCAM-specific phenotype. We interpret these
results to suggest that topographic targeting of OR-defined axon subclasses is
influenced by two distinct RNCAM-dependent mechanisms.
We find both isoforms of RNCAM expressed on axons in the nerve layer and
the distribution of alternatively spliced transcripts in OE suggests that
layers containing a high proportion of maturing OSNs express both isoforms. It
is thus plausible that genetic interaction of GpiRNCAM and TmRNCAM is a
consequence of their co-localization on axons in the nerve layer during axonal
outgrowth and navigation. Misrouted P2 axons in GpiRNCAM transgenic mice
bypass the correct glomerular target and turn caudally before defasciculating
at an incorrect choice point, i.e. at a glomerulus with an OR identity
different from P2. The resolution of the experimental system allows us to
determine that incorrectly targeted glomeruli show a similar location in
different individuals. This phenotype indicates that elevated levels of
GpiRNCAM reduce the specificity of OR-specific target selection. Genetic
analyses in Drosophila have shown that alteration in the degree of
Fas2-mediated axon-axon attraction directly influences the ability of motor
axons to respond to target-derived attractants and repellents
(Fambrough and Goodman, 1996;
Winberg et al., 1998
). For
example, the combined function of Fas2 and semaphorins is involved in
controlling selective defasciculation important for target selection during
the generation of neuromuscular connectivity
(Winberg et al., 1998
;
Yu et al., 2000
). The
possibility that the mechanism of target selection in Drosophila is
phylogenetically conserved, in order to be applicable for OR-specific axon
segregation in mammals, is appealing given that neuropilin 2 expression
correlates with OR zones (Cloutier et al.,
2002
; Norlin et al.,
2001
). A GpiRNCAM-mediated change in the balance between RNCAM and
neuropilin 2-mediated axon-axon attraction and repulsion, respectively, may
thus increase the probability of defasciculation of P2 axons in an region of
the OB that corresponds to an incorrect OR zone.
Errant axons in TmRNCAM transgenic mice, unlike those in GpiRNCAM
transgenic mice, terminate within incorrect glomeruli on the way towards the
correct target glomeruli. Moreover, targeting errors were located close to
medial and lateral P2 glomeruli within domains in which sporadic
semi-innervated glomeruli are found also in control mice. Previous studies
have shown that OSNs expressing related ORs belonging to the same zonal set
tend to form glomeruli in close proximity to each other
(Conzelmann et al., 2001;
Malnic et al., 1999
;
Tsuboi et al., 1999
). Analyses
of tagged OR genes indicate that axons of highly related OR identities
segregate from each other during a short postnatal phase and that only a few
misprojections can be detected in glomeruli close to the correct target
glomerulus at 1 week of age (Conzelmann et
al., 2001
). It is thus conceivable that regulated levels of
TmRNCAM influences precision of targeting within a domain of glomeruli with
highly related OR identities or, alternatively, glomeruli that belong to the
same zonal set of OR identities.
The finding that increased levels of TmRNCAM enhance one type, and suppress
another type, of OR-specific targeting error is intriguing in light of the
fact that presence of TmRNCAM on the axonal surface appears to be regulated.
Yoshihara et. al. have reported that odor stimulation causes disappearance of
immunoreactivity corresponding to an epitope (R4B12) of the rabbit ortholog of
RNCAM (Yoshihara et al.,
1993). Diminished RNCAM immunoreactivity is limited to axon
terminals within glomeruli. The fact that we find TmRNCAM to be the
predominant isoform in glomeruli opens up the possibility that TmRNCAM and not
GpiRNCAM is regulated as a consequence of odor exposure. Others have shown
that increased intracellular cAMP results in selective internalization of the
Tm form of apCAM because of phosphorylation of a sequence (PEST) in the
intracellular domain (Bailey et al.,
1997
). As TmRNCAM contains a PEST sequence
(Yoshihara et al., 1997
), it
is tempting to speculate that formation and/or maintenance of precise
OR-specific connections is influenced by dynamic changes of surface-expressed
TmRNCAM levels. Supporting this notion is the fact that the intensity of RNCAM
immunoreactivity varies slightly between individual glomeruli (data not
shown).
Axons of OSNs expressing a given OR take two routes that project to
glomeruli located in either the medial or the lateral OB. An equal
representation of OR-specific glomeruli and OR-zones in both the lateral and
medial division suggests that each bulb has two symmetrical sensory maps
(Nagao et al., 2000).
Interestingly, the GpiRNCAM-specific phenotype is restricted to the lateral
mirror map, while OSNs contributing to the medial mirror map was more
sensitive to the effect of TmRNCAM. Our result thus suggests that the two
mirror maps in each bulb are not formed or maintained with identical
mechanisms. We do not find evidence for an uneven distribution of RNCAM mRNA
splice variants or RNCAM immunoreactivity in glomeruli along dorsoventral,
mediolateral and rostrocaudal aspects of OE or OB. Medially and laterally
projecting P2 OSNs may be intrinsically different. Alternatively, differential
sensitivity to the effects of the two RNCAM isoforms can be a consequence of
interactions with signals having medial and lateral distribution. To address
such issue it will be important to identify molecular cues that divide OSN
projections in two mirror image maps, as well as proteins that directly
interact with RNCAM.
Taken together, we have uncovered three important functional characteristics of RNCAM. First, RNCAM does not determine the division of OSN projections into Z1 and Z2-4, respectively. Second, normal regulated levels of RNCAM are important for accurate OR-specific axon segregation of Z2-4 OSNs. Finally, we identify specific activities of RNCAM splice variants that influence formation of precise topographic axon connections in the vicinity and distant relative to the correct target.
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ACKNOWLEDGMENTS |
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