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The Journal of Neuroscience, November 15, 1999, 19(22):9856-9864
Development of P2 Olfactory Glomeruli in P2-Internal Ribosome
Entry Site-Tau-LacZ Transgenic Mice
Stephanie J.
Royal1 and
Brian
Key2
1 Neurodevelopment Laboratory, Department of Anatomy
and Cell Biology, University of Melbourne, Parkville, Victoria,
3052 Australia , and 2 Neurodevelopment Laboratory,
Department of Anatomical Sciences, University of Queensland, Brisbane,
Queensland, 4072 Australia
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ABSTRACT |
Primary olfactory neurons project their axons to the olfactory
bulb, where they terminate in discrete loci called glomeruli. All
neurons expressing the same odorant receptor appear to terminate in a
few glomeruli in each olfactory bulb. In the P2-IRES-tau-LacZ line of
transgenic mice, LacZ is expressed in the perikarya and axons of
primary olfactory neurons that express the P2 odorant receptor. In the
present study, we examined the developmental appearance of P2 neurons,
the topographical targeting of P2 axons, as well as the formation of P2
glomeruli in the olfactory bulb. P2 axons were first detected in the
olfactory nerve fiber layer at embryonic day 14.5 (E14.5), and
by E15.5 these axons terminated in a broad locus in the presumptive
glomerular layer. During the next 5 embryonic days, the elongated
cluster of axons developed into discrete glomerulus-like structures. In
many cases, glomeruli appeared as pairs, which were initially connected
by a fascicle of P2 axons. This connection was lost by postnatal day
7.5, and double glomeruli at the same locus were observed in 85% of
adult animals. During the early postnatal period, there was
considerable mistargeting of P2 axons. In some cases P2 axons entered
inappropriate glomeruli or continued to grow past the glomerular layer
into the deeper layers of the olfactory bulb. These aberrant axons were
not observed in adult animals. These results indicate that olfactory
axons exhibit errors while converging onto a specific glomerulus and
suggest that guidance cues may be diffusely distributed at target sites
in the olfactory bulb.
Key words:
olfactory; P2 receptor; glomerulus; development; topography; guidance
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INTRODUCTION |
Primary olfactory neurons reside in
the olfactory neuroepithelium lining the nasal cavity and project axons
via the olfactory nerve to the olfactory bulb in which they terminate
in discrete tufts of neuropil referred to as glomeruli. Each olfactory
neuron appears to stochastically express one of at least 1000 different types of odorant receptor proteins. All neurons expressing the same
protein are mosaically distributed within one of four longitudinally oriented zones in the nasal cavity (Ressler et al., 1993 ; Vassar et
al., 1993; Strotmann et al., 1994a,b). The current dogma is that axons
from primary olfactory neurons expressing the same receptor converge
onto one or two glomeruli of a possible 1800 glomeruli in each
olfactory bulb (Royet et al., 1988; Ressler et al., 1994).
In 1996, Mombaerts et al. developed a transgenic mouse line in which
the axons of a subpopulation of primary olfactory neurons expressing a
specific odorant receptor could be visualized by histochemical staining
for  -galactosidase. The coding region of the P2 odorant receptor
gene was replaced with a construct encoding the P2 receptor and a
recombinant fusion protein consisting of the tau microtubule-associated
protein and -galactosidase. Histochemical staining for
-galactosidase allowed the trajectory of axons to be traced
because the tau--galactosidase fusion protein was anterogradely
transported. The P2 odorant receptor is expressed exclusively by
neurons in zone three of the olfactory epithelium [according to the
nomenclature of Buck and Axel (1991)], and the axons of these neurons
project to one lateral and one medial glomerulus per olfactory bulb.
When the P2 gene was deleted by homologous recombination, P2 axons
failed to form glomeruli (Wang et al., 1998). Moreover, when the coding
region of the P2 gene was substituted with the coding region of M12, an
odorant receptor expressed in zone one, P2 axons formed glomeruli in
aberrant locations (Mombaerts et al., 1996). Together, these results
indicate that odorant receptors play a role in axon guidance; however,
it remains unclear whether they are directly or indirectly involved.
Despite our understanding of the topography of the primary olfactory
projection, we have no clear picture of the principal morphological or
cellular events involved in the development of glomeruli innervated by
axons expressing the same odorant receptor. In the present study, we
have analyzed the development of the P2 subpopulation of primary
olfactory neurons in the olfactory neuroepithelium, as well as their
projections to the olfactory bulb using the P2-internal ribosome entry
site (FRES)-tau-LacZ line of transgenic mice (Mombaerts et al., 1996).
We show that P2 axons begin to target a specific site in the olfactory
bulb as early as embryonic day 15.5 (E15.5) and that the P2 glomerulus arises from an initially broad locus that contains a mixed
subpopulation of axons. In many cases, two glomeruli arise from a
single site, and axons exhibit mistargeting by overshooting the
glomerular layer or by projecting into inappropriate neighboring
glomeruli. Thus, the P2 glomerulus emerges slowly and involves
considerable axonal reorganization to achieve the highly
topographical projection observed in adults.
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MATERIALS AND METHODS |
Animals. Homozygous P2-IRES-tau-LacZ transgenic mice
(Mombaerts et al., 1996) were mated overnight, and seven embryos were collected at daily intervals from E12.5 to E18.5. The day of a positive
sperm dam was designated E0.5. Seven mice were also collected on each
of the following postnatal (PD) days: PD0.5, PD3.5, PD5.5, PD7.5, and
PD14.5. The day of birth was designated PD0.5. Seven adults (12 weeks)
were also collected. Animals were killed by cervical
dislocation, and heads were fixed in 4% paraformaldehyde for 30 min at room temperature and then stored in 30% sucrose at 4°C for 48 hr. PD14.5 and adult tissue were fixed for 4 hr at room temperature and
decalcified in 20% EDTA at 4°C for 1 and 3 weeks, respectively.
Tissue was then embedded in O.C.T. compound (Sakura Finetek USA Inc.,
Torrance, CA) and frozen, and serial coronal 60 µm cryostat sections
were collected on 2% gelatin and 0.1% chrome alum-coated slides.
5-Bromo-4-chloro-3-indolyl--D-galactropyranoside
histochemistry. Sections were washed for three 20 min periods in
wash buffer (0.1 M phosphate buffer, 2 mM MgCl2, 5 mM EGTA, 0.02% Nonidet P-40, and 0.01% sodium
deoxycholate) and then incubated at 37°C for 1.5 hr with stain buffer
[0.1 M phosphate buffer, 2 mM MgCl2, 5 mM EGTA, 0.02% Nonidet P-40, 0.01% sodium
deoxycholate, 1 mg/ml 5-bromo-4-chloro-3-indolyl--D-galactropyranoside (X-gal)
(Austral, Victoria, Australia), and 5 mM
potassium ferrocyanide]. The reaction was stopped with three 5 min
washes of PBS. Because sections were 60-µm-thick, it was not
possible to visualize glomerular boundaries using a nuclear
counterstain. Consequently, after dehydration, sections were
counterstained in 0.1% eosin for 9 sec. Three animals from each age
were examined.
Immunohistochemistry. Serial sections from four animals at
each age were incubated for 30 min in 2% bovine serum albumin
(Sigma, St. Louis, MO) in Tris-buffered saline (TBS) containing
0.3% Triton X-100. Sections were then incubated overnight at 4°C
with rabbit anti--galactosidase antiserum (1:300, 5 Prime  3 Prime Inc., Boulder, CO) and goat anti-olfactory marker protein (OMP)
(Keller and Margolis, 1975) in TBS containing 0.3% Triton X-100.
Sections were washed in TBS containing 0.3% Triton X-100 (three 5 min
washes) and incubated for 2 hr at room temperature with donkey
anti-rabbit immunoglobulins conjugated to tetramethylrhodamine
isothiocyanate (TRITC) (1:100; Jackson ImmunoResearch, West
Grove, PA) and donkey anti-sheep immunoglobulins conjugated to FITC
(1:50; Jackson ImmunoResearch) in TBS containing 0.3% Triton X-100.
Sections were subsequently washed in TBS (three 5 min washes)
and then mounted in glycerol. Fluorescence images were collected using
a Bio-Rad (Hercules, CA) MRC 1024 confocal laser scanning microscope,
with the 40 and 63× oil immersion lenses. Z-series images for TRITC
and FITC were collected every 1.5 µm through the depth of the section
and merged. Optical sectioning revealed continuity of staining
throughout the depth of the section. A total of 12 glomeruli were
analyzed at each age.
Image analysis. Digital images of X-gal-stained sections
were collected using a SPOT cooled color digital camera and SPOT 32 software (Diagnostic Instruments Inc., Sterling Heights, MI) with a
10× objective lens. Because sections were 60-µm-thick, some regions
of the photographs appear out of focus. An estimate of the volume of P2
glomeruli in adults was obtained by summing the cross sectional area of
P2 glomeruli in serial sections using the Image Pro Plus computer
program (Media Cybernetics, Bethesda, MD).
The number of P2 neurons was quantified by analyzing serial
X-gal-stained sections of three animals at each age. Only P2 neuronal perikarya projecting a dendrite were counted to prevent counting the
same neuron twice. A P2 neuron was considered to be in a cluster if it
was less than one cell width (10 µm) from an adjacent P2 cell. A
cluster consisted of three or more neurons in this configuration.
To determine the percentage of glomeruli exhibiting exuberant growth,
confocal images were assessed for the presence of axons deep to the
glomerular layer. The boundary between the glomerular layer and
external plexiform layer boundary was defined by OMP, and even at the
earliest ages analyzed, there was a clear boundary between these layers.
The distribution of P2 neurons in the nasal cavity was depicted in
camera lucida drawings obtained using a 10× objective lens. Images
were traced and scanned into Adobe Photoshop version 5 (Adobe Systems,
San Jose, CA) and compiled in CorelDraw version 8 (Corel Corporation
Ltd., Dublin, Ireland).
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RESULTS |
Expression of P2 in the olfactory neuroepithelium
Topology
The distribution of primary olfactory neurons that express the P2
odorant receptor protein (P2 neurons) was analyzed in P2-IRES-tau-LacZ homozygous transgenic mice from E12.5 to adults. Serial coronal sections were cut through the heads of these mice and histochemically stained for -galactosidase to reveal P2 neurons. At E12.5, the nasal
cavity consists of a simple chamber and a single rudimentary turbinate,
ectoturbinate 2 (Fig. 1). There are few
P2 neurons at E12.5; approximately three to five perikarya are present
in each section of the nasal cavity. At E14.5, P2 neurons are clumped at the tips of both ectoturbinate 2 and the newly emerged and more
caudally located ectoturbinate 3 (Fig.
1B,C). Both of these turbinates
fuse caudally with the roof of the nasal cavity (data not
shown).
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Figure 1.
Camera lucida images detailing the distribution of
P2 neurons in the olfactory neuroepithelium at different rostrocaudal
levels. Cryostat sections were stained using X-gal histochemistry, and
sections were chosen based on morphological detail. Regions of P2
expression were demarcated by black bands on camera
lucida drawings. Each age is represented by the right nasal cavity
only. Scale bar, 500 µm. 1, 2,
3, and 4 refer to ectoturbinates 1, 2, 3, and 4, respectively; I, II, and
III refer to endoturbinates I, II, and III,
respectively.
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Between E14.5 and E16.5, the morphology of the nasal turbinates becomes
considerably more complex. At E16.5, four new turbinates have emerged.
Ectoturbinate 1 has protruded from the roof of the nasal cavity (Fig.
1D), resulting in the separation of P2 neurons into
two distinct clusters (Fig. 1E, arrows). A
rudimentary endoturbinate II has formed at E16.5; however, it does not
possess any P2 neurons. Endoturbinate III arises from the ventrolateral
wall of the nasal cavity, and ectoturbinate 3 now produces a small
lateral recess when it fuses with the roof of the nasal cavity (Fig.
1F, arrowhead). Consequently, P2 neurons
on the tip of ectoturbinate 3 (Fig. 1E) become
continuous with the lateral cluster of P2 neurons in the roof
neuroepithelium (Fig. 1E, open arrow).
Caudally, ectoturbinate 4 (Fig. 1F) has projected
into the nasal cavity. The P2 neurons on the tip of this turbinate fuse
with the medial cluster of P2 neurons (Fig. 1F,
open arrow), on the roof of the nasal cavity.
There is little change in either the morphology of the nasal cavity or
the distribution of P2 neurons between E16.5 and E18.5. The only
notable difference is that the medial end of ectoturbinate 2 has
flattened, splitting its cluster of P2 into two groups (Fig. 1G, arrowheads). At PD0.5, the roof of the nasal
cavity broadens, and the lateral cluster of P2 neurons has divided
(Fig. 1K, arrows). Ectoturbinate 2 has
protruded further into the nasal cavity and fuses caudally with the
roof of the nasal cavity (data not shown). The P2 neurons present on
the tip of this ectoturbinate then become continuous with the P2
neurons on the roof of the neuroepithelium. At PD7.5, ectoturbinate 3 and endoturbinate III have increased in size, resulting in the
separation of P2 clusters on both of these turbinates into two groups.
There is very little change in the distribution of P2 neurons between
PD7.5, PD14.5, and adults, except for separation of clusters on
ectoturbinate 2 (Fig. 1S,W, arrowheads), ectoturbinate 4 (Fig. 1U,
arrowheads), and endoturbinate II (Fig. 1W,
arrows). In summary, this spatiotemporal analysis has
revealed that the complex distribution pattern of P2 neurons observed
in adults emerges from a simple band of P2 neurons at E12.5. As the
nasal cavity enlarges and new turbinates are added, the P2 neurons
appear in individual sections to become partitioned into segregated
clusters. However, analysis of serial sections reveals that P2 neurons
are distributed in continuous bands that merge along the rostrocaudal
axis of the nasal cavity.
Quantification
The number of P2 neurons was determined by counting all LacZ
stained perikarya in serial sections throughout the length of the nasal
cavity in three animals at each time point from E12.5 to adults (Fig.
2). There were 50 P2 neurons at E12.5
when the rostrocaudal length of the nasal cavity was 0.57 mm. This
number gradually increased to 390 by E16.5, at which time the nasal
cavity was 2.75-mm-long. During this same time period, there was also a
considerable increase in the complexity of the nasal cavity (Fig. 1)
and hence cross-sectional area of the olfactory neuroepithelium. By
PD1.5, there were 1185 positive neurons present, which was greater than
a 20-fold increase from E12.5. Although the number of P2 neurons
continued to increase postnatally, the rate of increase was clearly
reduced. Between PD1.5 and PD7.5, P2 neurons had merely doubled in
number. In adults, there were 13,729 P2 neurons, representing an
~10-fold increase over levels at PD1.5. It is interesting to note
that, in the rat, there is a corresponding decrease in the total number
of proliferating neurons throughout the same period (Weiler and
Farbman, 1997).
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Figure 2.
Quantification of the total number of P2 neurons
in both the left and right nasal cavities at different ages. The number
of neurons was seen to increase with age, the largest rise occurring
during embryogenesis. Three animals were counted at each age. Error
bars represent SEM. Where error bars appear to be absent, errors
were too small to be depicted.
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P2 neurons are restricted to a discrete annular band in the nasal
cavity, referred to as zone three, from as early as E12.5 (Fig.
3A). Previous studies have
indicated that primary olfactory neurons expressing a specific receptor
are randomly dispersed as individual neurons within zones (Buck and
Axel, 1991; Ressler et al., 1993; Vassar et al., 1993). However, our
qualitative analysis throughout the entire nasal cavity revealed that,
although P2 neurons are widely dispersed, they are either present as
individual neurons (Fig. 3B,E) or
located in small clusters (Fig. 3C,D). We
semiquantified the presence of clusters by counting P2 neurons found in
a group of three or more and whose perikarya were directly apposed or
less than one cell diameter apart (Fig. 3C). It should be
noted that our values may be underestimated because it was not possible
to maintain continuity in counts between adjacent sections.
Nonetheless, this analysis revealed that there were developmental and
maturational changes in the spatial relationship between P2 neurons
across the tangential surface of the neuroepithelium. For instance, 4%
of all P2 neurons were clustered at E14.5, whereas this value increased
to 12% at PD3.5. The level of clustering subsequently decreased,
reaching an adult-like level of 6% (Table 1).
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Figure 3.
Analysis of the distribution of P2 neurons and
their axonal projections. Coronal cryostat sections were stained using
X-gal immunohistochemistry (A) and X-gal
histochemistry (B-F, I).
A, P2 was expressed in neurons
(arrowheads) found in zone three (indicated by
broken line) of the olfactory neuroepithelium at E12.5.
B, A lone P2 neuron with its cell body at the base of
the olfactory neuroepithelium in adult. C, A cluster of
P2 neurons at PD3.5 (arrow) are distinct from adjacent
individual neurons (arrowheads). Individual neurons were
identified by focusing through the thick section. D,
Axons expressing P2 do not fasciculate (arrowheads) as
they pass through the olfactory neuroepithelium (PD14.5).
E, Once P2 axons have exited the olfactory
neuroepithelium, they form numerous small bundles within the submucosa
(arrows) in adult. Arrowheads depict
widely separated perikarya. F, Adjacent P2 neurons send
their axons in opposite directions (arrow) in the
submucosa (adult). G, H, P2 neurons in
other sensory neuroepithelia. Coronal cryostat sections were stained
using X-gal histochemistry. G, Two neurons expressing P2
are seen in the vomeronasal organ (PD3.5). H, At PD7.5,
a P2 neurons is present in the organ of Masera (arrow).
Scale bar, 100 µm. C, Cartilage; OE,
olfactory epithelium; S, nasal septum;
SM, submucosa; VNO, vomeronasal organ;
2, ectoturbinate 2.
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Table 1.
Percentage of neurons found in clusters and the percentage
of glomeruli with axons deep to the glomerular layer in three animals
at each age
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P2 neurons were also observed in small numbers in other regions of the
nasal cavity. Two to three P2 neurons were observed in the vomeronasal
organ of all animals from E16.5 (Fig. 3G) and in the organ
of Masera from E14.5 (Fig. 3H).
Projection of P2 axons in the olfactory nerve
We next examined the trajectory of the P2 axons between the nasal
cavity and the olfactory bulb. P2 axons did not fasciculate as they
exited the basal layers of the olfactory neuroepithelium, even when
they arose from neurons in close apposition (Fig. 3D, arrowheads). There did not appear to be any correlation
between position of perikarya in the neuroepithelium and the trajectory of axons in the submucosa. In some cases, axons clearly diverged into
different fascicles (Fig. 3F, arrow). P2 axons
penetrated the cribriform plate at numerous points along the
anteroposterior axis of the nasal cavity. Axons arising from the nasal
septum passed through the medial surface of the cribriform plate,
whereas those emerging from the lateral neuroepithelium entered the
nerve fiber layer through the lateral region of the cribriform plate.
Targeting of P2 axons to glomeruli
Topology
To investigate the formation of glomeruli by P2 axons, serial
sections of embryonic tissue were double-labeled with antisera to
-galactosidase and OMP and then analyzed by confocal microscopy. P2 axons were first observed in the nerve fiber layer at E14.5. By
E15.5, P2 axons had targeted a specific region on both the medial and
lateral surfaces of the olfactory bulb (Fig.
4A,B). However, unlike at E14.5, these axons had exited the nerve fiber layer
and penetrated the underlying glomerular layer in which they terminated
diffusely. Double labeling with OMP antiserum revealed that P2 axons
were intermingled with axons expressing other receptors in the
presumptive glomerular layer (Fig. 4B, arrow). At E17.5, the P2 axons began to cluster and target a
specific region in the glomerular layer (Fig. 4C). By E18.5,
the P2 axons had formed two to three tufts that were interconnected by
bundles of axons (Fig. 4D, arrows).
Between E18.5 and PD7.5, these tufts either condensed and formed a
single glomerulus or instead separated into two discrete glomeruli that
were connected by a bundle of axons (Fig. 4E). By
PD14.5, all interconnected glomeruli had separated and formed discrete
glomeruli (Fig. 4F). Mombaerts et al. (1996) reported
that <5% of adult mice possess two glomeruli innervated by P2 axons
at the same locus. In contrast, we found that interconnected glomeruli
were present at all ages between PD0.5 and PD7.5 and that double
glomeruli were found in 85% of adults examined. These glomeruli
appeared as an adjacent pair or were separated by up to five glomerular
widths from their partner. Double glomeruli were also three times more
likely to be present on the medial rather than on the lateral surface
of the bulb. In some adults, extra glomeruli formed in the posterior
ventromedial region of the olfactory bulb (Fig.
5A,B).
These glomeruli were only partially innervated by P2 axons (Fig.
5C,D). The P2 axons terminated in a discrete
subregion of the glomerulus, either with a diffuse (Fig. 5C)
or dense (Fig. 5D) distribution.
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Figure 4.
Development of glomeruli in the
olfactory bulb. A-D, Double-label
immunohistochemistry of coronal cryostat sections stained for
-galactosidase (green) and OMP
(red). A single image for -galactosidase is presented
in A, and merged images for OMP and -galactosidase
are presented in B-D. B is the same
section as A. E, F, Coronal cryostat sections stained
with X-gal histochemistry. A, Confocal image of
-galactosidase at E15.5 reveals P2 axons are diffusely dispersed
within a localized area of the olfactory bulb (dotted
lines). B, Identical tissue section as
A, disclosing both OMP and -galactosidase, shows
dispersed axons in the glomerular layer (arrow).
C, By E17.5, P2 axons have begun to condense in the
glomerular layer. D, At E18.5, individual glomeruli
become visible (arrows) but remain connected by large
bundles of axons. E, A glomerulus adopts a rounded
morphology by PD0.5, although it remains joined by a band of axons.
F, In the adult, glomeruli are completely separated.
G, Schematic representation of double-glomerular
development. Scale bar, 50 µm. EPL, External plexiform
layer; GL, glomerular layer; MCL, mitral
cell layer; NFL, nerve fiber layer.
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Figure 5.
A-D, Partial innervation of
glomeruli by P2 axons. Coronal cryostat sections stained for X-gal
histochemistry. C and D are high-power
images of A and B, respectively.
A, C, P2 axons are seen to completely
innervate the target medial glomerulus (filled
arrow), whereas P2 axons are dispersed throughout a subregion
of the extra glomerulus (open arrow). B,
D, A subregion of the extra glomerulus is innervated by
P2 axons (the glomerular border is defined by a broken
line). Scale bar, 100 µm.
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Some primary olfactory axons have been observed to extend beyond the
boundary of the target glomerulus and inappropriately penetrate the
external plexiform layer during both the embryonic and early postnatal
periods (Key and Akeson, 1993; Tenne-Brown and Key, 1999). To
investigate whether P2 axons display similar aberrant trajectories,
serial sections of olfactory bulb were double-labeled with antisera
against -galactosidase and OMP. Between E16.5 and PD7.5, individual
P2 axons were observed to project through the glomerular layer and into
the external plexiform layer. These axons were always OMP-negative and
either bypassed their target glomerulus or passed directly through the
glomerulus. Analysis of compiled scans collected through the thickness
of the section at PD3.5 revealed that some misguided P2 axons coursed over the surface of adjacent P2-negative glomeruli (Fig.
6A,B, arrows). At PD7.5, misguided axons were observed to branch
(Fig. 6C,D, arrows), some axons
coursed radially toward the mitral cell layer (Fig.
6E,F, arrowheads),
whereas others appeared to turn around and project back toward the P2
glomerulus (Fig. 6A-D, arrowheads). As
animals increased in age from PD0.5 to PD7.5, the extent of exuberant
growth decreased until it was no longer present in adults (Table 1).
Our analysis of compiled scans through the topographically fixed
glomeruli revealed that P2 axons arborized throughout the whole width
and depth of glomeruli.
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Figure 6.
A-F, Behavior of axons targeting
glomeruli in the early postnatal period. Coronal cryostat sections
immunostained for -galactosidase (green) and
OMP (red). A, C, and
E are single images of -galactosidase staining only;
B, D, and F are merged
images of -galactosidase and OMP. A,
B, At PD3.5, the growth cone of a P2 axon
(arrow) is seen at the surface of an adjacent
P2-negative glomerulus. Axons loop back into the target glomerulus
(arrowhead). C, D, At
PD7.5, a P2 axon exits the target glomerulus
(arrowhead), enters the mitral cell layer, and turns
back to the glomerular layer. This axon also branches deep to the
glomerular layer (arrow). E,
F, At PD7.5, a P2 axon (arrowhead) passes
through the glomerular layer and heads toward the mitral cell layer.
Scale bars, 100 µm. EPL, External plexiform layer;
GL, glomerular layer; NFL, nerve fiber
layer.
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Mombaerts et al. (1996) suggested that the topographical position
of P2 glomeruli along the rostrocaudal axis remained constant between
animals. We determined the position of the P2 glomeruli in both the
left and right olfactory bulbs of three adult animals by measuring the
distance from the rostral pole of the bulb to the beginning of the
lateral glomerulus and from the rear of this glomerulus to the
beginning of the medial glomerulus. This analysis revealed that the
positions of the P2 glomeruli were topographically fixed within
~180 µm; the lateral glomerulus was 1440 ± 75 µm from the
front of the bulb, and the medial bulb was 770 ± 90 µm caudal
to this position.
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DISCUSSION |
Although P2 axons target specific loci in the olfactory bulb as
early as E15.5, we show that discrete glomeruli emerge slowly and that
navigational errors occur for up to the next 10 d of development.
In addition to terminating in two topographically fixed glomeruli,
there are many instances in which P2 axons also innervate seemingly
inappropriate extra glomeruli. In some cases, P2 axons converge onto
subregions of these extra glomeruli, indicating that an individual
glomerulus is innervated by distinct subpopulations of axons expressing
different odorant receptor genes.
Previous studies have reported that primary olfactory axons expressing
the same odorant receptor project to one of two topographically fixed
glomeruli in the olfactory bulb of adult mice (Ressler et al., 1994;
Mombaerts et al., 1996; Wang et al., 1998). Real-time analysis of the
developing olfactory nerve in zebrafish embryos demonstrated that
olfactory axons navigate directly to specific loci in the olfactory
bulb (Dynes and Ngai, 1998). This has led to the suggestion that the
targeting of these axons to specific glomeruli during development is
precise and without error. However, tracing of individual axons in both
rats and mice during the formation of glomeruli revealed there are
considerable targeting errors (Tenne-Brown and Key, 1999). Our results
here clearly demonstrate that the homing of functionally discrete
subpopulations of olfactory axons to their glomerular targets is not
without error during development. Interestingly, the topographical
localization of extra glomeruli that form as a result of aberrant
targeting are not fixed in space. This projection pattern has
implications for our understanding of olfactory perception because it
indicates that the glomerular coding of odorants is not preserved
between individuals.
Temporal regulation of P2 receptor expression
Few neurons express P2 in the olfactory neuroepithelium at E12.5,
but the number rapidly increased up to E18.5, rising approximately twofold to threefold every 2 d. Although this increase is
consistent with the generation of new P2 neurons over time, it is also
possible that the P2-IRES-tau-LacZ construct is gradually expressed in a preexisting population of neurons generated early in development. However, the expression of P2 in growing axons in vitro
(our unpublished observations) suggests that P2 is expressed in
differentiating neurons, which are constantly being produced in the
olfactory neuroepithelium. Moreover, the large number of these neurons
in the adult supports the de novo generation of P2 neurons
throughout development and into adulthood. The number of P2 neurons
increased postnatally, doubling between PD1.5 and PD7.5 and again from
PD7.5 to PD14.5, which is in agreement with the overall increase in the
size of the olfactory bulb in mice and rats during this period (Pomeroy
et al., 1990). This continual increase in P2 neurons during the
postnatal period may not be typical of all olfactory neuron
subpopulations. For instance, the subpopulation of neurons expressing
the odorant receptor OR3 remains relatively stable between PD0 and PD20
(Nef et al., 1992).
Non-stochastic mechanisms for neuronal specification
The number of neurons in the adult has been determined for the
murine olfactory receptor subfamilies K4, K7, and K18 (Ressler et al.,
1993). Each of these subfamilies contains between 10 and 15 different
receptor genes. The total number of neurons in both sides of the animal
expressing these receptors varied from ~4500 to 8200 and was
considerably lower than the 13,729 we observed in adult P2 mice. Thus,
there appears to be considerable variability in the number of neurons
expressing an individual receptor. This variability is not caused
by differences between zones because the K4 and K7 subfamilies
and the P2 receptor are all expressed in the same zone in the nasal
cavity. Moreover, the variability is not a result of interanimal
variability because the number of positive P2 receptor neurons present
was relatively constant between animals.
What are the mechanisms that control the specification of receptor
phenotype? One possibility is that inductive signals from the
underlying mesenchyme delineate zonal boundaries in the olfactory neuroepithelium during early embryogenesis. Although the regulatory mechanisms are unknown, it is clear that there are independent processes controlling expression of receptor genes within zones and the
selection of one of the four zones within the neuroepithelium (Qasba
and Reed, 1998). The choice of zone probably then determines which set
of olfactory genes are competent to be expressed. Each neuron within a
zone may stochastically express one receptor from a restricted
repertoire. However, a purely stochastic mechanism is unlikely because
of the wide variability in size of receptor subpopulations within a
zone. One would predict that stochastic mechanisms would instead
generate subpopulations of similar size, at least within the same zone.
Alternatively, it is possible that differences in final number arise
because receptors begin to be expressed at different embryonic ages.
Perhaps the repertoire is developmentally regulated (Barth et al.,
1996) and the size of the receptor population decreases as the onset of
expression begins at later embryonic ages. In this case, receptor type
could still be stochastically selected, but it would be from a
repertoire that changes during development. However, Barth et al.
(1996) have shown that the final adult receptor number is independent of when it was first expressed in the olfactory neuroepithelium of
zebrafish. Moreover, the K4 subfamily of receptors begins to be
expressed at the same age (Sullivan et al., 1995) as the P2 receptor,
indicating that final receptor number is not correlated with the time
of onset of expression. In summary, it appears that stochastic
mechanisms are not operating to choose the receptor expressed from a
zonal repertoire.
Formation of glomeruli
Valverde suggested that protoglomeruli delimited by periglomerular
cells are apparent at E17 in rat (Santacana et al., 1992; Valverde et
al., 1992). These protoglomeruli were defined by anterogradely tracing
axons to small defined loci in the olfactory bulb. However, by
specifically examining the terminations of P2 axons in the olfactory
bulb, we were able to define, for the first time, the major
morphological events in the development of a chemically identifiable
glomerulus (Fig. 4G). Initially, at E15.5, axons randomly
penetrate the presumptive glomerular layer in the region destined to
contain the target glomerulus. By E17.5, the axons have compacted, and
at E18.5, the outline of single or numerous glomeruli can be recognized
in the glomerular layer. When multiple glomeruli are present at the
same site, they continue to be interconnected by a thick band of axons
up to PD7.5. Individual discrete glomeruli are only observed from this
age on. It appears that glomerular cues are initially widespread, which
is consistent with recent reports of targeting errors in early
postnatal animals (Tenne-Brown and Key, 1999).
Errors in axon targeting
In the present study, we have shown that P2 axons clearly exhibit
errors in laminar growth during the early postnatal period, as well as
during embryogenesis. At E16.5, P2 axons were observed growing beyond
the glomerular layer into the deeper layers of the olfactory bulb. This
exuberant growth was present in 55% of specific target sites examined
and persisted until PD3.5. After this age, the prevalence of
overshooting axons sharply declined. Most notably, mistargeting dropped
after P7.5 from 20% to no evidence of mistargeting in adult animals.
Although these results suggest that P2 neurons generated during this
period do not exhibit as much mistargeting as younger axons, it should
be pointed out that fewer axons are probably entering each glomerulus
in adults than at early ages. Thus, we may need to examine more animals
at these later stages to observe mistargeting.
Our detailed serial section analysis has now revealed that the
innervation pattern of P2 axons in the olfactory bulb is more complex
than previously described. We have found that P2 axons typically
terminate in two to four glomeruli per olfactory bulb. In the P2 mice,
there was no consistency in the presence or location of extra
glomeruli. A few animals had no extra glomeruli in either bulb, some
mice exhibited an extra glomerulus only at the lateral surface of one
bulb, whereas other animals possessed extra glomeruli at all sites. The
extra glomeruli were more likely to occur in the medial position than
the lateral position; however, there was no preference shown for either
the left or right bulb. In some cases, extra glomeruli were immediately
adjacent, whereas others were separated by at least several glomeruli.
The extra glomeruli vary in size, and analysis of the area of glomeruli
in each position revealed the total glomerular area was consistent
across both bulbs, suggesting P2 neurons must create a fixed glomerular
volume independent of how many glomeruli they form. Previous work has
shown glomeruli are innervated by distinct subsets of olfactory neurons
(Treloar et al., 1996), and we have confirmed this observation for
axons expressing P2 receptors. We observed glomeruli in which P2 axons
terminated in subregions. This is the first demonstration that more
than one subpopulation of axons expressing different receptors
innervate the same glomerulus. It is possible that these glomeruli
represent the specific targeting of axons expressing more than one
receptor or that the guidance cues for another receptor are so similar
to those for P2 that some P2 axons terminate inappropriately.
| |
FOOTNOTES |
Received May 21, 1999; revised Aug. 13, 1999; accepted Aug. 18, 1999.
This work was supported by a National Health and Medical Research
Council Grant 970333 to B.K. and by a University of Melbourne Research
Scholarship to S.J.R. We thank Dr. P. Mombaerts for providing the
P2-IRES-tau-lacZ mice and Dr. F. Margolis for OMP antiserum.
Correspondence should be addressed to Brian Key, Neurodevelopment
Laboratory, Department of Anatomical Sciences, University of
Queensland, Brisbane, Queensland, 4072 Australia. E-mail:
brian.key{at}mailbox.uq.edu.au.
| |
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TOP
ABSTRACT
INTRODUCTION
