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 Previous Article | Next Article 
The Journal of Neuroscience, November 1, 1998, 18(21):8919-8927
A Transient Population of Neurons Pioneers the Olfactory Pathway
in the Zebrafish
Kathleen E.
Whitlock1 and
Monte
Westerfield2
1 Section of Genetics and Development, Cornell
University, Ithaca, New York 14853-2703, and 2 Institute of
Neuroscience, University of Oregon, Eugene, Oregon 97403-1254
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ABSTRACT |
Mechanisms guiding the first axons from the olfactory placode of
the peripheral nervous system (PNS) to the olfactory bulb in the
vertebrate CNS are unknown. We analyzed the initial outgrowth of
axons from the olfactory placode in zebrafish and found a precocious transient class of pioneer neurons that prefigure the primary olfactory
pathway before outgrowth of olfactory sensory axons or expression of
olfactory receptor genes. Not only are the pioneers antigenically,
morphologically, and spatially distinct from olfactory sensory neurons,
they are also developmentally distinct; via fate mapping, we show that
they arise from a more anterior region of the lateral neural plate than
do the first sensory neurons. After the axons of the sensory neurons
grow into the CNS, the pioneer neurons undergo apoptotic cell death.
When we ablated the pioneers before axonogenesis, the following sensory
axons showed severe misrouting. We propose that the pioneers provide
the first necessary connection from the PNS to the CNS and that they
establish an axonal scaffold for the later-arriving olfactory sensory
neurons.
Key words:
fate map; cell death; olfactory neurons; axon guidance; olfactory receptors; cell lineage
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INTRODUCTION |
The olfactory organ is a unique
cellular population that arises outside the CNS from the ectodermally
derived olfactory placode (Farbman, 1992 , 1994; Crews and Hunter,
1994). Initially, the olfactory placode is an apparently
homogeneous population of cells. As the placode differentiates into the
olfactory epithelium, stratified cell types characteristic of the adult
epithelium appear, including basal cells, sensory neurons, and support
cells. Throughout life, the basal cells generate olfactory sensory
neurons that migrate toward the apical surface of the epithelium while
extending axons and dendrites (Menco and Farbman, 1985; Farbman, 1994).
The cell bodies of the olfactory sensory neurons remain in the
epithelium, and their axons grow through the olfactory nerve into the
differentiating CNS. Once within the CNS, the axons segregate to form
glomeruli, clustered grape-like structures in the olfactory bulb
(Shepherd, 1972). The glomerular organization of olfactory sensory
afferents is characteristic of both invertebrates and vertebrates
(Christensen and Hildebrand, 1987; Farbman, 1992). Within a species,
the glomeruli are organized in a stereotyped pattern in the olfactory
bulb (Graziadei and Graziadei, 1979; Baier and Korsching, 1994; Byrd
and Brunjes, 1995; Rupp et al., 1996).
Little is known about how the axons of the first olfactory sensory
neurons navigate to the CNS and initiate patterned connections with the
developing olfactory bulb. Dye labeling has shown that individual
glomeruli in the olfactory bulb receive inputs from sensory neurons
scattered throughout the olfactory epithelium (Riddle and Oakley,
1991). Recent studies indicate that the axons of sensory neurons
expressing the same olfactory receptor converge on the same glomerulus
(Mombaerts et al., 1996), express receptor transcript in their
axon terminals (Ressler et al., 1993; Vassar et al., 1994), and respond
to the same small subset of odorants (Zhao et al., 1998). This has led
to the suggestion that olfactory receptors may play a role in sensory
axon guidance (Singer et al., 1995). These studies did not examine the
first connections between the olfactory placode and the telencephalon;
thus, whether receptors play a role in the initial connection from the
periphery to the bulb is unknown.
Because of their rapid development and the accessibility of early
developmental stages, we have used zebrafish to analyze the initial
events that connect the axons of the olfactory sensory neurons with the
developing bulb. We have discovered a precocious class of neurons, the
pioneer neurons, which precede the olfactory sensory neurons, establish
initial contact with the telencephalon, and then die. The pioneer
neurons are identifiable by their unique location and morphology within
the olfactory epithelium, their antigenicity, and their origin from a
more anterior region of the neural plate than that which gives rise to
the first sensory neurons. By ablating the pioneers, we show that they
are necessary to target the incoming axons of the sensory neurons to
the developing olfactory bulb. Because the pioneer neurons appear not
to express any of the known olfactory receptor genes, it is unlikely
that olfactory receptors participate in the initial connection between the olfactory placode and the developing telencephalon.
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MATERIALS AND METHODS |
Animals. Zebrafish embryos were obtained from natural
spawnings of wild-type (AB) fish. Fish were staged by morphology, and times of development are expressed as standard stages (Kimmel et al.,
1995) or as hours after fertilization (h) at
28.5°C.
Antibodies and immunocytochemistry. Embryos were fixed
overnight at 4°C in 4% paraformaldehyde in 0.1 M
phosphate buffer with 0.15 mM CaCl2 and 4%
sucrose, pH 7.2. After washes in 0.1 M phosphate buffer and
dH2O, embryos were permeabilized in acetone (7 min at
 20°C), washed in dH2O followed by 0.1 M
phosphate buffer, and then preadsorbed in 2% normal goat serum (NGS)
in PBDT [0.1 M phosphate buffer, 2% bovine serum
albumen, 1% dimethylsulfoxide (DMSO), and 0.5% Triton X-100]. A
mouse monoclonal antibody, zns-2 (Trevarrow et al., 1990), was used
(1:300) in PBDT with 2% NGS to label the pioneer neurons. In addition
to pioneer neurons in the olfactory placodes, zns-2 also labels neurons
in the trigeminal ganglia. Embryos were incubated in this primary
antibody overnight at 4°C. They were then rinsed three times over 2 hr and placed in secondary goat anti-mouse antibody (1:200; Sternberger
Monoclonals Inc.) or FITC-conjugated goat anti-mouse (1:200; Sigma, St.
Louis, MO) in PBDT with 2% NGS overnight at 4°C. FITC-labeled
preparations were then mounted in PBS, viewed using a Zeiss
(Oberkochen, Germany) confocal microscope, and analyzed with the Voxel
View program (Vital Images). Embryos processed for
HRP-diaminobenzadine (DAB) coloration were washed five times over 2 hr
and incubated in peroxidase-mouse antiperoxidase at 1:500 in PBDT with
2% NGS (Sternberger Monoclonals Inc.) overnight at 4°C. Embryos were
then rinsed in 0.1 M phosphate buffer and 1% DMSO three
times for 1 hr. The coloration reaction was done using 0.02% DAB in
1:1 dH2O/PBS with 1% DMSO and 0.003% hydrogen peroxide.
Fixed embryos were prepared for sectioning by dehydrating in an alcohol
series (30, 50, 70, 95, 2 × 100%), embedding in Epon
(Westerfield, 1995), and cutting 7.5 µm serial sections, which were
then covered in Permount and coverslipped. A variety of
antibodies known to label vertebrate olfactory neurons, including
neural cell adhesion molecule (NCAM) and olfactory marker protein, were tried with no success. In addition, the antibody reported to mark pioneers in the mouse trigeminal (Stainier and Gilbert, 1990) does not work in zebrafish.
RNA in situ hybridizations. RNA in
situ hybridizations were performed using digoxigenin-labeled RNA
probes (Thisse et al., 1993) for the olfactory receptors. Probes were
generated from zebrafish olfactory receptor cDNAs 2, 4, 9, and 13 (Barth et al., 1996). Based on the number and timing of cells
expressing receptor mRNA, the sensitivity of the digoxigenin-labeled
probes appeared equal to that of the radio-labeled probes used by Barth
et al. (1996). Each preparation was drawn using a camera lucida,
and the cells expressing olfactory receptors were scored as being in
the basal part of the olfactory organ if they were within two cell
diameters (~20 µm) of the basal surface. Otherwise, they were
scored as apical.
Double labeling immuno-RNA in situ
hybridization. These procedures were combined by first performing
immunocytochemistry with DAB coloration using the zns-2 antibody as
described above, except that RNAsin (Boehringer Mannheim, Indianapolis,
IN) was added to all blocking-incubation solutions. This was
followed by RNA in situ hybridization. It should be noted
that the quality of the signal when double labeling is dependent on the
antibody. The zns-2 antibody shows a decrement in signal after the RNA
in situ hybridization, and for some antibodies there is a
total loss of signal.
Dye labeling. Olfactory neurons were labeled with the
carbocyanine dye DiI (DiIC18(3); Molecular Probes, Eugene,
OR) by adding a 0.5% solution of the dye to the embryo medium. The
fish were then rinsed three to five times in embryo medium and placed
in a clean dish. The live fish were anesthetized (Westerfield, 1995), mounted ventral side up in methyl cellulose, and viewed by fluorescence confocal microscopy. Images were collected on a confocal microscope (Zeiss) and analyzed with the Voxel View program (Vital Images).
Terminal deoxynucleotidyl transferase-mediated biotinylated UTP
nick end labeling (TUNEL). Embryos were fixed
overnight in 4% paraformaldehyde, rinsed several times in PBS, and
dehydrated in a graded methanol series. The preparations were then
treated with MeOH/H202 (5:1) and rehydrated
into 10× Tris-buffered saline (TBS) (1 M Tris base and 1.5 M NaCl, pH 7.5), followed by two washes in TBS for 5 min
each. After a 30 min digestion in Proteinase-K (1 µg/ml) at 37°C,
they were washed three times for 5 min each in 10× TTBST (10×
TBS, 2% Triton X-100, and 5% Tween-20) and two times for 5 min each
in 5× TTase buffer (125 mM Tris buffer, 1 M Na Cacodylate, 1.25 mg/ml BSA, 1% Tween-20, and 1 mM CoCl2). Reaction solution was then added [100 µl of
TTase buffer, 0.5 µl of TTase (Boehringer Mannheim), and 0.25 µl of
fluorescein dUTP (Boehringer Mannheim)], and the preparations were
preincubated on ice for 1 hr, incubated at 37°C for 1 hr, washed two
times in TTBST, and washed overnight at 4°C. The preparations were
mounted in PBS and viewed with the confocal microscope.
Cell labeling and lineage tracing. Embryos at the four
somite stage were embedded in agar with a window cut to expose the lateral edge of the neural plate. These blocks were mounted on a
microscope slide and covered with physiological saline. Microelectrodes (~300 M resistance) were pulled (Sutter Instruments), backfilled with a mixture of Rhodamine dextran dye [2%, molecular weight (MW)
3000; Molecular Probes] and Fluorescein dextran dye (2%, MW 3000;
Molecular Probes) in 0.2 M KCl. Cells were visualized with
a 40× water immersion lens using a fixed-stage microscope (Zeiss).
After penetration of a single cell, dye was passed into the cell by
using capacitance compensation to create small voltage changes
(Westerfield, 1995). Successful filling of the cell was judged by a
rapid check of the Rhodamine fluorescence. Preparations for
lineage tracing were allowed to develop to the desired age, and then
fluorescent images were collected on a confocal microscope (Zeiss) and
reconstructed using the Voxel View and Voxel Math programs (Vital
Images).
Ablations. Animals were anesthetized and then rinsed and
mounted in agarose blocks ventral side up, exposing the anteroventral aspect of the olfactory placode. At the time when the olfactory placodes were first evident, 18-20 h, the pioneer cells were carefully cut out of the anteroventral part of the placode under a 40×
dissecting microscope using glass microneedles. Glass microelectrodes
were used because laser ablation of all cells in the 15-20 cell
cluster was unreliable, and removal by suction destroys the integrity of the olfactory placode. The cells of the olfactory placode are separate from the developing telencephalon, allowing the removal of the
pioneer cells without damaging the telencephalon. The pioneer cells
were ablated from a single olfactory placode per animal. The intact
side served as a control for assessing both the general health of the
embryo after ablation and the quality of the zns-2 labeling (used to
score the presence of pioneer neurons). The embryos were fixed at 48 h
and labeled with DiI to reveal sensory neuron morphology. Of the 35 total preparations operated on at 18-20 h and analyzed for sensory
neuron misrouting, 17 were successfully labeled with zns-2 (Table
1). In all experiments, there were some
preparations with general growth defects and/or poor DiI labeling;
these were not included in the analysis.
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RESULTS |
In zebrafish, the olfactory placode first appears at 17 h after
fertilization as a thickening of the ectoderm [Fig.
1A, H, (A,B)], and this thickening later invaginates to form the
naris by 32 h (Whitlock and Westerfield, 1995, 1997). We found
that the zns-2 antibody (Trevarrow et al., 1990) recognized neurons whose axons first emerged from the olfactory placode at 24 h. At this
time and during the second day of development, there was an average of
11 pioneer neurons (± 2; n = 23 olfactory placodes) labeled in each olfactory placode. These neurons had large round cell
bodies (Fig. 1B, arrowhead) and axons that
extended along the lateral edge of the telencephalon (Fig.
1B, arrow). No dendrites were apparent in
live embryos using Nomarski optics or using the antibody (Fig.
1B, arrowhead) in fixed preparations.
Because these are the first neurons seen with axons emerging from the
olfactory placode, thus initiating the connection between the olfactory placode and the developing telencephalon, we identified them as pioneers. The region in which the zns-2-positive axons (Fig.
1C,D, black arrows) meet the
developing telencephalon expresses emx1 (Fig.
1C,E, white arrows), a gene that
is expressed in the developing telencephalon (Simeone et al., 1992;
Morita et al., 1995). By 30 h, the axons of the pioneer neurons had
extended into the developing telencephalon and had started to
defasciculate, and by 38 h, axonal condensations of zns-2-positive
axons (shown at 38 h in Fig. 1F, white
arrow) started to appear in the presumptive olfactory bulb. These axons penetrated the surface of the telencephalon and extended into its most anterior region. By 52 h, distinct condensations of
zns-2-positive axons were apparent in the developing bulb (Fig. 1G, white arrow, arrowhead;
I, arrowhead. These axonal condensations had a
very fibrous appearance in both whole mounts (Fig. 1G,
arrowhead) and sectioned material (Fig.
1I, arrowhead). The zns-2 labeling of cell
bodies in the olfactory epithelium was greatly diminished by 42-44 h
and was no longer detectable after 48 h, although axonal labeling
remained strong.
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Figure 1.
The pioneer neurons initiate the formation
of the olfactory nerve and establish glomerular-like structures in the
telencephalon. A, B, Frontal views with
dorsal to the top. C-F, Dorsal views
with anterior to the top. G, Ventral view
with dorsal to the top. H, Diagrams
labeled with letters corresponding to
panels to show orientation. The second diagram is a side
view, anterior to the left at a stage equivalent to the
frontal view in H (A, B).
The olfactory placodes are in red, and the eyes are in
gray. I, A transverse section with
anterior to the top. A, Frontal view of
the head of a 24 h live zebrafish embryo. The olfactory placodes
(op) are paired thickenings lying dorsoanteriorly. The
broken line outlines the placode on the
left. B, Enlargement of a frontal view of
a placode labeled with the zns-2 antibody at 24 h. Dendrites are absent
on the apical surface (arrowhead), and the axons
(arrow) exit basally and grow along the telencephalon.
The broken line demarcates the edge of the placode.
C, The pioneer axons (brown-blue,
black arrow) meet the telencephalon in the region
expressing the gene emx1 (purple,
white arrow) in a 26 h embryo. D,
E, A 30 h preparation showing two focal planes, labeled
only with zns-2. The axons exit the placode (black
arrows) and defasciculate in the telencephalon (white
arrows). The broken line demarcates the
posterior edge of the placode. F, At 38 h, the pioneer
axons (white arrow) start to form condensations in the
telencephalon. G, Axons labeled with zns-2 (black
arrow) extend into the developing olfactory bulb in a 52 h
embryo. Note the axonal condensations (white arrow)
evident in the CNS. H, The developing olfactory
placode has moved from a dorsal to a more anterior location in front of
the developing eye (see A, B). As a
result of this morphogenetic movement, the zns-2-labeled axons are more
easily viewed from the ventral side (G,
I) as they project anteriorly into the
telencephalon. I, A 7.5 µm Epon section through
the olfactory organ and bulb at the same developmental stage as
G, with the axonal condensations indicated by the
arrowhead. Scale bars: A, 40 µm;
B, 25 µm; C-F, 35 µm;
G, I, 40 µm.
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During the second and third days of development, the olfactory nerve
became prominent, and the zns-2-labeled axons of the pioneer neurons
(Fig. 2A,
arrow) formed more complex axonal condensations (Fig.
2A, arrowhead) within the bulb. A second
wave of neurons, distinct from the pioneers, was visualized by 44 h,
after the naris had opened. These later-developing neurons had
dendrites, which projected to the naris and, thus, could be labeled by
external application of DiI (Fig. 2B). In contrast,
the pioneer neurons had no obvious dendrites and were unlabeled by this
technique (Fig. 2B, asterisk). Because
these later-developing cells had the morphology of sensory neurons and
dendrites in contact with the outside world, we identified them as
sensory neurons. The first of these later-developing sensory neurons
(Fig. 2B, arrow) extended axons into the
developing olfactory bulb (Fig. 2B,
arrowhead) after the pioneer neurons had formed axonal
condensations within the olfactory bulb. Some of the cells in the
initial cluster of four to six sensory neurons (Fig.
2B) had dendrites but had not extended axons. These
neurons lie adjacent to the eye in the apical-lateral region of the
olfactory organ (Fig. 2B, arrow), distinct
from the position of the pioneer neurons whose cell bodies occupy the basal-medial region (Fig. 2B,
asterisk).
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Figure 2.
The axons of the pioneer neurons and first sensory
neurons colocalize in the CNS. A, A 44 h embryo labeled
with zns-2. The pioneer axons converge to form the olfactory nerve
(arrow) and then defasciculate to form axonal
condensations in the olfactory bulb (arrowhead).
Double cross indicates the region occupied by the
(unlabeled) sensory neurons. B, Lateral-oblique view of
the nose of a live 50 h fish labeled with DiI. Two cell bodies
(arrow) with axons and several without axons are evident
lying next to the eye. The axons extend into the developing olfactory
bulb (arrowhead). Asterisk marks the
region occupied by (unlabeled) pioneer neurons. C-E,
The axons of primary olfactory sensory neurons colocalize with the
pioneer axons in a single 1.5 µm optical section. Confocal
micrographs of ventral view of an olfactory organ
(outlined in purple) at 44 h showing
sensory neurons labeled with DiI (arrows in
C and D, red) and pioneer
neurons labeled with zns-2 (D and E,
green). The growth cones of the sensory neurons in the
differentiating olfactory bulb colocalize (arrowhead)
with the axons of the pioneer neurons. e, Eye. Anterior
is to the top. This preparation is a ventral view as
depicted in Figure 1H, (C-F).
Scale bars: A, 30 µm; B, 20 µm;
C-E, 40 µm.
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We labeled the sensory neurons with DiI (Fig.
2C,D, red) and the pioneer neurons
with zns-2 in the same preparation (Fig. 2D,E, green) and used
confocal microscopy to show that the DiI-labeled sensory axons and the
zns-2-positive pioneer axons overlap with one another in the
telencephalon (Fig. 2C-E, arrowheads). The axons
of the sensory neurons are in the proximity of the axons of the pioneer
neurons at the exit of the olfactory nerve, and the axons of both cell
types are colocalized at their terminations within the presumptive
olfactory bulb. These results suggest that, once leaving the olfactory
organ, the growth cones of the later developing sensory neurons follow
the pioneer axons.
The pioneer neurons are a transient population of cells. We visualized
their death using terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) (Gavrieli et al., 1992). Cell death in the developing olfactory organ peaked at ~48 h and was
localized primarily in the basal-medial region (Fig. 3,
magenta) occupied by the pioneer neurons. Preparations
double labeled for both TUNEL (Fig. 3, magenta) and zns-2
(Fig. 3, green) showed that the majority (80% of the cells;
n = 25 animals examined) of the TUNEL-labeled cells
within the olfactory organ were also zns-2-positive (Fig. 3,
arrowhead) at this time, confirming that the dying cells
were the pioneer neurons. Thus, the pioneer neurons undergo apoptosis
as the axons of the first sensory neurons reach the differentiating
olfactory bulb.
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Figure 3.
The olfactory pioneer neurons die.
Preparation labeled for zns-2 (green) and TUNEL
(magenta) at 42 h shows pioneer neurons dying at this
time. The TUNEL-labeled cells are primarily located in the basal-medial
part (arrowhead) of the olfactory organ.
o, Olfactory organ; ob, olfactory bulb;
double cross indicates the location of the unlabeled
sensory neurons. Ventral-oblique view. Serial optical sections from the
olfactory organ into the bulb were superimposed. Scale bar, 20 µm.
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To determine the origins of the pioneer neurons and the olfactory
sensory neurons, we labeled single cells (Fig.
4, A, bottom inset)
at the edge of the anterior neural plate at the four somite stage
(~12 h) (Fig. 4A, top inset) in the
domain we know gives rise to the olfactory placode (our unpublished
observations). Single cells labeled anteriorly at the lateral
edge of the neural plate (Fig. 4A,
1) gave rise to clones of cells that included olfactory pioneer
neurons (Fig. 4B), identified by their round cell
bodies and lack of dendrites, when scored at 36 h. As was observed with
zns-2 labeling (Fig. 1B), pioneer cells labeled with
lineage tracer dyes did not show dendrites and occupied the position
characteristic of pioneer neurons, in the basal-medial region of the
olfactory placode. The clones arising in the anterior region (Fig.
4A, 1) never contained both pioneer
neurons and sensory neurons (n = 13 clones). Moreover,
when we labeled cells in this region (Fig. 4A,
1) and examined the animals at 48 h, after the wave of cell
death, we never observed labeled pioneer cells (n = 22 animals).
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Figure 4.
The pioneer neurons and the olfactory sensory
neurons arise from different regions of the neural plate.
A, Dorsal view of the neural plate in a live 12 h embryo
before the formation of the olfactory placode. Arrows 1
and 2 indicate the regions in which single cells were
labeled. The location of their progeny is shown in B and
C, respectively. Top inset is a lateral
view of an embryo, with arrowhead indicating the region
shown in the micrograph. Bottom inset is a lateral view
of a live embryo with a single cell labeled in position 2 at 12 h,
viewed with both bright-field and fluorescence optics.
B, Ventral view of live embryo at 36 h, before the onset
of olfactory pioneer cell death. Single cells, labeled at the anterior
end of the neural plate
(A,1), gave rise to pioneer
neurons, recognized by their basal-medial location in the developing
olfactory organ. C, Ventral view of a live embryo at 48 h. Single cells, labeled farther posterior in the neural plate
(A, 2) adjacent to the
central region of the developing eye
(A, e), gave rise to
olfactory sensory neurons lying apically at the posterior edge of the
olfactory organ. Anterior is to the top. Scale bars:
A, 40 µm; B, C, 20 µm.
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In contrast to the fate of lateral anterior cells, cells labeled more
posteriorly at the lateral edge of the neural plate, next to the lens
placode (Fig. 4A, 2), gave rise
exclusively to clones containing early olfactory sensory neurons (Fig.
4C) when scored at 48 h. None of these clones contained
olfactory pioneer neurons (n = 12). These observations
demonstrate that olfactory pioneers and sensory neurons arise from
precursors located in different regions of the neural plate, suggesting
that they have distinct origins.
To examine the function of the olfactory pioneers, ablations of the
pioneers were conducted in animals at a time just after the formation
of a discernible olfactory placode, 18-20 h, yet 4 h before the
outgrowth of the axons of the pioneer neurons and ~1 d before axonal
outgrowth of the first olfactory sensory neurons. Animals with ablated
pioneers were allowed to develop to 50 h; we then examined the
morphology of sensory neurons using DiI and the presence or absence of
pioneers using zns-2. Complete ablation of the pioneers, as judged by
loss of zns-2 labeling (Fig.
5A, compare
asterisk, arrowhead), resulted in misrouting of
the sensory axons (Fig. 5B,C). The
misrouted sensory axons typically extended posteriorly (Fig.
5B) toward the anterior or postoptic commissures, where they
wandered extensively in the dorsoventral plane, a trajectory never
observed in normal embryos. In other embryos with incompletely ablated
pioneers, as judged by zns-2 labeling, the sensory axons split between
the normal anterior and abnormal posterior directions (Fig.
5C). Preparations labeled with both DiI and zns-2 (Table 1)
showed that when no pioneers were present sensory axons were misrouted
(Fig. 5B, Table 1) and that when at least some pioneer neurons survived the ablation (data not shown) some sensory axons grew
in the correct direction (Fig. 5C, Table 1). If the pioneers were ablated at 38 or 46 h, after they had established axonal condensations in the telencephalon and the axons of the sensory neurons
had started growing into the CNS, there was no effect on axonal
routing. These results strongly suggest that the pioneers are necessary
for sensory axon guidance from the olfactory placode to the region of
the telencephalon in which the olfactory bulb will develop.
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Figure 5.
Ablation of pioneer neurons results in misrouting
of olfactory sensory axons. Ventral views at 50 h with anterior to the
top. A, The absence of zns-2 labeling on
the ablated side (right, asterisk)
indicates that all the pioneer neurons were removed. Control side
(left, arrowhead) shows normal pattern of
zns-2 labeling. B, Olfactory sensory neurons labeled
with DiI show posterior misrouting in which axons
(arrow) grow posteriorly in the absence of pioneer
neurons. C, Olfactory sensory neurons labeled with DiI
show split misrouting in which axons grow both posteriorly
(arrow) and anteriorly (arrowhead) toward
telencephalon. Scale bars: A, 80 µm;
B, C, 40 µm.
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Putative odorant receptor gene transcripts are localized in the cell
bodies of sensory neurons (Buck and Axel, 1991) and in sensory
axon terminals in the olfactory bulb (Ressler et al., 1993; Vassar et
al., 1994). This has led to the suggestion that odorant receptors may
play a role in guiding the axons of olfactory sensory neurons in
addition to their role in detecting odorants (Singer et al., 1995;
Mombaerts et al., 1996). We examined the expression of olfactory
receptor messages (Fig.
6A) to learn whether or
not they are expressed in the pioneer neurons. Of the receptors that
have been cloned to date in zebrafish (Barth et al., 1996; Byrd et al.,
1996), only four are expressed during the time that olfactory pioneer
neurons are alive (Barth et al., 1996). We used a mixture of the four
olfactory receptor gene probes and found no expression of receptor RNA
in the olfactory placode before 24 h. Receptor expression was first
detectable at 24 h in 50% of the animals (n = 46 olfactory organs), and by 30 h all preparations contained at least two
to three cells per olfactory organ expressing receptor genes. At 52 h
(Fig. 6A), an average of 7.7 cells per olfactory
organ (n = 52 olfactory organs) expressed olfactory receptors (Fig. 6B). Throughout the first 2.5 d
of development, receptor gene expression remained preferentially
located in cells in the apical region of the olfactory organ in which
the sensory neurons are located, not in the basal part of the
epithelium in which the pioneer neurons lie (Fig.
6A,B). These results suggest that
the sensory neurons, not the pioneer neurons, express putative odorant
receptors and that receptor expression starts after the arrival of the
pioneer axons in the telencephalon.
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Figure 6.
Sensory neurons, but not pioneer neurons, express
olfactory receptor genes. A, Ventral view of a 52 h
olfactory organ showing the combined expression of four olfactory
receptor gene transcripts localized in the cytoplasm; axons are
unlabeled. Labeled cells (dark blue) are located near
the apical surface of the olfactory organ in which the sensory neurons,
but not the pioneer neurons, are located. The basal edge of the
olfactory organ is demarcated by the broken line. Scale
bar, 20 µm. B, Cells expressing olfactory receptors
are apically located. Bars represent the average (± SE) number of cells expressing olfactory receptor genes in each
olfactory organ as a function of developmental time in the apical
(ap) or basal (ba) region. Both olfactory
organs from 22 to 28 animals were scored for each time point.
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DISCUSSION |
Although neurons in the PNS of other vertebrates have been termed
"pioneers" based only on their early appearance during development (Stainier and Gilbert, 1990; Gong and Shipley, 1995), we have described here for the first time in vertebrates a class of PNS neurons
that are both early born and required for establishing axonal pathways
to the CNS. These olfactory pioneers are morphologically and temporally
distinct from the olfactory sensory neurons, undergo programmed cell
death, are essential to target sensory axons to the developing
olfactory bulb, and arise from a distinct region of the neural
plate.
The axons of the pioneer neurons form the initial necessary link
between the olfactory placode of the PNS and the differentiating telencephalon of the CNS (Fig. 7,
green). The pioneer axons grow to the area of the developing
telencephalon that expresses emx1 (Fig. 7,
stipple). The pattern of olfactory receptor message
expression (Fig. 7, orange) correlates with the
location of the sensory neurons and not the pioneer neurons. After the
arrival of the first sensory neuron axons (Fig. 7,
red) within the developing telencephalon, the pioneer
neurons undergo apoptotic cell death (Fig. 7,
purple).
View larger version (24K):
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|
Figure 7.
A transient population of pioneer neurons
establishes the olfactory pathway. Summary diagram of the developing
olfactory system from 24 to 54 h. A, The zns-2-positive
pioneer neurons (green) are distinguished by
their round dendriteless cell bodies. The stippled area
indicates the region of emx1 gene expression in which
the pioneer neurons contact the differentiating telencephalon. The
sensory neurons (red) extend their axons into the CNS,
following the axons of the pioneer neurons. The pioneer neurons become
positive for TUNEL (purple), indicating that they
die. The cells expressing olfactory receptor mRNAs
(orange) are located near the apical surface in the same
region that contains the olfactory sensory neurons. B,
Time line showing the temporal sequence of developmental events. The
colors correspond to the coding used in
A; the arrow indicates when the olfactory
placode becomes apparent with Nomarski optics in the live embryo. The
dashed lines indicate that not all preparations show
labeling at the indicated time.
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|
In the embryonic olfactory epithelium, mitotic figures initially appear
at the apical rather than basal surface. As the animal develops, the
epithelium becomes stratified, and the pattern of cell division shifts
basally, with the daughter cells migrating apically as they extend
dendrites and axons (Farbman, 1994). The pioneer neurons are located
basally early in development, consistent with their being a nondividing
population (Fig. 1B,C).
Furthermore, it is unlikely that the pioneer neurons later become
sensory neurons, because this would require that they sprout dendrites
after their axons are in the CNS and then migrate apically, something
we have never observed.
Our fate mapping studies support the idea that the pioneer neurons are
a developmentally distinct population of neurons by two criteria;
clones containing pioneers never contain sensory neurons, and the
region of the neural plate giving rise to pioneers is distinct from the
region giving rise to the first sensory neurons. The clones resulting
from labeling single cells in the anterolateral neural plate, 6 hr
before the appearance of the olfactory placode, contain only pioneer
neurons. In contrast, cells lying more posteriorly at the edge of the
neural plate give rise to the first olfactory sensory neurons (Fig. 4).
Thus, our fate map shows that early in development, cells destined to
give rise to olfactory pioneer neurons and sensory neurons are
lineage-restricted and already occupy distinct positions at the edge of
the neural plate. Therefore, these two classes of neurons may be
derived from different types of precursor cells.
The first axons to exit the olfactory placode have been described
previously by electron microscopic analysis at 20 h (Hansen and Zeiske,
1993), and neurons with axons emanating from the olfactory placode have
been identified using horseradish peroxidase labeling at 24 h (Wilson
et al., 1990). The axons of the pioneer neurons visualized with zns-2
exit the olfactory placode at the same time (Fig. 1B)
as the axons described in previous studies (Wilson et al., 1990; Hansen
and Zeiske, 1993), thus supporting their identification as the
first axons. In rats, neurons termed pioneers have been described using
a GAP-43 antibody (Gong and Shipley, 1995) that recognizes many axon
types within the nervous system. In the absence of other information on
the identity of these neurons, it thus remains unclear whether they
correspond to the mammalian equivalents of the olfactory pioneer
neurons described here for the zebrafish. Dynes and Ngai (1998) have
recently shown the presence of "unipolar" neurons in the olfactory
placode, which may correspond to the pioneers we describe.
When the axons of the zebrafish pioneer neurons leave the olfactory
placode, they extend along the surface of the telencephalon and then
defasciculate in the region of the presumptive olfactory bulb (Fig. 1).
It is currently unknown how these initial axons project to the correct
region of the telencephalon. Studies in other systems, such as
Xenopus (Stout and Graziadei, 1980), suggest that arrival of
axons from the olfactory placode helps trigger olfactory bulb
development. Our data show that the pioneer axons project specifically
to the region of the telencephalon in which cells express the
emx1 gene (Fig. 1D). Expression of
emx1 begins before the formation of the olfactory
placode (Simeone et al., 1992; Morita et al., 1995) and arrival of the
pioneer axons, suggesting that emx1 may help the pioneer
axons recognize their target region. The emx1 protein has also been
reported to be localized to the axons of olfactory sensory neurons
(Briata et al., 1996). These observations are consistent with the
hypothesis that borders of regulatory gene expression mediate axon
guidance (MacDonald et al., 1994) and that emx1 may regulate
expression of instructional cues recognized by the growth cones of the
olfactory pioneer neuron axons.
The zebrafish olfactory pioneer neurons provide the initial link
between the PNS and the CNS. We have shown that ablation of the pioneer
neurons severely disrupts axonal guidance of the sensory neurons,
suggesting that these pioneers provide essential guidance cues for
pathfinding by later-developing sensory neuron axons (Fig. 5, Table 1).
In addition, the pioneer neurons die after they have established the
initial pathway to the developing olfactory bulb. Pioneer neurons play
a similar required role in several other systems. Within the CNS of
mammals, cortical subplate neurons pioneer the connection between the
thalamus and the visual cortex, and their ablation results in abnormal
axon projections (McConnell et al., 1989). The T1 pioneer neurons in
the limb of grasshoppers first establish the path from the PNS to the
CNS, and when they are removed, the following sensory neurons fail to
reach the CNS (Klose and Bentley, 1989). Furthermore, the zebrafish olfactory pioneer neurons undergo programmed cell death (Fig. 3), like
both the T1 (Kutsch and Bentley, 1987) and subplate pioneers (Ghosh et
al., 1990).
The olfactory pioneer neurons we have described in zebrafish may
correspond to an apparently specialized group of cells previously observed at the base of the developing olfactory placode in mammals. In
rats, Lejour (1967) noted that the formation of the olfactory nerve was
preceded by an aggregation of placodally derived neuroblast-like cells.
This "blastema" appears between the olfactory placode and the
telencephalon, and neural fibers of the nasal epithelium and terminal
nerve converge on this blastema before contacting the developing
forebrain. More recently, Schwanzel-Fukuda et al. (1992) reported a
similar blastema in mice that expresses NCAM and precedes the
appearance of the olfactory, vomeronasal, and terminal nerves. In
zebrafish, the pioneer neurons provide a bridge through which later
developing axons pass as they exit the developing placode and extend to
the telencephalon. Thus, the pioneers may serve a guidance function
analogous to the blastema of mammals in bridging the peripheral and CNS
early in development.
The olfactory system of the adult zebrafish is organized similarly to
that of other vertebrates (Byrd and Brunjes, 1995; Rupp et al., 1996),
with an invariant pattern of glomeruli in the olfactory bulb (Baier and
Korsching, 1994). Olfactory sensory axons may affect cell cycle
kinetics in the telencephalon (Gong and Shipley, 1995), although how
the specific pattern of glomeruli is specified is currently unknown. In
insects, in which the formation of glomeruli is much better understood,
they appear to arise from an interaction of the incoming sensory axons
with the glia of the olfactory bulb (Oland et al., 1990; Tolbert and
Sirianni, 1990). When the development of glia, but not the
sensory neurons, is blocked, the glomeruli form less distinctly (Oland
et al., 1988). Our results show that zebrafish olfactory pioneer
neurons provide the first axons from the olfactory placode to the
telencephalon and that these pioneer axons form a axonal scaffold in
the developing olfactory bulb (Fig. 1). At the developmental times
examined here, the number of axonal condensations (15 per bulb) is
slightly greater than the number of pioneer neurons, indicating that a
pioneer neuron may target axons to more than one axonal condensation.
These axonal condensations may be the first olfactory glomeruli, but
many more glomeruli must be added later to form the number (80 per
bulb) observed in adults (Baier and Korsching, 1994). We currently do not know whether there is a continuous addition of glomeruli or perhaps
a metamorphosis-like abrupt addition that leads to the adult glomerular
number.
Our results reveal that an axonal scaffold is established by the
pioneers and that these pioneers do not appear to express odorant
receptors, suggesting that these receptors do not provide the main
guidance cue that allows the sensory axons to reach the telencephalon.
Likewise, in mice (Mombaerts et al., 1996; Wang et al., 1998), receptor
expression does not appear to confer specific target identity on
olfactory sensory axons, which supports the hypothesis that other cues,
such as cell-surface glycoproteins, guide axons within the CNS
(Schwarting et al., 1992a,b; Riddle et al., 1993; Bargman,
1996). However, receptors expressed in later-developing sensory
neurons may contribute to regional segregation of afferent axons within
the bulb.
| |
FOOTNOTES |
Received April 21, 1998; revised Aug. 17, 1998; accepted Aug. 19, 1998.
This work was supported by grants from the Muscular Dystrophy
Association (K.E.W.), National Institutes of Health/National Institute
on Deafness and Other Communication Disorders (K.E.W.), National
Institutes of Health (M.W.), and the W. M. Keck
Foundation. We thank C. Bargman, J. Eisen, J. Ewer, C. Kimmel, and M. Schwanzel-Fukuda for helpful discussions.
Correspondence should be addressed to Kathleen Whitlock, Section of
Genetics and Development, 445/449 Biotechnology Building, Cornell
University, Ithaca, NY 14853-2703.
| |
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Top
Abstract
Introduction
