A G protein/cAMP signal cascade is required for
axonal convergence into olfactory glomeruli
Alexander T. Chesler, Dong-Jing Zou, Claire E. Le Pichon, Zita A. Peterlin, Glennis A. Matthews, Xin Pei,
Michael C. Miller, and Stuart Firestein†
Department of Biological Sciences, Columbia University, 1212 Amsterdam Avenue, New York, NY 10027
Edited by Linda M. Bartoshuk, Yale University School of Medicine, New Haven, CT, and approved November 7, 2006 (received for review October 20, 2006)
The mammalian odorant receptors (ORs) comprise a large family of
G protein-coupled receptors that are critical determinants of both
the odorant response profile and the axonal identity of the
olfactory sensory neurons in which they are expressed. Although
the pathway by which ORs activate odor transduction is well
established, the mechanism by which they direct axons into proper
glomerular relationships remains unknown. We have developed a
gain-of-function approach by using injection of retroviral vectors
into the embryonic olfactory epithelium to study the ORs’ contri-
bution to axon guidance. By ectopically expressing ORs, we dem-
onstrate that functional OR proteins induce axonal coalescence.
Furthermore, ectopic expression of G? mutants reveals that acti-
vation of the signal transduction cascade is sufficient to cause
axonal convergence into glomeruli. Analysis of G? subunit expres-
sion indicates that development and odorant transduction use
separate transduction pathways. Last, we establish that the gen-
eration of cAMP through adenylyl cyclase 3 is necessary to estab-
lish proper axonal identity. Our data point to a model in which
axonal sorting is accomplished by OR stimulation of cAMP produc-
tion by coupling to G?s.
axon guidance ? development ? olfaction
the olfactory bulb (OB), where they coalesce into glomeruli.
Strikingly, any given glomerulus is populated exclusively by
axons of cells expressing the same odorant receptor (OR) (1, 2).
This feat is all the more remarkable because, with ?1,000 ORs
in mouse (3, 4) and each neuron expressing only one of those
receptors (5), there are effectively ?1,000 populations of neu-
to be a functional unit of odor coding, surprisingly little is known
of the factors critical to its formation.
Genetic lesions of a cyclic nucleotide-gated channel subunit
CNGA2 (6–8) or a G protein subunit G?olf (9) effectively
silence the odor response but do not appear to severely affect the
organization of glomeruli in neonates. On the other hand, even
minor changes in the OR protein cause shifts in glomerular
position (10). There are two distinct processes at work: coales-
cence of like axons into a glomerulus and determination of a
specific position for that glomerulus. Since the initial finding a
decade ago that ORs are critical determinants in OSN axonal
convergence (1), there has been little further insight into the
mechanisms of glomerular formation. Many factors have been
implicated in glomerular positioning (11) but not directly in the
coalescence of axons into specific glomeruli. In the absence of
projection neurons, interneurons, or even much of the OB itself,
axons from OSNs expressing OR P2 still coalesce (11). This
result has led to the notion that glomerular coalescence does not
require a preexisting ‘‘target,’’ is intrinsic to OSN axons, and is
mediated by the ORs themselves (10).
However, there is mounting evidence that activity-generated
processes might affect glomerular formation. We recently found
that the mature organization of the olfactory system cannot
occur in the absence of cellular activity (12). Similarly, in
n mammalian olfactory systems, axons from olfactory sensory
neurons (OSNs) in the olfactory epithelium (OE) project to
adenylyl cyclase type 3 (AC3) knockout mice, glomerular struc-
and synaptic activity seem to be essential for the establishment
and maintenance of proper connectivity (15).
Thus, in contrast to the visual system in which patterned
activity plays a clear role in the establishment of precise pro-
jections, there are conflicting data concerning the effect of
activity on glomerular development. Here we address the pos-
sibility that a critical feature of activity in the olfactory system is
the production of cAMP by activation of the G protein cascade.
Such a model would explain why CNGA2 knockout mice, which
retain G protein signaling despite odor-evoked electrical silence,
achieve largely normal glomerular formation, but AC3 knockout
mice do not. Why then do knockouts of G?olf, which couples
ORs to AC3 in mature OSNs, also appear to have normal
glomeruli for OR P2 at birth (9)? Here we show that an
alternative G protein, G?s, likely fulfills this essential role during
development. The direct involvement of G protein/cAMP sig-
naling in the formation of olfactory projections provides a
mechanism in the formation of sensory maps.
In Utero Imaging and Injection of the Developing Olfactory System.
We have developed the use of retroviral vectors as an alternative
approach to transgenic mice for studying the molecular genetics
of mammalian olfactory development in vivo. To deal with the
small target size of the embryonic olfactory system, we modified
previously published protocols for imaging embryonic ventricles
with an ultrasound biomicroscope (16). We constructed a ret-
roviral vector encoding tauGFP (tGFP) that, when injected into
the embryonic OE, was expressed robustly in the developing OE
and persisted into adulthood. Examination of postnatal day 21
(P21) mice injected with the tGFP retroviral vector at embryonic
day 11 (E11) revealed widespread ectopic expression specifically
throughout the OE (Fig. 1A). We had similar success injecting
at various stages (E10.5–E16) and also extended our study to rats
Injection of the retroviral vector resulted in numerous clusters
of tGFP?cells composed of multiple cell types, including basal
cells, OSNs, and apical sustentacular cells (data not shown). In
addition to labeling cell bodies in the OE, tGFP-labeled axons of
Author contributions: A.T.C. and D-J.Z. contributed equally to this work; A.T.C., D-J.Z., and
S.F. designed research; A.T.C., D-J.Z., C.E.L.P., Z.A.P., G.A.M., X.P., and M.C.M. performed
A.T.C., D-J.Z., C.E.L.P., Z.A.P., and X.P. analyzed data; and A.T.C., D-J.Z., and S.F. wrote the
The authors declare no conflict of interest.
This article is a PNAS direct submission.
OR, odorant receptor; AC3, adenylyl cyclase type 3; Pn, postnatal day n; En, embryonic day
n; GPCR, G protein-coupled receptor; tGFP, tauGFP.
†To whom correspondence should be addressed. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2007 by The National Academy of Sciences of the USA
January 16, 2007 ?
vol. 104 ?
no. 3 ?
newborn OSNs were readily observed in the OB. After birth, a
network of numerous tGFP? axons covered the OB (Fig. 1B).
These axons entered many glomeruli, where they branched (Fig.
1 C and D). In sum, we have taken advantage of the fact that
retroviral vectors productively infect only dividing cells to de-
velop a technique that allows us to ectopically express genes in
newly born OSNs and to study OSN axon projections as they
extend into the OB.
Ectopic Expression of ORI7 in Developing OSNs Induces Their Axons to
Coalesce. Current models posit that OSN axonal identity is
determined by OR expression. With the retrovirus, we could
directly ask whether OR expression alone was sufficient to
determine OSN axonal identity by forcing the precocious ex-
pression of an ectopic OR. Injection of a retroviral vector
encoding an N-terminally tagged rat ORI7 construct (HA-
ORI7) followed by IRES-EGFP resulted in mosaic expression
throughout the OE. The OR protein was detected by using the
HA epitope tag in the cell body, dendrites, and cilia of OSNs
(Fig. 1E), indicating proper OR trafficking (17, 18). The OSNs
ectopically expressing HA-ORI7 appeared phenotypically nor-
mal, expressing the various cell-type-specific markers, including
OMP and endogenous ORs (data not shown). Overall, ectopic
expression resulted in expression in many more neurons (?2-
fold) than is typically seen for endogenous ORs.
ORI7 is activated by octanal and structurally related ligands
(19). We therefore asked whether exogenous HA-ORI7 was
sufficient to alter the odorant response properties of OSNs in
which it was ectopically expressed. Calcium imaging with a
diagnostic panel of four ligands was used to distinguish ORI7
from the ?50 other ORs thought to be sensitive to octanal (Fig.
1F) (19). One hundred percent of octanal-responsive GFP?
OSNs displayed the expected ORI7 profile, thus confirming that
HA-ORI7 is functional (n ? 6). For comparison, similar to our
previous study (20), only 13% of the octanal-responsive GFP?
alters the odorant response properties of OSNs in an expected
We next tested whether the exogenous OR protein was
sufficient to alter axonal identity by examining the projection of
transduced OSNs. Whole-mount images and serial sections of
P21 rat OBs (infected at E15) revealed that ectopic HA-ORI7
expression resulted in the convergence of axons into multiple
distinct glomeruli throughout the OB (Fig. 1G). Such coales-
cence was never observed in tGFP controls (Fig. 1 B and D). We
noted a large variation in the number, position, and size of the
information (SI) Fig. 4]. However, despite the variation in
position, convergence of OSN axons was observed in all animals
with broad infections (n ? 50; covering ?20% of the visible OE
area in bisected nasal cavity), ranging from two to eight glo-
meruli per half-bulb. Among them, we observed homogenous
(Fig. 1I), partially coalesced (Fig. 1 H and J, open arrowheads),
and compartmentalized glomeruli (Fig. 1J Inset; SI Fig. 4 B and
C) with a relative ratio of 2:5:3. Animals with less robust
infection were not analyzed, because it had been reported that
a minimum number of OSN axons are needed to support a
glomerulus (21). Overall, these data clearly demonstrated that
the expression of a functional OR was sufficient to induce axonal
sorting into glomeruli.
ORI7 Function Correlates with the Axonal Guidance Phenotype. Ex-
pression of an ectopic OR both altered odorant response profiles
and induced coalescence of OSN axons, raising the possibility
that OR function, not just expression, was important. Serendipi-
tously, we found that a different N terminus modification, the
addition of a Myc tag, disrupted ORI7 function, allowing us to
internal ribosome entry site results in widespread ectopic expression. (A) Medial
view of olfactory turbinates in a P21 mouse that had been injected at E11.5.
Numerous GFP?clusters of cells (green) are apparent throughout the OE. (B)
Whole-mount image of a P21 rat OB showing that numerous GFP?axons dif-
indicate glomeruli. (Scale bar, 100 ?m.) (D) Confocal projection of a 60-?m
cryosection from a rat OB showing how these fibers enter and branch within
odorant responses and axonal projections. (E) As shown by immunohistochem-
istry, HA-ORI7 protein (red) localizes to OSN cell bodies, dendrites, and cilia
injected at E15. Two ORI7 agonists [octanal (OAL) and trans, trans2,4-octadienal
(2,4-OD)] and two odorants known not to activate the receptor [octanol (OOL)
and hexanal (HEX)] are selected as a diagnostic panel for ORI7 function. The
correct response profile largely identifies ORI7 from the other ORs that also
respond to OAL. All odorants were presented at 30 ?M. (Vertical scale bar, 10%
shows a heterogeneous glomerulus (open arrowhead) within 200 ?m of a ho-
axonal convergence. (Inset) A higher-magnification view of the glomerulus
fibers do not appear to fill the entire glomerulus. (Scale bar, 100 ?m.) (K–N)
Ectopic expression of Myc-I7 in OSNs (Myc-I7-i-tGFP). (K) (Upper) Myc-ORI7 (red)
traffics to OSN cilia (white arrowhead) and axons (open arrowhead). OSNs
coexpress tGFP (green). (Scale bar, 12.5 ?m.) (Lower) A higher magnification of
the cilia. (L) As indicated by calcium imaging, expression of Myc-ORI7 does not
to 3-isobutyl-1-methylxanthine (IBMX), indicating an intact transduction path-
way. (Vertical scale bar, 10%?F/F; horizontal scale bar, 30 s.) (M) Western blots
rat OE indicate the expression of a full-length Myc-ORI7 protein. (N) Ectopic
(green) in a P21 rat (compare to ectopic functional HA-ORI7 expression in G–J).
(Scale bar, 120 ?m.)
Glomerular convergence of axons from OSNs expressing the functional
www.pnas.org?cgi?doi?10.1073?pnas.0609215104 Chesler et al.
test this proposition. Despite proper trafficking to OSN cilia
(Fig. 1K), Myc-ORI7 expression in OSNs did not confer respon-
siveness to OAL (0/5 IBMX-responsive GFP? OSNs; Fig. 1L).
the production of a truncated OR protein (Fig. 1M). Calcium
imaging experiments further indicated that the Myc-ORI7-
expressing OSNs were still physiologically functional (Fig. 1L).
However, the nonfunctional Myc-ORI7 protein in OSN axons
(Fig. 1K, open arrowheads) failed to cause these axons to
coalesce (Fig. 1N). Instead, the axons entered many glomeruli in
a manner indistinguishable from tGFP controls (compare Fig. 1
D and N). These results suggest that OR function is necessary for
inducing axonal convergence.
Ectopic Activation of G Protein Signaling Results in Axonal Coales-
cence. Based on our tagged ORI7 experiments, we hypothesized
that activation of the transduction cascade encodes critical
information about the OR expressed by a given OSN. We
therefore tested the influence of downstream signaling by di-
rectly activating transduction among a subset of OSNs. OR-
induced activation of G?olf stimulates the production of the
second-messenger cAMP by AC3 (13) and is required for
of G?olf (Q214L) that possesses reduced GTPase activity, thus
increasing its basal level of activity (G?olf*; ref. 22). We first
determined that G?olf* could productively couple to ORs by
using a defined OR in a heterologous expression assay. Further-
more, the elevated basal level was not saturating, and odorant
responsiveness appeared similar to that of G?s (SI Fig. 5).
Although the cell bodies of OSNs ectopically expressing
G?olf* were distributed throughout the OE in a manner similar
to that for tGFP (Fig. 1A), remarkably, their axons coalesced
into glomeruli in the OB (Fig. 2 A and B). The axonal sorting
phenotype was very robust and was observed in all mice and rats
examined (n ? 21) ranging in age from P3 to P21. Like with
HA-I7, the widespread expression of G?olf* across the zones in
the OE resulted in the formation of numerous ectopic glomeruli
(2–10 per half-bulb). From these experiments, we conclude that
increased G?olf signaling is sufficient to create an axonal
phenotype resulting in axonal sorting and coalescence into
As a negative control, we expressed an additional mutant of
G?olf* that decreases the G protein’s overall GTP affinity
(G?olf-Q214L/D282N), thus reducing its basal level of activity
(SI Fig. 5B; ref. 22). We found G?olf*282 coupled very poorly
to heterologously expressed ORs (SI Fig. 5B). More critically,
when expressed in developing OSNs in vivo, the double mutant
(G?olf*282) failed to induce axonal coalescence (Fig. 2C).
Indeed, axons from OSNs expressing G?olf*282 looked indis-
tinguishable from axons expressing tGFP alone (Fig. 1 B–D).
From these data, we conclude that G?olf must be sufficiently
active to induce OSN axons to sort into glomeruli.
B) Ectopic expression of a constitutively active G?olf mutant (G?olf*-i-tGFP)
induces axonal sorting. (A) A whole-mount sagittal view of the OB from a P7
rat injected with a G?olf* retroviral vector at E15. One large tGFP conver-
(B) A 20-?m cryosection of a different OB reveals axons from OSNs expressing
G?olf* coalescing into a glomerulus (arrowhead) in a P7 rat. TOTO3 (blue)
labels all of the nuclei in the OB. (Scale bar, 100 ?m.) (C) Axons from OSNs
expressing a mutant of G?olf* with reduced activity (G?olf*282-i-tGFP) no
longer sort into glomeruli. The resulting pattern is indistinguishable from
tGFP controls (Fig. 1D). (Scale bar, 100 ?m.) (D and E) Two-color in situ
expression in the OE. (D) Two-color ISH of G?s (Left) or G?olf (Right; both red)
at E15 with two markers for OSN maturation (GAP43 on top and OMP on
bottom; both green). G?s expression is more widespread than G?olf in both
immature cells (GAP43?; arrowheads) and mature cells (OMP?; arrowheads).
can still be found expressed in a few immature OSNs (arrowheads). G?s is
absent from the mature population. Conversely, G?olf expression is largely
for mature OSNs. Dashed lines indicate the basal lamina. (Scale bar, 25 ?m.)
(F–J) Ectopic expression of constitutively active G?s mutant (G?s*-i-tGFP). (F)
Similar to G?olf*, ectopic expression of G?s* also results in axonal conver-
(Scale bar, 200 ?m.) (G) Axons from OSNs ectopically expressing G?s* sort and
coalesce into a glomerulus (arrowhead). (Scale bar, 100 ?m.) (H) A magnified
view of the glomerulus in G (arrowhead). (Scale bar, 50 ?m.) (I) (Upper)
as assayed by OMP expression (red, arrowheads). (Lower) Expression of G?s*
also does not inhibit endogenous OR expression, as seen by immunostaining
with anti-GFP (green) and a mixture of antibodies for OR37 and OR256–17
(red) in a P7 mouse. Double-labeled cell is indicated by white arrowhead.
Importantly, not all GFP? OSNs express the same OR. Open arrowheads
indicate OSNs not expressing either OR37 or OR256–17. (Scale bar, 25 ?m.) (J)
(Upper) The axon terminals of OSNs expressing G?s* (labeled by GFP, green)
are enriched for the synaptic marker synaptophysin (Synph; red) even in
by the dendrites of MAP2?bulbar neurons (red). (Scale bar, 25 ?m.)
Chesler et al. PNAS ?
January 16, 2007 ?
vol. 104 ?
no. 3 ?
Embryonic Activation of G?s Induces Axonal Coalescence. Although
the targeted deletion of G?olf resulted in anosmia and signifi-
cantly reduced peripheral odorant responses, the formation of
the glomeruli for OR P2 appeared unperturbed at P0 (9). This
suggests that, at least in embryos, G?olf is not necessary for the
convergence of some populations of OSN axons. However, it
remained possible that ORs still coupled to another G protein
We therefore examined the expression patterns of G?s and
G?olf at E15 and P5 (Fig. 2 D and E, respectively). At E15, when
OSN axonal sorting begins, we found widespread expression of
G?s throughout the OE. G?s was expressed not only in pro-
genitor cells (data not shown) but also in GAP43?immature
OSNs and OMP?mature OSNs. G?olf expression, on the other
hand, was very sparse and largely restricted to the few mature
OSNs present at this point (Fig. 2D). In contrast, at P5, when
many glomeruli have emerged, G?s was still highly expressed by
progenitor cells but was now only weakly expressed by some
immature neurons and not detected in mature OSNs. G?olf
expression was largely absent from immature OSNs, being
expressed almost exclusively by OMP?neurons (Fig. 2E). In
summation, our results suggest G?s may partner with ORs in
embryonic and immature OSNs during the process of axon
extension and convergence, whereas G?olf is expressed in
mature OSNs whose main function is odor-evoked transduction
(summarized in SI Fig. 6).
We thus investigated whether G?s activation underlies axon
sorting. Unfortunately, disruption of the widely expressed G?s
gene results in embryonic lethality by E10.5 (23), thwarting
loss-of-function studies. We therefore took an alternative ap-
proach to testing the role of G?s by using our gain-of-function
assay. We expressed a mutant, G?s-Q227L (G?s*), which pos-
sesses increased basal signaling (SI Fig. 5; ref. 22). As was the
case with G?olf*, G?s* productively coupled to heterologously
expressed ORs where, despite having elevated activity, the
odorant-induced dose–response curves had similar saturation
kinetics as wild-type G?s (SI Fig. 5).
Ectopic expression of G?s* in developing OSNs was sufficient
to induce the coalescence of their axons in every animal exam-
that OSNs ectopically expressing G?s* still expressed ORs
normally. Importantly, staining with OR-specific antibodies
revealed that not all of the G?s*-positive OSNs expressed the
same OR (Fig. 2I), thus indicating that elevated G?s activity
does not promote the expression of a particular OR. Many of
these OSNs appeared to mature fully (Fig. 2I Upper). Further-
more, the sites of convergence were enriched with markers of
synapse formation and contained the dendrites of projection
neurons (Fig. 2J), suggesting they are functional glomeruli. Our
results support the view that the activity of stimulatory G?
subunits underlies OSN axonal sorting and subsequent coales-
cence into glomeruli.
Functional Nonolfactory G Protein-Coupled Receptor (GPCR) Induces
Axonal Coalescence. Our results pointed to a model where OR
activation of a stimulatory G protein cascade induced axonal
sorting. One prediction of this model is that nonolfactory GPCRs
should induce the axonal convergence if they can activate G?s/
G?olf signaling in OSNs. It was shown that targeted replacement
creation of novel glomeruli (24). However, that study did not
examine whether ?2AR was functionally coupled to OSN trans-
duction. We ectopically expressed a flag-rho tagged human ?2AR
by using a retroviral vector and found that, like HA-ORI7 and
Myc-ORI7, ectopic ?2AR was localized in OSN cilia and axons
(Fig. 3A). When stimulated with the ?2AR agonist isoproterenol,
5/5 (100%) OSNs expressing ectopic ?2AR responded in calcium
imaging (Fig. 3B). In contrast, virtually none of the GFP?[yet 3-
isobutyl-1-methylxanthine (IBMX)-responsive] OSNs (3/144; 2%)
from the same field of cells responded to isoproterenol (Fig. 3C).
As predicted, the ectopic expression of ?2AR resulted in OSN
axons coalescing into multiple glomeruli throughout the OB (Fig.
3 D and E), although we noted that, in many cases, the axons often
did not fully enter the glomerulus (i.e., Fig. 3E). It is unclear why
it may reflect differences in basal signaling, coupling efficiency, or
protein levels. Conversely, when we ectopically expressed V1Rb2,
a GPCR thought not to couple to G?s but rather to G?o, we failed
to detect either calcium responses to its ligand 2-heptanone (25) or
coalesced axons (data not shown).
Disruption of the cAMP Pathway Perturbs Axonal Projection. We
hypothesized that a critical outcome of G?s signaling in the OSNs
contributing to axonal sorting was the stimulation of cAMP pro-
coalescence. (A–E) Ectopic expression of functional human ?2AR in OSNs
(FR-?2AR-i-tGFP) results in axonal convergence. (A) Immunohistochemical
staining reveals that ?2AR protein (red) traffics to OSN cilia in a P7 mouse OE
injected with a retroviral vector at E13. (Scale bar, 10 ?m.) (B) A GFP?OSN
GFP?OSNs in the same field of cells are not activated. (Vertical scale bar,
10%?F/F; horizontal scale bar, 2 min.) (D and E) Axons (green) of OSNs
ectopically expressing ?2AR converge in the OB but do not fully enter a
glomerulus (white arrowhead) in a P21 rat. TOTO3 (blue) labels all of the
of AC3 activity perturbed axonal sorting. M71G and M72Z mice are crossed
with AC3 knockout mice to generate compound mutants. At P20, M72Z axon
projection in the bulb is observed in the whole-mount stained with X-Gal.
M71G and M72Z axonal innervation patterns in glomeruli are studied by
immunohistochemistry. (F) Convergence of the M72 axons into a single glo-
merulus in the posterior medial half-bulb of AC3 wild-type mice. (G) Highly
perturbed projection of M72 axons in an AC3?/?littermate. (H) In the AC3?/?
background, M71G (green) and M72Z (red) axons converge into distinct
M71G and M72Z axons intermingle within the same glomerulus. (Scale bar, A
and B, 500 ?m; C and D, 25 ?m.)
Activation of cAMP pathway underlies GPCR induction of axonal
www.pnas.org?cgi?doi?10.1073?pnas.0609215104 Chesler et al.
duction. We therefore decided to analyze mice where the function
of AC3 was genetically disrupted. AC3 is required for odorant
transduction and is activated by both G?olf and G?s (13). In the
AC3?/?mice, odor-evoked electrical activity was completely abol-
ished, and forskolin-induced cAMP generation was greatly re-
duced. Moreover, glomerular structures in the OB of AC3?/?mice
have gross morphological defects (14). By crossing into an AC3?/?
background gene-targeted mice that coexpresses the marker tauL-
of cAMP signaling in the precise projection of M72 axons. In
AC3?/?mice at P20, M72 axons normally converged into a single
glomerulus in either the medial or lateral posterior half-bulb (Fig.
axonal projection was highly disrupted with M72 axons found in
numerous atypical locations in both the anterior and posterior OB
(n ? 3 OBs, Fig. 3G).
The disorganized projection pattern suggested that M72 axons
had lost their unique identity in the AC3?/?background. We
thus examined another hallmark of M72Z axons, their ability to
sort from the axons of OSNS expressing M71, a highly homol-
ogous OR (10). By using mice with M71 and M72 receptors
differentially marked [M71-IRES-tGFP (M71G) and M72Z], in
the AC3?/?background, we observed, as expected (10), that
these two populations sorted from each other into homogenous
populations (Fig. 3H). However, in an AC3?/?background, M71
and M72 axons now intermingled (Fig. 3I). Mixed M71/M72
glomeruli were found in 83% of the three broad projection
regions analyzed per OB (anterior, posterior lateral, and poste-
rior medial; n ? 12 OB projection regions) in the AC3?/?mice.
These data strongly implicate cAMP signaling as a requirement
for the establishment of a unique OSN axonal identity.
The key event in the development of ordered connections
between the OE and the OB is the coalescence of ?1,000
populations of axons into homogenous glomeruli. This remark-
able sorting process is guided by the expression of a specific OR
that appears to serve a double duty, conferring ligand recogni-
tion in the periphery and axonal identity in the OB. Whether the
OR itself is both necessary and sufficient and what molecular
steps underpin the OR’s function in guidance have remained
open questions since the initial observation (1).
We have used a retroviral-based technique to manipulate
expression of the key genes in the pathway coupling the OR to
cellular activity to explore the axonal sorting mechanism. The
technique offers a complementary approach to the use of
transgenic mice in that gene expression is mosaic, avoids poten-
tial issues of lethality, and provides temporal and spatial control.
In keeping with previous work, we found that ectopic expression
of only functional receptors drove OSN axons to coalesce in the
OB. Additionally, we have demonstrated that a feature universal
to GPCRs, downstream coupling to intracellular G protein
cascades, functions in axonal sorting. Last, through the exami-
nation of gene-targeted animals, we have linked a requirement
for cAMP-dependent signaling pathways to the formation of
Formation of Glomeruli. A hallmark of a mature glomerulus is the
homogeneous innervation by axons from cells expressing the
same OR (2) and largely stereotyped positioning in the OB.
Although many factors have been shown to influence the
positions of glomeruli (11), the sorting of axons and their
coalescence into homogenous fascicles seemed to have a single
determinant, the ORs. There is strong evidence that OR amino
acid sequence, level of OR protein expression, and timing of OR
expression are all important determinants of sorting (10). We
now suggest that receptor activation and downstream signaling
by stimulatory G? subunits are also critical determinants of
axonal coalescence into glomeruli.
Based on our expression studies, we find that G?s is expressed
at the right time and place to participate in OSN axon guidance.
Furthermore, we demonstrate that activation of the G protein
cascade is sufficient to induce axonal sorting. We observe OSN
axons sorting apparently independently of the expression of a
specific OR, relying instead on G protein activity. Although our
interpretation differs, our finding is consistent with those of
Feinstein and Mombaerts (10), who demonstrated that the
G?s-coupled ?2-adrenergic receptor sufficiently substituted for
an OR, but a presumably G?o-coupled Vomeronasal receptor
(V1Rb2) was largely insufficient to engender glomerular for-
mation (24). Last, we identify the generation of cAMP as a key
player in conferring OSN axonal identity. Examination of the
OBs of AC3?/?mice, and in particular of the fate of OSN
populations expressing two closely related ORs (M71 and M72),
further underscores that cAMP is a key player in the axonal
Sorting Machinery. Although the downstream targets of the G
protein signaling remain unknown, several models can be envi-
sioned (SI Fig. 6). There are several examples of GPCR signaling
affecting growth cone turning through alterations of cAMP,
cGMP, and/or Ca2?levels (26, 27). Alternatively, cAMP and/or
Ca2?-dependent gene transcription may indirectly control sort-
ing through the regulation of guidance genes (SI Fig. 6D). cAMP
and Ca2?are both well established potent modulators of gene
expression for numerous genes in diverse pathways (28). The
expression of ORs before OSN maturation and the coexpression
of G?s with ORs in the embryonic OE are consistent with such
a function. These models are consistent with the finding that
modulation of PKA and CREB activity alters gene transcription
profiles and glomerular position (29). However, ectopic expres-
sion of an active PKA mutant in a mosaic of OSNs by using a
retroviral vector was insufficient to promote coalescence
(A.T.C., unpublished data). Thus, although G protein stimula-
tion of cAMP is clearly involved in axonal coalescence, there are
likely multiple downstream targets of this pathway.
Establishing Axon Identity Through an OR Code. It remains chal-
lenging to explain how G protein signaling from ?1,000 ORs
results in such apparently precise wiring in the adult. For
example, ORs M71 and M72 that differ by only 11 amino acids
(96% identity) and have overlapping response profiles are able
to sort into two distinct glomeruli in close proximity (10).
Because it is unlikely there exist ?1,000 ligands in the OB acting
as guidance cues for the ?1,000 ORs, activation of the ORs had
not previously been considered necessary for their role in axon
guidance. However, we show that, without AC3 activity, OSN
populations are no longer able to maintain their individuality.
It has been suggested that the ORs function in axon guidance
by homotypic interactions (10). However, if OR interactions
alone were both necessary and sufficient to instruct axonal
sorting (10), it is difficult to imagine how the ectopic constitu-
tively active G?olf or G?s could induce axons to coalesce. One
would have to envision the unlikely scenario that increasing the
basal level of G protein signaling causes either up-regulation of
a specific OR in these cells or the selective survival of a single
population of OSNs. Based on our immunohistochemistry and
OR in situ experiments (A.T.C. and C.E.L.P., unpublished
observations), this seems highly unlikely.
An alternative possibility is that homotypic interactions mod-
ulate G?s activity, thus providing a link between interacting ORs
and intracellular pathways. Ectopic activation of G?s could have
allowed OSN axons expressing ORs with only weak homotypic
interactions to remain associated. Conversely, disruption of
cAMP signaling could serve to destabilize strong homotypic
Chesler et al. PNAS ?
January 16, 2007 ?
vol. 104 ?
no. 3 ?
interactions. Last, in the absence of OR-mediated homotypic
interactions, differences in ligand-independent basal signaling
could be sufficient to generate OR-specific signatures, because
many GPCRs are believed to have high levels of constitutive
Although the notion that the ORs themselves provide axonal
identity is very appealing, no mechanism by which the receptors
could perform this function has been demonstrated. Despite
data supporting activity-independent sorting (6–9), our results
and those of others challenge this notion (12–15). These dis-
crepancies can be reconciled by a model in which the informative
role of activity begins with the G protein/cAMP cascade. The
mechanisms by which specific ORs activate the G protein
Materials and Methods
Animals. Timed pregnant Swiss–Webster mice and Sprague–
Dawley rats were from Taconic Farms (Germantown, NY).
AC3?/?mice were from D. R. Storm (13). M71-IRES-tGFP and
M72-IRES-taulacZ mice were from P. Feinstein and P. Mom-
Ultrasound-Guided Injection. This method was adapted from Ga-
iano et al. (16). Embryos were imaged by using a Paradigm P45
OE was injected using a Drummond PCR micropipette with a
30° beveled tip and a 20- to 40-mm opening, controlled by a
Picospritzer III (General Valve) with pressures of 6–9 psi (1
psi ? 6.89 kPa) and 50- to 100-ms durations.
Viral Constructs. All viruses were made using the Pantropic
Retroviral Expression System (Clontech, Mountain View, CA)
with the pLNCX2 vector according to the manufacturer’s pro-
tocols. For construct details, see SI Text.
In Situ Hybridization and Immunostaining. Two-color fluorescent in
situ hybridization was performed as described in Ishii et al. (31).
Immunostaining was performed as reported (12). For probe
sequences and antibody specifications, see SI Text.
Functional Assays. Calcium imaging was performed as reported
(20). Dual luciferase assays were performed per the manufac-
turer’s instructions (Promega, Madison, WI). For details, see SI
We thank members of the S.F. laboratory for comments; P. Feinstein
(The Rockefeller University, New York, NY) and M. Rogers for
comments and materials; and C. Zhang (Columbia University), T. Ishii
(The Rockefeller University), F. Margolis (University of Maryland,
Baltimore, MD), K. Touhara (University of Tokyo, Tokyo, Japan), H.
Breer (University of Hohenheim, Stuttgart, Germany), and D. Storm
(University of Washington, Seattle, WA) for materials. This work was
supported by the National Institute on Deafness and Other Communi-
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www.pnas.org?cgi?doi?10.1073?pnas.0609215104 Chesler et al.