A G protein/cAMP signal cascade is required for axonal convergence into olfactory glomeruli - PubMed (original) (raw)

A G protein/cAMP signal cascade is required for axonal convergence into olfactory glomeruli

Alexander T Chesler et al. Proc Natl Acad Sci U S A. 2007.

Abstract

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' contribution to axon guidance. By ectopically expressing ORs, we demonstrate that functional OR proteins induce axonal coalescence. Furthermore, ectopic expression of Galpha mutants reveals that activation of the signal transduction cascade is sufficient to cause axonal convergence into glomeruli. Analysis of Galpha subunit expression indicates that development and odorant transduction use separate transduction pathways. Last, we establish that the generation of cAMP through adenylyl cyclase 3 is necessary to establish proper axonal identity. Our data point to a model in which axonal sorting is accomplished by OR stimulation of cAMP production by coupling to Galphas.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Glomerular convergence of axons from OSNs expressing the functional ectopic rat ORI7. (A_–_D) Injection of a retroviral vector encoding tGFP following an 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 diffusely project to the OB but do not invade the cortex (dashed line). (Scale bar, 200 μm.) (C) Confocal projection of a 20-μm cryosection from a rat OB showing GFP+ axons entering many glomeruli. TOTO3 (blue) labels all of the nuclei. Arrowheads indicate glomeruli. (Scale bar, 100 μm.) (D) Confocal projection of a 60-μm cryosection from a rat OB showing how these fibers enter and branch within glomeruli. (Scale bar, 25 μm.) (E_–_J) Ectopic expression of functional HA-ORI7 alters odorant responses and axonal projections. (E) As shown by immunohistochemistry, HA-ORI7 protein (red) localizes to OSN cell bodies, dendrites, and cilia (arrowhead) and overlaps with EGFP (green) in a P7 rat injected at E15. (Scale bar, 6.25 μm.) (F) Calcium imaging of dissociated GFP+ OSNs from 3- to 4-week-old rats 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 μM. (Vertical scale bar, 10% ΔF/F; horizontal scale bar, 2 min.) (G) Whole-mount image of a P21 rat injected at E15 with HA-ORI7 retroviral vector results in numerous GFP+ axons converging at multiple locations in the OB. Arrowheads indicate four observable convergences. (H_–_J) Sections of OB from an infected P21 rat reveal HA-ORI7 axons converging in the OB. (H) A section of an OB from a 3-week-old rat injected with HA-ORI7 at E15 shows a heterogeneous glomerulus (open arrowhead) within 200 μm of a homogeneous glomerulus (white arrowhead). (I) Higher-magnification view of the homogeneous glomerulus shown in H. (Scale bar, 50 μm.) (J) Another example of axonal convergence. (Inset) A higher-magnification view of the glomerulus (white arrowhead) showing that many of the fibers have coalesced. Note that the fibers do not appear to fill the entire glomerulus. (Scale bar, 100 μm.) (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 μm.) (Lower) A higher magnification of the dendrite of a transduced OSN (green) showing Myc immunoreactivity (red) in the cilia. (L) As indicated by calcium imaging, expression of Myc-ORI7 does not confer responsiveness to octanal (OAL) at 30 μM. However, the OSNs still respond to 3-isobutyl-1-methylxanthine (IBMX), indicating an intact transduction pathway. (Vertical scale bar, 10%ΔF/F; horizontal scale bar, 30 s.) (M) Western blots using the Myc antibody from the retrovirus-producing GP2 cell line and infected rat OE indicate the expression of a full-length Myc-ORI7 protein. (N) Ectopic expression of nonfunctional Myc-ORI7 in OSNs does not result in coalesced axons (green) in a P21 rat (compare to ectopic functional HA-ORI7 expression in G_–_J). (Scale bar, 120 μm.)

Fig. 2.

Unique axonal identities encoded by OSN signal transduction. (A and B) Ectopic expression of a constitutively active Gαolf mutant (Gαolf*-i-tGFP) induces axonal sorting. (A) A whole-mount sagittal view of the OB from a P7 rat injected with a Gαolf* retroviral vector at E15. One large tGFP convergence, as well as two smaller ones, is visible (arrowheads). (Scale bar, 100 μm.) (B) A 20-μm cryosection of a different OB reveals axons from OSNs expressing Gαolf* coalescing into a glomerulus (arrowhead) in a P7 rat. TOTO3 (blue) labels all of the nuclei in the OB. (Scale bar, 100 μm.) (C) Axons from OSNs expressing a mutant of Gαolf* with reduced activity (Gαolf*282-i-tGFP) no longer sort into glomeruli. The resulting pattern is indistinguishable from tGFP controls (Fig. 1_D_). (Scale bar, 100 μm.) (D and E) Two-color in situ hybridization (ISH) reveals developmental dynamics of stimulatory Gα subunit expression in the OE. (D) Two-color ISH of Gαs (Left) or Gαolf (Right; both red) at E15 with two markers for OSN maturation (GAP43 on top and OMP on bottom; both green). Gαs expression is more widespread than Gαolf in both immature cells (GAP43+; arrowheads) and mature cells (OMP+; arrowheads). (E) Two-color ISH at P5. At this stage, Gαs is more restricted to the basal OE but can still be found expressed in a few immature OSNs (arrowheads). Gαs is absent from the mature population. Conversely, Gαolf expression is largely restricted to the apical OE, where it completely overlaps with OMP, the marker for mature OSNs. Dashed lines indicate the basal lamina. (Scale bar, 25 μm.) (F_–_J) Ectopic expression of constitutively active Gαs mutant (Gαs*-i-tGFP). (F) Similar to Gαolf*, ectopic expression of Gαs* also results in axonal convergence (arrowhead) in the whole-mount OB of a P7 rat that was infected at E15. (Scale bar, 200 μm.) (G) Axons from OSNs ectopically expressing Gαs* sort and coalesce into a glomerulus (arrowhead). (Scale bar, 100 μm.) (H) A magnified view of the glomerulus in G (arrowhead). (Scale bar, 50 μm.) (I) (Upper) Expression of Gαs* (indicated by GFP, green) does not inhibit OSN maturation as assayed by OMP expression (red, arrowheads). (Lower) Expression of Gαs* 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 μm.) (J) (Upper) The axon terminals of OSNs expressing Gαs* (labeled by GFP, green) are enriched for the synaptic marker synaptophysin (Synph; red) even in heterogeneous glomeruli. (Lower) The ectopic Gαs* glomeruli are innervated by the dendrites of MAP2+ bulbar neurons (red). (Scale bar, 25 μm.)

Fig. 3.

Activation of cAMP pathway underlies GPCR induction of axonal coalescence. (A_–_E) Ectopic expression of functional human β2AR in OSNs (FR-β2AR-i-tGFP) results in axonal convergence. (A) Immunohistochemical staining reveals that β2AR protein (red) traffics to OSN cilia in a P7 mouse OE injected with a retroviral vector at E13. (Scale bar, 10 μm.) (B) A GFP+ OSN dissociated from a P21 rat infected at E15 responds to 10 μM isoproterenol. (C) GFP− OSNs in the same field of cells are not activated. (Vertical scale bar, 10%ΔF/F; horizontal scale bar, 2 min.) (D and E) Axons (green) of OSNs ectopically expressing β2AR converge in the OB but do not fully enter a glomerulus (white arrowhead) in a P21 rat. TOTO3 (blue) labels all of the nuclei in the OB. [Scale bar: D, 100 μm; E (enlarged view of D), 25 μm.] (F_–_I) Lack 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 glomerulus 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 glomeruli within close proximity of one another. (I) In the AC3−/− background, M71G and M72Z axons intermingle within the same glomerulus. (Scale bar, A and B, 500 μm; C and D, 25 μm.)

References

1. Mombaerts P, Wang F, Dulac C, Chao SK, Nemes A, Mendelsohn M, Edmondson J, Axel R. Cell. 1996;87:675–686. - PubMed
1. Treloar HB, Purcell AL, Greer CA. J Comp Neurol. 1999;413:289–304. - PubMed
1. Buck L, Axel R. Cell. 1991;65:175–187. - PubMed
1. Zhang X, Firestein S. Nat Neurosci. 2002;5:124–133. - PubMed
1. Malnic B, Hirono J, Sato T, Buck LB. Cell. 1999;96:713–723. - PubMed

A G protein/cAMP signal cascade is required for axonal convergence into olfactory glomeruli - PubMed (original) (raw)