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First published online 30 May 2007
doi: 10.1242/dev.006346

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Adenylyl cyclase-dependent axonal targeting in the olfactory system

Julien A. Dal Col^1,*, Tomohiko Matsuo^1,*, Daniel R. Storm² and Ivan Rodriguez^1,

¹ Department of Zoology and Animal Biology, and NCCR Frontiers in Genetics, University of Geneva, Geneva, Switzerland.
² Department of Pharmacology, University of Washington, Seattle, WA 98195-7280, USA.

View larger version (89K): [in this window] [in a new window]   Fig. 1. Ac3 expression in the mouse olfactory system. (A) RT-PCR showing that Ac3 transcripts are present during development of the main olfactory and vomeronasal epithelia. (B) Western blot analysis of Ac3 expression in the main olfactory epithelium and olfactory bulb. The closed arrow indicates the position of the expected 200-210 kD Ac3 band. The open arrow indicates the Ac3 variant found in the olfactory bulb (155-170 kD). A weak non-specific band of 200 kD is present in wild-type and Ac3-/- olfactory bulb extracts. (C) In situ hybridization of the olfactory epithelium shows Ac3 mRNA expression in OSNs of a P15 mouse. A control hybridization on a similar section from an Ac3-null mouse is shown in the insert. (D) Expression of Ac3 in the vomeronasal organ of a P0 mouse (insert shows the corresponding in situ hybridization of an Ac3-null animal). The signal was weaker than the one observed in the main olfactory epithelium. (E) Expression of Ac3 in the olfactory system of E12.5, 16 and 19 embryos. The two lower images represent antibody controls: ctr1, Ac3-/- section; ctr2, pre-adsorbed antibody. (F-H) Immunohistochemistry (F, DAPI; G, OMP) on coronal sections shows that Ac3 (H) is highly enriched on OSN dendritic endings, but is also expressed on OSN axonal bundles and in glomeruli. Arrowheads in F show individual glomeruli. Scale bars: 60 µm. Cp, cribriform plate; Gl, glomerular layer; MOE, main olfactory epithelium; NL, nerve layer; OB, olfactory bulb; Sm, septum; VNE, vomeronasal epithelium.

View larger version (121K): [in this window] [in a new window]   Fig. 2. Aberrant OSN projections in Ac3-null mice. (A-F) Whole-mount analysis of P15 Omp-lacZ bulbs in which all mature OSNs are stained blue. (A,B) Control and Ac3-/- mice, respectively. A clear reduction in the number of caudal glomeruli is observable in the mutant when compared with the control. (C,D) Dorsal view of the olfactory bulb of two animals. (E,F) Corresponding dorsal view of Ac3-/- animals, revealing a drastic reduction of dorsocaudally located glomeruli. Arrows indicate the position of necklace/Grueneberg glomeruli, the organization of which appears affected in the mutants. (G,H) Sagittal sections of control (G) and Ac3-null (H) P15 mice. Omp is stained in green. The complete lack of caudal Omp-positive glomeruli on the dorsal part of the bulb is clearly visible. Inserts correspond to high magnifications of wild-type and Ac3-/- dorsorostral glomerular layers, and show a glomerular disorganization in Ac3-/- animals. Scale bars: 0.5 mm. AOB, accessory olfactory bulb; C, caudal; D, dorsal; L, lateral; M, medial; MOE, main olfactory epithelium; OB, olfactory bulb; R, rostral; V, ventral; VNO, vomeronasal organ.

View larger version (77K): [in this window] [in a new window]   Fig. 3. Axonal projections of P2-, M72-, MOR23- and V1rb2-expressing Ac3-/- sensory neurons. (A-K) Whole-mount views of X-Gal-stained (blue) OSNs (except for H and I, in which the green color reflects the expression of GFP). (A,B) No glomerulus is observed in B. Inserts show the tip of the largest right turbinates of wild-type and Ac3-/- mice. The number of P2-expressing OSNs is reduced in mutant animals. (C,D) Lateral view of P15 M72-lacZ control and Ac3-/- animals. Closed arrows indicate the position of the wild-type medial glomeruli. The open arrow indicates the position of the novel, rostromedial glomerulus. Analysis of turbinates (inserts) shows no differences in the number of M72-expressing neurons. (E) The two medial glomeruli persist in Ac3-/- M72-lacZ P50 mice. (F,G) Dorsal views of P15 M72-lacZ (F) control, and (G) Ac3-/- left bulbs. Open and closed arrows indicate the topographical position of mutant and wild-type M72 glomeruli, respectively. (H,I) Medial views of right bulbs of P15 animals, in which all axonal projections corresponding to MOR23-expressing OSNs are GFP fluorescent. A novel glomerulus is formed on the rostroventral side of the bulb in Ac3 mutant animals (open arrow). (J,K) Dorsal views of the accessory olfactory bulb of V1rb2-lacZ control (J) and Ac3-null (K) 3-week-old animals. External boundaries of the left accessory olfactory bulb are shown as a dotted line in J. No obvious alteration of the typical V1rb2 map was observed. Scale bar: 1 mm. C, caudal; D, dorsal; L, lateral; M, medial; R, rostral; V, ventral.

View larger version (66K): [in this window] [in a new window]   Fig. 4. Anteroposterior targeting shift in P15 Ac3-null mice. (A) Schematic diagram showing the medial portion of the right bulb. White dots correspond to the topographical glomerular coordinates innervated by M72-expressing OSNs. Blue dots indicate the innervation of M72-expressing OSNs in an Ac3-/- background. (B) Schematic showing a dorsal view of a left olfactory bulb. The color code is similar to that in A, and circles indicate lateral glomeruli. (C) Similar lateral view to that in A, but with coordinates corresponding to glomeruli innervated by MOR23-expressing OSNs. (D) Lateral schematic view of the olfactory system, in which is indicated the zonal limit reached on the septum by M72-expressing OSNs in Ac3+/+ mice; in blue is the corresponding limit reached in Ac3-/- animals. C, caudal; D, dorsal; L, lateral; M, medial; R, rostral; V, ventral.

View larger version (52K): [in this window] [in a new window]   Fig. 5. Altered structure and non-exclusive innervation of glomeruli in Ac3-/- glomeruli. (A,B) Sagittal sections through olfactory bulbs of P17 Ac3-/- MOR23 and M72 homozygous mice. Arrows indicate the presumed external glomerular limits, which are difficult to identify due to the loose ensheathing of the glomeruli by periglomerular cells. The glomerular structures are irregular, and the glomeruli corresponding to M72 and MOR23 are innervated by fibers stained with NCAM (in red) that do not express M72 or MOR23. Control sections of wild-type glomeruli innervated by MOR23 (C) or M72 fibers show exclusive innervation and regular structure of the glomeruli (data not shown) (Feinstein and Mombaerts, 2004; Vassalli et al., 2002). Scale bars: 100 µm. OB, olfactory bulb.

View larger version (49K): [in this window] [in a new window]   Fig. 6. Ac3/OR co-expression in the olfactory neuroepithelium. (A-C) In situ hybridizations of main olfactory epithelium sections (M72-lacZ P5 mouse), with an Ac3 probe (red) and a probe recognizing M72-expressing OSNs (ß-gal, green). Two M72-expressing OSNs are shown in C, one positive and the other negative for Ac3 expression. (D) Graph indicating the percentage of MOR23-, M72- and P2-expressing OSNs reactive (yellow) or not (green) with the Ac3 probe. (E,F) In situ hybridizations corresponding to Ac3 (red) and Omp (green) expression. A clear correlation of expression can be observed. Scale bars: 30 µm. MOE, main olfactory epithelium.

View larger version (98K): [in this window] [in a new window]   Fig. 7. Neuropilin expression in Ac3-/- and Ac+/+ OSN axonal projections. (A) Sagittal section through the olfactory system of a P20 control mouse shows a strong neuropilin 1 expression in OSN axonal bundles (AB) and in the nerve layer (NL) of the olfactory bulb. The cribriform plate is shown with white dots. (B) Similar staining as in A, on an Ac3-/- P20 mouse. No neuropilin 1 was detected in OSN axonal bundles and projections to the bulb. (C,D) Anti-neuropilin 1 staining (green) of Ac3+/+ (C) and Ac3-/- (D) olfactory bulb coronal sections. No neuropilin 1 staining is observed in Ac3-/- nerve layer or glomeruli. NCAM-expressing OSN projections are in red. (E-J) Coronal sections of wild-type bulbs (P11) showing neuropilin 1 expression (red) in axonal projections corresponding to OSNs expressing the MOR23 odorant receptor (green). (E,H) DAPI staining. Both lateral (E-G) and medial (H-J) glomeruli are immunoreactive. (K-N) M72 (M) and P2 (K) glomeruli are respectively positive and negative for neuropilin 1 expression shown in N and L, respectively. Scale bars: 120 µm. Cp, cribriform plate; D, dorsal; Gl, glomerular layer; L, lateral; M, medial; NL, nerve layer; OB, olfactory bulb; V, ventral.