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First published online 17 October 2007
doi: 10.1242/dev.008722

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Axonal wiring of guanylate cyclase-D-expressing olfactory neurons is dependent on neuropilin 2 and semaphorin 3F

The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA.

View larger version (13K): [in this window] [in a new window]   Fig. 1. Targeted mutagenesis of the Gucy2d, Sema3f and Sema3b loci. (A) Genomic structure of the Gucy2d locus on chromosome 7E1 according to the NCBI database. The coding region is spread over 19 exons, with at least another three exons at the 5' untranslated region. No other genes are predicted within the Gucy2d locus. (B) GC-D knock-in mutations. A PacI site (P) was created 3 bp downstream of the stop codon in exon 22 by PCR subcloning between NcoI (N) and SacI (S). The targeting vector encompasses the region from an upstream EcoRI (R) site to a downstream XhoI (X) site. Left, targeting vector. Right upper, targeted mutation in ES cells. Right lower, targeted mutation after germline transmission. Diagram is not to scale. (C) Genomic organization of the Sema3f and Sema3b loci on chromosome 9F2. The coding regions comprise 17 exons spanning 22 kb and 6 kb, respectively. At least three other genes (Gnai2, Slc38a3 and Gnat1) are predicted within the 80 kb between Sema3f and Sema3b. (D) Targeting strategies to create knockout mutations of Sema3f (left) and Sema3b (right). Gene targeting replaces exons 2-15 (Sema3f) or exons 1-17 (Sema3b) with the ACNF cassette. During germline transmission in male chimeras, the ACNF cassette excises itself, removing most (Sema3f) or all (Sema3b) of the ORF. Diagrams are not to scale.

View larger version (116K): [in this window] [in a new window]   Fig. 2. Characterization of GC-D+ neurons in the MOE. (A) Whole-mount view of the turbinates of a GCD-ITL homozygous 3-week-old (3 wk) mouse after X-Gal staining. Labeled cell bodies can be seen on all turbinates, with a greater number situated towards the ventral aspects. The greatest accumulation is located at the caudal end of the MOE (arrow). Anterior. left, dorsal up. (B) Whole-mount view of the septal area of a GCD-ITL homozygous mouse (3 wk). A similar distribution pattern as in the turbinates is apparent. Labeled axons take a straight route towards the cribriform plate. A few labeled cells reside within the septal organ (asterisk). (C) Sections through the MOE of a GCL-ITL homozygous mouse (P10). Labeled cell bodies (arrows in left and middle panels) reside within the lower layers of the epithelium. The morphology of labeled cells is similar to that of conventional OSNs. Cells extend dendrites towards the lumen (asterisks in right panels) and axons towards the lamina propria (arrows in right panels). (D) High-magnification view of a GFP+ cell in a whole-mount of the MOE of a GCD-ITG homozygous mouse (3 wk). A typical neuronal morphology is apparent with a dendrite and dendritic knob (asterisk) and a long axon (arrows). Insert shows en face view of a dendritic knob with GFP+ cilia. (E) Sections through the MOE of a GCL-ITG/GCL-ITL double heterozygous mouse (P7). The cell body is positive for the intrinsic fluorescence of GFP (left panel) and ß-galactosidase immunofluorescence (middle panel). The combined image is shown on the right. Asterisk in middle and right panels denotes the dendritic knob. Scale bars: 1000 µm A,B; 40 µm C, left two panels; 20 µm C, right panel; 20 µm in D,E.

View larger version (119K): [in this window] [in a new window]   Fig. 3. GC-D+ glomeruli in the necklace area of the olfactory bulb. (A) Whole-mount view of the medial OB in an adult GCD-ITL homozygous mouse, stained with X-Gal. Axons enter the bulb through the cribriform plate (asterisk) on the ventral side, and project towards the caudal end of the OB where they form interconnected glomeruli. Anterior, left, dorsal up. (B) The lateral OB. A greater number of axons are entering on the lateral side, and larger glomeruli are formed. Asterisks denote the MOE turbinates. (C,D) The dorsal (C) and ventral (D) OB. Glomeruli are arranged as a closed necklace of beads on a string. Axons projecting from the MOE, glomeruli, and axonal connections between glomeruli are visible ventrally, whereas only glomeruli and axonal connections are visible dorsally. Anterior is up. (E) Three examples of lightly X-Gal stained glomeruli on the lateral OB of GCD-ITL homozygous 6-week-old (6 wk) mice. No obvious pattern of glomerular positions is apparent. Larger glomeruli tend to form on the ventral aspects of the bulb. Some glomeruli form a cluster, at a consistent position (arrows). Anterior, left; dorsal, up. (F) Section of necklace area in adult GCD-ITG homozygous mice. There are superficially located GFP+ glomeruli in the dorsal aspect (upper left), ventral aspect (lower left) and lateral aspect (right). A cluster of glomeruli can be seen (arrows). Dorsal is up. Scale bars: 1000 µm in A-D; 500 µm in E; 50 µm in F.

View larger version (19K): [in this window] [in a new window]   Fig. 4. Numbers of GC-D+ glomeruli. (A) Graph of glomerular size in adult GCD-ITL mice (n=8 bulbs). The longest diameter of a given GC-D+ glomerulus was measured. Three classes of glomeruli can be distinguished: large, 236.2±41.4 µm (n=24); medium, 132.9±20.2 µm (n=53); small, 66.6±10.9 µm (n=75). (B) Graph of developmental series of the number of GC-D+ glomeruli per bulb, at 2 weeks postnatal (2 wk; n=9 bulbs), 4 wk (n=12), 6 wk (n=10) and 12 wk (n=14). (C) Graph of numbers of glomeruli in NP2- homozygous, GCD-ITL homozygous mice compared to GCD-ITL homozygous (`wild-type') mice.

View larger version (92K): [in this window] [in a new window]   Fig. 5. Axonal wiring of GC-D+ neurons depends on Npr2 and Sema3f. (A) Whole-mount view of the medial OB of GCD-ITL homozygous mice visualized by X-Gal staining in wild-type (+/+), NP2- homozygous (NP2- -/-), F homozygous (F -/-), and B homozygous (B -/-) backgrounds. The innervation pattern in B homozygous mice is similar to that in wild type. NP2- and F homozygous mice have axons that extend not only in the ventral areas, but also in the dorsal and anterior aspects of the MOB where they form ectopic glomeruli (arrows). Several of these glomeruli form in the deeper layers of the MOB (arrows with asterisks). Anterior, left; dorsal up. (B) Whole-mount view of the dorsal OB of GCD-ITL homozygous mice in the same backgrounds as in A. Labeled axons and glomeruli (arrows) are detectable in NP2- and F homozygous mice in areas that are not occupied in wild-type and B homozygous mice. Anterior left, lateral up. (C) Whole-mount view of the lateral OB of GCD-ITL homozygous mice in the same backgrounds as in A and B. As seen medially, ectopic glomeruli (arrows) are observed that reside sometimes in deeper layers (arrows with asterisks). Anterior left, dorsal up. Scale bar: 1000 µm.

View larger version (110K): [in this window] [in a new window]   Fig. 6. Immunohistochemistry of necklace and ectopic glomeruli. (A) Sections (18 µm) through the caudal OB at the level of the necklace area of a GCD-ITG homozygous mouse stained with an antibody to PDE2A. All GFP+ axons (green) are also PDE2A+ (red), and are thus yellow in the overlay image. Right-most panel: large glomerulus. Dorsal is up. (B) Sections (18 µm) through the caudal OB at the level of the necklace area of a GCD-ITL homozygous, NP2- heterozygous mouse stained with an antibody to ß-galactosidase (red). A subset of Nrp2+ (GFP; green) glomeruli is also positive for ß-galactosidase (arrows); other necklace glomeruli are only Nrp2+ (asterisks). All GC-D+ glomeruli are also Nrp2+ (yellow in overlay). Right-most panel: high magnification. Dorsal is up. (C) Sections (18 µm) through the OB of a GCD-ITG homozygous, F homozygous mouse stained with antibodies to OMP (red). Green is the intrinsic green fluorescence of GFP. Three left panels: a high-magnification view of an ectopic glomerulus that is filled by GC-D+ axons (yellow in overlay), at the midlevel of the OB outside the necklace area. Right-most panel: example of glomeruli in the necklace area. A GC-D+ glomerulus (red and green) above a GC-D- glomerulus (red only). Ectopic glomeruli reside deeper than glomeruli in the necklace area. Dorsal is up. (D) Sections (18 µm) through the midlevel OB outside the necklace area of a GCD-ITL homozygous, NP2- homozygous (GFP; green) mouse stained with antibodies to MAP2 (red) and ß-galactosidase (blue). MAP2 stained dendrites fill the glomeruli in the glomerular layer (GL) including the ectopic GC-D+/Nrp2+ glomerulus (light blue in overlay image), but are absent in the outer nerve layer (ONL). Scale bars: 100 µm in A,B,D; 50 µm in C.

View larger version (110K): [in this window] [in a new window]   Fig. 7. Aberrant VSN axonal projections in NP2- and F mutant mice. (A) Whole-mount view of the medial surface of the MOB. X-Gal staining of adult V1rb2-IRES-taulacZ homozygous mice that are otherwise wild-type (wt). Dorsal is up, anterior left. (B) Whole-mount view of the medial surface of the MOB. X-Gal staining of adult V1rb2-IRES-taulacZ homozygous mice that are B homozygous (B-/-). No differences are observed in the axon tracts of V1rb2+ VSNs compared with A. (C) Axonal projections of V1rb2+ VSNs across the MOB in a NP2- homozygous, V1rb2-IRES-taulacZ homozygous background. V1rb2+ axons penetrate the cribriform plate (left side) and form tightly packed bundles, but then become much less fasciculated and spread out over a greater area of the MOB. Axons eventually reach and innervate the AOB, which is located at the posterior (right) side of the MOB. (D) A pattern similar to that in C is seen for V1rb2+ VSN axons in a F homozygous, V1rb2-IRES-taulacZ homozygous background. (E-H) Whole-mount view of the left caudodorsal OB. (E) GFP fluorescence of Nrp2+ VSN axons of a NP2- heterozygous, Sema3f wild-type (wt) mouse at 3 weeks postnatal (3 wk). The heterozygous NP- mutation is used as a marker for apical VSNs. Axons innervate only the anterior AOB (aAOB). No Nrp2+ axons occupy the posterior AOB (pAOB). Anterior is top, medial to the right. (F) GFP fluorescence of Nrp2+ axons of a Sema3c homozygous mouse at 4 wk. The heterozygous NP2- mutation is used as a marker for apical VSNs. Axon tracts arrive medially to innervate the aAOB. (G) GFP fluorescence of Nrp2+ VSN axons in a NP2- homozygous mouse. VSN axons also cover the medial half of the pAOB (asterisk). (H) GFP fluorescence of Nrp2+ VSN axons in a NP2- heterozygous, F homozygous mouse. The heterozygous NP2- mutation is used as a marker for apical VSNs. Similar to G, there are misprojections to the pAOB (asterisk). (I-L) Axonal innervation of the aAOB by V1rb2+ VSNs (V1rb2-IRES-taulacZ homozygous background) as visualized by X-Gal staining of whole mounts. All axons remain anterior in a wild-type adult mouse (I). In a NP2- homozygous mouse, V1rb2+ axons also project to the pAOB (arrow in J). A similar phenotype is seen in a F homozygous background (arrow in K). A less severe misrouting can be seen in some F heterozygous mice, with a few axons veering to the pAOB forming small glomerular structures (arrow in L). Innervation patterns in the anterior AOB are comparable in all backgrounds. The red line demarcates the aAOB from the pAOB. Anterior is top, medial to the right. Scale bars: 500 µm in A-D; 150 µm in E-H; 100 µm in I-L.