Article Figures & Data
Figures
- Figure 4.
Correlation between the DiI-stained areas in the OE and locations of DiI spots in the OB. A, The retrograde staining of OSN cell bodies was analyzed along the endoturbinate II and ectoturbinate 2′ of the OE. This region includes all zones from 1 to 4 and is reproducibly stained with DiI from any point on the lateral side of the OB. B, Staining signals were plotted on the unrolled maps of the OE region along endoturbinate II and ectoturbinate 2′. The midpoint of the signal distribution on the map was determined, and its location was represented by one of the different colors as defined by a color gradation diagram. C, Eighty different DiI spots analyzed in the present study are colored and located in the schematic diagram of the OB. A, Anterior; D, dorsal; P, posterior; V, ventral.
- Figure 1.
In situ hybridization of the mouse OE sections. A, Conventional OE zones are represented by the following standard OR genes: MOR 265-1 (zone 1), 277-1 (zone 2), 103-1 (zone 3), 103-15 (zone 4a), and 244-1 (zone 4b). Zone 4 was subdivided into two subzones, 4a and 4b, to calculate zone indices for the zone 4 OR genes. Four cursor lines, 1-4, were placed between the two neighboring zones or subzones as shown. The expression of the OMACS gene that codes for the olfactory-specific medium-chain acyl-CoA synthetase (Oka et al., 2003) was used as a marker for zone 1. Zone 1 is also defined as an OCAM-negative zone by in situ hybridization. Scale bar, 100 μm. B, Distribution of hybridization signals. Five OR genes, MOR 103-1, 253-9, 157-1, 253-7, and 103-15, expressed in zone 3 and/or zone 4 are analyzed. The zone 3-4 regions (boxed in the diagram of A) in every sixth coronal section (8 μm thick) were examined from the anterior-to-posterior direction (left to right). Hybridization signals were plotted on the unrolled maps of the OE region along endoturbinate II and ectoturbinate 2′. The zone index of each OR gene was calculated as a ratio of hybridization signals in two neighboring zones. For example, MOR 253-9, for which the zone index was 3.2, showed 20% of total signals in zone 4a and 80% in zone 3. C, Distribution of zone indices. A total of 80 different OR genes were analyzed. Zone indices for the standard OR genes are indicated by arrows. The scale does not reflect the physical distances between the neighboring zone boundaries.
- Figure 2.
Identification of OSN projection sites in the OB. Glomerular locations are compared for four OR genes (right). Distribution of hybridization signals in the OE was analyzed for each OR gene (left), and the zone index was calculated as described in Figure 1 B. Projection sites for 235-1 (A16), 236-1 (MOR18), 244-3 (MOR83), and 244-1 (MOR28) were determined by in situ hybridization of OB sections (Tsuboi et al., 1999) and by fluorescent staining of the OB with the differently tagged OR transgenes (Serizawa et al., 2003) (K. Miyamichi and S. Serizawa, unpublished result). B, Projection sites for the transgenic OR genes are shown. To visualize the glomeruli for the GFP-tagged HA16 and MOR10 (244-2) transgenes, the whole-mount OBs were observed for GFP by fluorescence microscopy. Projection sites for the endogenous A16 (235-1) and MOR10 (244-2) genes (indicated by a triangle and a square, respectively) were determined by in situ hybridization of OB sections using the OR gene sequences as probes (Tsuboi et al., 1999). Projection sites for the transgenes (green triangles for the Tg-HA16 and blue squares for the Tg-MOR10) are shifted compared with those for the endogenous genes, as shown in the schematic diagram. Scale bar, 500 μm. C, OE sections of the BAC-HA16, BAC-220T, and non-Tg mice were analyzed by in situ hybridization with OR gene probes. Hybridization signals were plotted schematically, and the zone indices were calculated as described for Figure 1 B. Expression of the GFP-tagged transgenes was detected with the GFP coding sequence as a probe. Distributions of hybridization signals across the boundary of zones 4a and 4b are schematically shown with the zone indices. A, Anterior; D, dorsal; P, posterior; V, ventral.
- Figure 3.
Retrograde DiI staining of OSN axons. A, The OB was marked with a felt-tip pen to determine the location of the target spot for DiI labeling (a-c). The posterior portion of the OB was sliced off with a microslicer until the target glomerulus appeared on the sliced surface of the remaining OB. DiI crystal was placed on the target spot, which usually covered three to five glomeruli. The sample was then embedded in agarose gel and incubated for 2-3 weeks at 37°C to allow the DiI to diffuse from the glomeruli to the OSN cell bodies through their axons. B, Coronal sections of the OE were analyzed for the distribution of DiI fluorescence. Two neighboring DiI spots on the OB gave similar retrograde staining patterns in the OE, whereas spots that were far apart usually yielded different patterns. C, A pair of DiI spots (1:2), located on the same level along the dorsal/ventral axis, were analyzed for the staining patterns in the OE. Note that their patterns are quite similar, despite the large separation of the DiI spots on the OB along the anterior/posterior axis. Similar examples are shown for three other sets of DiI spots (3:4, 5:6, and 7:8). Scale bars: A, 500 μm; B, C, 100 μm. A, Anterior; D, dorsal; P, posterior; V, ventral.
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