Article Figures & Data
Figures
- Fig. 1.
Genetic approach. A, Targeted mutagenesis of the OMP locus. a,OMP-GFP-LTNL targeting vector. The green box (GFP) represents the coding sequence of theGFP gene. The gray box(tk-neo) represents the negative selectable markerHSV-tk followed by the positive selectable markerpgk-neo, flanked by loxP sites (red triangles). The relevant restriction sites are indicated asX (XhoI), R(EcoRI), and S (SphI).b, Wild-type OMP locus. The pink box (OMP) indicates the coding sequence of theOMP gene and 150 nucleotides of the 3′ noncoding region. The black bar on the left represents the 5′ external probe used to detect homologous recombination at this locus by Southern blot analysis. c, OMP locus after homologous recombination. d, OMP locus after Cre recombination. B, Targeted mutagenesis of theM72 locus. a,M72-IRES-tauGFP-LNL targeting vector. The blue box (M72) represents the coding sequence of theM72 OR gene. The white box(i) represents the IRES sequence. The green box (tauGFP) represents the coding sequence of the tauGFP fusion. The gray box(neo) represents the selectable markerpgk-neo flanked by loxP sites (red triangles). The relevant restriction sites are indicated as N (NdeI), RV(EcoRV), and H (HindIII).b, Wild-type M72 locus. c,M72 locus after homologous recombination.d, M72 locus after Cre recombination.C, Diagram of bicistronic design. When a neuron chooses, by an unknown process, the mutant M72-IRES-tauGFP allele for expression, a bicistronic transcript is produced in the nucleus that is exported to the cytoplasm. Ribosomes translate two polypeptides from this message: the M72 OR protein (a 7-transmembrane protein) and the tauGFP fusion protein (a fluorescent axonal marker). Cotranslation of both the receptor and the reporter is mediated by theIRES sequence. The M72 OR protein is targeted to the plasma membrane. The tauGFP marker binds to microtubules, which are present abundantly in axons and axon terminals.
- Fig. 2.
Olfactory epithelium of OMP-GFPmice. A, Medial whole-mount view of a half-head of an adult mouse. Olfactory epithelium (left) and olfactory bulb (right) are intensely fluorescent. Images were photographed using an epifluorescence stereomicroscope (Leitz MZ12; Leitz, Stuttgart, Germany). Image width, 6 mm.B, Close-up whole-mount view of the olfactory epithelium. Green dots represent OSNs; dark areas correspond to the non-GFP-expressing supporting cells. Images were photographed using a Leitz MZ12 stereomicroscope. Image width, ∼1 mm. C, Low-power view of a histological section through the nose. Green fluorescent OSNs line the convoluted surface of the turbinates. Their axons are assembled in bundles underneath the epithelium. Images were photographed using an epifluorescence wide-field microscope (Zeiss Axioplan 2). Image width, ∼1.4 mm. D, Medium-power view of a histological section through the nose. Thehorizontal white lines indicate the approximate levels of the optical sections shown in E–H. Images were photographed using a confocal laser scanning microscope (Zeiss LSM 510). Image width, ∼256 μm. E, Optical section through OSN dendrites produced by TPLSM from a 4-d-old mouse. Individual dendritic knobs are visible. Image width, ∼256 μm.F, Optical section at the level of the cell bodies of OSNs produced by TPLSM. Image width, ∼256 μm. G, Optical section at the level of the axons of the cell bodies produced by TPLSM. Single axons are clearly visible, even when imaged through the brightly labeled cell body layer, which saturated the detector. Image width, ∼256 μm. H, Optical section at a level below the epithelium produced by TPLSM. OSN axons coalesce to form ribbon-like fascicles. Image width, ∼256 μm.
- Fig. 3.
Olfactory bulb of OMP-GFP mice.A, Whole-mount view of the dorsal surface of both olfactory bulbs. Top is posterior; bottomis anterior. The outer nerve layer and the glomerular layer produce intense fluorescence that is easily detectable with an epifluorescence stereomicroscope (Zeiss Stemi SV11). Image width, 5 mm.B, Close-up whole-mount view of glomeruli in the olfactory bulb, photographed with an epifluorescence stereomicroscope (Zeiss Stemi SV11). Image width, ∼1.2 mm. C, Optical section at the level of the glomerular layer (right) and the outer nerve layer (left) produced by TPLSM. Glomeruli are discrete globose structures covered with a thick mat of fibers running across the surface of the bulb without obvious direction or stereotyped organization. Image width, ∼256 μm.D, Optical section at the level of the glomeruli, below the outer nerve layer, produced by TPLSM. The nonfluorescent areas inside the glomeruli presumably correspond to the dendrites of interneurons and second-order neurons and to glia and blood vessels, which do not express OMP and thus do not express GFP in these mice. Image width, ∼256 μm.
- Fig. 4.
M72 glomeruli in M72-IRES-tauGFPmice. A, Whole-mount view of the dorsal surface of the olfactory bulbs of a mature M72-IRES-tauGFP mouse. Orientation is identical to that in Figure 3A. A pair of medial and lateral green fluorescent glomeruli can be discerned within each bulb. This study concentrated on the lateral M72 glomeruli because they reside in a flattened region of the dorsal surface of the olfactory bulb and are readily accessible for imaging. The dark lines are blood vessels within the meningi. Images were photographed using an epifluorescence stereomicroscope (Zeiss Stemi SV11). Image width, 5 mm. B, Schematic representation of image shown in A. P, Posterior;M, medial; A, anterior; L, lateral, R, right; L, left. The interbulbar symmetry of the positions of the M72 glomeruli is apparent. The intrabulbar symmetry of the positions of the M72 glomeruli is along a plane that intersects with the midline at an ∼30° angle, such that the lateral glomeruli are more anterior and more dorsal than the medial glomeruli. C, Stereo pair of three-dimensional TPLSM reconstruction of the right lateral glomerulus of a PD18M72-IRES-tauGFP mouse (strain T15/loxP). A few major fascicles terminate in the glomerulus. Image width, ∼256 μm.D, Stereo pair of the left lateral glomerulus of the mouse shown in C. There is no bilateral symmetry; the pattern of fascicles is different from that in C. Image width, ∼256 μm. E, Stereo pair of a lateral glomerulus from a PD18 M72-IRES-tauGFP mouse, a littermate of the mouse shown in C and D. Multiple small fascicles converge onto the glomerulus from widely varying angles. Image width, ∼256 μm.
- Fig. 5.
Glomeruli in mature M72-IRES-tauGFPmice. In these projections, the dorsolateral surface of the olfactory bulb is oriented toward the viewer, and the rostral end of the nose is pointed toward the bottom. Each projection depicts an example of M72 glomeruli imaged in different individuals of PD18 or older. Image width, ∼256 μm; scale bar, 40 μm.A–C, Examples of mature M72 glomeruli in the lateral hemisphere of the right bulb. D–F, Examples of mature M72 glomeruli in the lateral hemisphere of the left bulb. These glomeruli receive their axonal input predominantly in the form of smaller fascicles. In some instances (D, F), fascicles loop back onto the glomerulus (top right).
- Fig. 6.
Glomeruli in mature M71-IRES-tauGFPmice. Three examples are shown. The architecture of the axonal plexuses and glomeruli is complex and variable.
- Fig. 7.
Postnatal development of M72 glomeruli. The montage shows coalescing M72 axons in four littermates at successive postnatal days. From a tangle of fibers at birth (PD1), a glomerular-like structure develops between PD2 andPD3.
- Fig. 8.
Right–left pairs of developing M72 glomeruli. The right and left lateral M72 glomeruli within different individual mice were imaged on PD2 (A), PD3 (B), or PD5 (C). The dorsolateral surface of the olfactory bulb is oriented toward the viewer, and the rostral extreme of the nose is pointed toward thebottom. Image width, 256 μm; scale bar, 80 μm.A, Lateral M72 glomeruli in three PD2 mice. Eachrow represents the right and left lateral M72 glomeruli within an individual mouse. Converging M72 axons are clearly visible; however, a glomerular-like structure is not obvious and may not have stabilized by this stage. B, Lateral M72 glomeruli in three PD3 mice. M72 glomerular-like structures become visible as M72 axons converge and stabilize their target glomeruli. C, Lateral M72 glomeruli in three PD5 mice. A glomerular-like structure has formed and is clearly visible.
Additional Files
Animation 7: Rocking reconstruction of an M72 glomerulus of a mature M72-IRES-tauGFP mouse.
Same glomerulus as shown in Fig 4E. Numerous smaller fascicles arrive from directions spread over 360 degrees.
Animation 8: Rocking reconstruction of an M72 glomerulus of a mature M72-IRES-tauGFP mouse.
Same glomerulus as shown in Fig 5D. Most fascicles arrive from down under, but one fascicle (top right) approaches the glomerulus from a very different orientation.
Animation 1: Section series through the olfactory epithelium of a PD2 OMP-GFP mouse.
Same specimen as shown in Fig. 2E-H. The optical sections are taken from superficial to deep. Dendritic knobs are followed by dendrites, cell bodies, axons, and axon fascicles. Only with TPLSM it is possible to see dim features as single axons underneath a layer of extremely bright structures such as the cell bodies.
Animation 2: Section series through the olfactory bulb of an adult OMP-GFP mouse.
Same specimen as shown in Fig. 3 C,D. The optical sections are taken from superficial to deep. The outer nerve layer is followed by the glomerular layer.
Animation 3: Section series through the olfactory bulb of a PD4 OMP-GFP mouse.
The optical sections are taken from superficial to deep. The outer nerve layer is followed by the glomerular layer. The glomeruli are smaller and less-well formed as in Animation 2.
Animation 4: Section series through an M72 glomerulus of a mature M72-IRES-tauGFP mouse.
Same glomerulus as shown in Fig 4C. The images are taken from deep to superficial.
Animation 5: Rotating reconstruction of an M72 glomerulus of a mature M72-IRES-tauGFP mouse.
Same glomerulus as shown in Fig 4C. The rotation provides a better appreciation of the various levels at which axons and axon fascicles approach the glomerulus.
Animation 6: Rocking reconstruction of an M72 glomerulus of a mature M72-IRES-tauGFP mouse.
Same glomerulus as shown in Fig 4D. It is the lateral glomerulus in the left bulb of the same mouse as the glomerulus shown in Animation 5.
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