Table 1.
Primary Antisera Used.
Fig 1.
OEC-rich areas of the olfactory system express β-galactosidase (β-gal) in α7lacZ/+ and α7lacZ/lacZ mice.
A: In the central (olfactory bulb, OB) and peripheral (nasal cavity, NC) olfactory areas, sagittal sections from α7+/+ mice have no β-gal reaction. B, C: Mice with one (α7lacZ/+, B) or two copies (α7lacZ/lacZ, C) of the α7lacZ allele have similar patterns of β-gal histochemistry. α7/β-gal is within the olfactory nerve layer (ONL) and the lamina propria (LP), two areas heavily populated with OECs. Blood vessels also express α7/β-gal. D, E: Arrows point to the β-gal-labeled olfactory nerve (cranial nerve I) as it courses through the cribriform plate (C) that separates the nasal cavity from the olfactory bulbs. F: A horizontal section of α7lacZ/lacZ olfactory mucosa shows intense β-gal expression in the LP (arrows) and light reactivity in the olfactory epithelium (OE). Scale bars for A-C: 200 μm; D-E: 50 μm; F: 100 μm.
Fig 2.
Expression of α7 integrin and β-gal in the olfactory nerve.
A-C: Anti-α7 integrin (ITGA7, red) and Hoechst nuclear stain (blue) show the presence of α7 protein in α7+/+ (A) but not α7lacZ/lacZ (B) olfactory nerve (ON, arrowheads), olfactory nerve layer (ONL) and glomerular layer (GL). Adjacent α7lacZ/lacZ section (C) has α7/β-gal reactivity (green) in the ON and ONL (arrowheads). D-F: OECs in the ON are marked with arrowheads and show antibody expression in α7+/+ (D) or α7/β-gal in α7lacZ/lacZ nerve (F). The α7lacZ/lacZ negative control shows only Hoechst-labeled nuclei (E). Scale bar A-C: 100 μm; D-F: 20 μm.
Fig 3.
α7 integrin colocalizes with OEC markers SOX10, S100β, and AQP1 in the olfactory bulb.
A-C: Expression of α7/β-gal (green) in α7lacZ/lacZ olfactory bulbs overlaps with SOX10 expression (red nuclei) at arrows. D-F: A confocal image (4.0 μm-thick Z-stack) of the olfactory nerve shows α7/β-gal immunofluorescence (green) within the OEC cell bodies (arrows) and their thin processes. SOX10 (red) and β-gal staining colocalize within OEC nuclei (arrows). G-I: The α7/β-gal product (green) appears to colocalize in the α7lacZ/lacZ olfactory nerve layer (ONL) with the glial marker S100β (red). OEC processes extend into the glomerular layer (GL; arrowheads) but don’t enter the glomeruli. J-L: The water channel Aquaporin 1 (AQP1, red) is highly expressed in the ONL and colocalizes with α7/β-gal fluorescence (green). Arrowheads mark AQP1 and light β-gal expression around but not within the glomeruli, the typical distribution of OECs. Scale bars for A-C, G-L: 50 μm; D-F: 20 μm.
Fig 4.
α7/β-gal is not expressed by mature olfactory receptor neurons.
A-C: The α7lacZ/lacZ olfactory bulb expresses α7/β-gal (green) in the olfactory nerve layer (ONL), and is closely associated with the olfactory marker protein (OMP, red). In axons of mature olfactory receptor neurons, OMP immunoreactivity fills their synaptic target, the glomeruli (G). α7/β-gal expression and OECs are excluded from the glomeruli. D-F: Fascicles of the olfactory nerve within the lamina propria (LP) have distinct areas of β-gal (green, arrows) and OMP-positive (red) immunoreactivity. β-gal-labeled OECs surround the OMP-labeled axon bundles. The olfactory epithelium (OE) contains OMP-labeled (red) olfactory receptor neurons. Scale bars for A-C: 50 μm; D-F: 20 μm.
Fig 5.
β-Dystoglycan is expressed on the outer OEC membrane.
A-C: In the ONL of the olfactory bulb, β-dystroglycan (β-DG, green) is concentrated on the perimeters of large nerve fascicles, whereas α7/β-gal (red) is distributed broadly. α7/β-gal strongly stains OEC cell bodies (arrows) while β-DG immunoreactivity is restricted to the outer OEC membranes. D-F: In the ONL, β-DG reactivity is excluded from mature olfactory receptor neurons identified with OMP (red). G-I: In the nasal cavity β-DG (green) and α7/β-gal (red) are both strongly expressed around the perimeter of cross sections of olfactory nerve fascicles (arrowheads). J-L: Laminin α1 reactivity (red) colocalizes with β-DG on olfactory nerve fascicles. Scales bars for A-C, D-F, G-I, and J-L: 20 μm.
Fig 6.
α7 integrin does not facilitate OEC-induced neurite outgrowth.
A-H: Representative images from the experimental groups. A-D show cultures seeded with α7+/+ cortical neurons, whereas E-H contained α7lacZ/lacZ neurons. Neurons were cultured on laminin (A, E), PLL (B, F), PLL + α7+/+ OECs (C, G), and PLL + α7lacZ/lacZ OECs (D, H). Neurons were visualized with β3-tubulin, and OECs marked with Cell Tracker Green. I: Total neurite outgrowth per well was normalized to culture-matched outgrowth on PLL (normalized mean ± SEM is plotted; See S1 Table for raw data). Neuronal genotype did not affect neurite outgrowth on laminin or PLL (n = 3, p = 0.838). The laminin substrate induced more neurite growth than PLL (1.99 ± 0.07 units normalized; ***p<0.0001), and OECs enhanced neurite outgrowth lengths at a level that did not differ from laminin. The addition of OECs significantly increased the neuronal outgrowth compared to PLL only levels (α7+/+ OECS: 1.60 ± 0.11 **p = 0.0003; α7lacZ/lacZ OECs: 1.71 ± 0.14, ***p<0.0001). J: For neuron-OEC co-cultures we sorted individual neurite lengths by the type of association they made with OECs (i.e., aligned, cross, no contact) and averaged them. The lengths of neurites that aligned with OECs were compared to neurites grown on laminin, PLL, and different OEC associations. (See S2 Table for averages, further description, and p-values). Scale bar A-H: 50 μm.
Fig 7.
α7 integrin mediates OEC migration on a laminin substrate.
A-F: Immunopurified OECs from all three genotypes migrated through transwell inserts coated with laminin. Enlargements of boxed areas A, C, and E are shown in B, D, and F. OECs have numerous membrane protrusions (arrowheads) that interact with other OECs. G: The migratory-potential ratio was used to assess the number of p-75-NGFR-labeled OECs that migrated through the laminin-coated insert divided by those that migrated on PLL-coated inserts. The mean migratory-potential ratio for α7+/+ OECs was 2.14 ± 0.76, and for α7lacZ/+ OECs was 1.45 ± 0.23; α7lacZ/lacZ OECs did not have a substrate preference (0.92 ± 0.19). Many more α7+/+ OECs migrated through the laminin-coated transwell membranes than α7lacZ/lacZ OECs (**p<0.01). Scale A, C, E: 50 μm; B, D, F: 50 μm.