Sequential Maturation of Olfactory Sensory Neurons in the Mature Olfactory Epithelium

Cell proliferation in the septal wall of the OE in P25 mice. A, B, Maximum intensity projections of confocal z-stacks showing Ki67-expressing cells (red) in the dorsal (A) and ventral (B) domains of the septum. Note the low density of Ki67⁺ cells in the dorsal zone (arrows). Ki67-labeled cells at the luminal surface of the ventral septum are sustentacular cells. C, Confocal image showing cCasp3-expressing cells in the ventral septal OE (green). Note the low number of apoptotic cells (arrow). D, Illustration of the OE from a coronal perspective. Black and red rectangles represent dorsal and ventral zones of the septal OE, respectively. E, F, Histograms show the number of BrdU⁺ cells per millimeter (E) and Ki67⁺ cells per millimeter (F) in the dorsal and ventral zones of the septal OE at different following time points after BrdU injections: DPI-B-1, DPI-B-3, DPI-B-5, and DPI-B-7. The mean ± SEM values are plotted (n = 3). G, Histogram representing the number of BrdU⁺ and Ki67⁺ cells in the ventral septal OE. The mean ± SEM values are plotted. **p < 0.01, ***p < 0.001. Dashed white lines delineate the surface of the OE (top) and basal lamina (bottom). DRAQ-5 is in blue. Scale bars, 25 μm.

Cell death in the OE in P25 mice. A, Semipanoramic view of the OE including the septum and turbinates. a′, Caspase 3⁺ cells (green) are mainly spread across the turbinates (arrows). Almost no caspase 3⁺ cells were observed along the septal wall. B, C, High magnification of the septal wall showing the absence of caspase 3⁺ cells. D, E, High magnification of different turbinates showing the presence of several caspase 3⁺ cells (arrows). Scale bars: A, 400 μm; a′, 200 μm; B–E, 50 μm.

Migration and maturation of P25-born OSNs in the septal OE. A, Newborn OSNs migrate radially in the OE. Graph showing relative radial position of BrdU⁺ cells in the dorsal (gray) and ventral (red) septal OE at different time points after BrdU injections. B, Graph showing the distance between BrdU⁺ cells and the basal lamina; distance is represented in micrometers. C, Relative position of P25-born OSNs in the ventral zone of the septum at six different time points during a 12 d timecourse after BrdU administration. The mean ± SEM values are plotted (n = 3). *p < 0.05, **p < 0.01. D, Coimmunolabeling for BrdU (magenta) and markers of maturation (green): GAP 43, AC3, and OMP at different time points after BrdU administration. D1–D3, Confocal images at low magnification representing flattened stacks of 10–12 1-μm-thick optical sections. D4–D9, High-magnification confocal images representing flattened stacks of seven to nine 1-μm-thick optical sections. GAP 43 and BrdU costaining at DPI-B-5 (D4, D5). AC3 and BrdU costaining at DPI-B-8 (D6, D7). OMP and BrdU costaining at DPI-B-12 (D8, D9). In D4a–c to D9a–c, square cells (a) are magnified in b showing BrdU labeling, and in c showing the corresponding markers GAP 43, AC3, and OMP. In both b and c, colabeled cells are marked with an asterisk. E, Line graph showing the percentage of colabeled (marker/BrdU) cells of the total number of BrdU⁺ cells counted. Transition from immature to mature OSN occurs at 10 d after basal cell division. OSNs first express AC3 at 5 d post-basal cell division. OMP expression is not evident until 8 d post-basal cell division. The mean ± SEM values are plotted (n = 3). **p < 0.01, ***p < 0.001. Dashed white lines delineate the surface of the OE (top) and basal lamina (bottom). DRAQ-5 is in blue. Scale bars: D1–D3, 25 μm; D4–D9, 10 μm.

Summary correlating migration and maturation of P25-born OSNs in the ventral zone of the septal OE. Top diagram shows the relative radial position of BrdU⁺ cells throughout the OE during a 12 d timecourse following BrdU administration. Pie charts located on the bottom half of the diagram show the percentage of the following costaining cells: BrdU⁺/GAP 43⁺ (blue), BrdU⁺/AC3⁺ (red), and BrdU⁺/OMP⁺ (gray) of the total number of BrdU⁺ cells counted. BrdU⁺ cells not expressing any other marker are represented in green. Dotted vertical columns represent each time point of analysis after BrdU administration: DPI-B-1, DPI-B-3, DPI-B-5, DPI-B-8, DPI-B-10 and DPI-B-12.

BrdU/AC3/OMP triple immunostaining at 10 and 12 d following BrdU administration. A, Confocal image at low magnification representing flattened stacks of seven to nine 1-μm-thick optical sections. BrdU⁺ (green) OSNs coexpressing AC3 (red) and OMP (blue) at day 12 after BrdU injections (arrows). B, Pie charts quantifying BrdU⁺ cells expressing AC3 (maroon) or OMP (gray), coexpressing AC3 and OMP (yellow), or not expressing either AC3 and/or OMP (green) at DPI-B-10 and DPI-B-12. C, D, Higher-magnification confocal images showing examples of BrdU⁺ cells (green) coexpressing AC3 (red) and/or OMP (blue). C1–C4, Dotted circle denotes a BrdU⁺ cell (C2, green) coexpressing OMP (C1, blue) and AC3 (C3, red), while a dotted square highlights a BrdU⁺ cell (C2, green) expressing OMP (C1, blue), but not AC3 (C3, red); C4 shows a merged image. D1–D4, A dotted circle shows a BrdU⁺ cell (D2, green) coexpressing OMP (D1, blue) and AC3 (D3, red); D4 shows a merged image. E1–E4, Arrow pointing to a BrdU⁺ cell (E2, green) expressing OMP (E1, blue), but not AC3 (E3). Next to the BrdU⁺ cell, there is a BrdU⁻ cell (arrowhead) coexpressing OMP (E1, blue) and AC3 (E3, red); E4 shows a merged image. Dashed white lines delineate the surface of the OE (top) and basal lamina (bottom). Scale bars: A, 25 μm; C–E, 10 μm.

Inducible Cre-recombinase strategy for labeling P25-born OSNs in the OE following basal cell division. A, Ascl1^CreERT2/R26R^ZsGreen double transgenic mice were obtained by crossing an Ascl1^CreERT2/+ transgenic mice line with an R26R^{ZsGreen/ZsGreen} transgenic mice line. 4OH-Tx induces Cre recombination in Ascl1⁺ cells, which results in the expression of ZsGreen in Ascl1^CreERT2/R26R^ZsGreen double transgenic mice. B–D, Cre-recombinase strategy controls: Ascl1^CreERT2/R26R^ZsGreen double transgenic mice show olfactory sensory neuron-specific labeling 12 d after 4OH-Tx injection (B), but no specific labeling after sunflower oil injection, although some nonspecific labeling is present deep in the OE and beneath the basal lamina (C); Ascl1⁺/R26R^ZsGreen transgenic mice show no labeling after 4OH-Tx injection (D). Scale bars, 300 μm.

In vivo genetic fate-mapping strategy reveals spatiotemporal morphologic features of P25-born OSNs. A–D, ZsGreen⁺ cell bodies migrate radially toward the surface of the OE during a 12 d time course following basal cell division, revealing that most of them are differentiated as OSNs with characteristic morphologic features. OSN apical dendrites and axons can be detected as early as 2 d following 4OH-Tx injection (B–D), but not earlier (A). E–H, ZsGreen⁺ axons form fascicles starting at 3 d following 4OH-Tx injection (E) and travel toward the OB (F), and cross the cribriform plate at DPI-Tx-4/5 (G, arrow), and some of them reach the oONL as early as DPI-Tx-5 (H). I, By DPI-Tx-6, the oONL is heavily innervated by ZsGreen⁺ axons. DRAQ-5 is in blue. Dashed white lines delineate the basal lamina in A–G, and the limit between the glomerular layer (GL) and the ONL in H. Dotted circles delineate glomeruli in G, and each glomerulus is designated by the letter “G.” Scale bars: A–D, 20 μm; E, F, H, I, 200 μm; G, 300 μm.

In vivo genetic fate-mapping strategy reveals no evidence of polarity in P25-born OSNs. A, B, ZsGreen⁺ cell bodies lying on the basal lamina (dashed line) at DPI-Tx-1. Initial extension of the apical dendrite (arrowhead) and axon (arrow). Scale bar, 20 μm.

Tracking ZsGreen⁺ axon extension in Ascl1^CreERT2/R26R^ZsGreen double transgenic mice at different time points following 4OH-Tx injection. A, B, F, ZsGreen⁺ axons innervate iONL profoundly at 7-DPI-Tx (A), and they surround ventral glomeruli closely at DPI-Tx-8; some scarce axons enter the GL (B, F). Panoramic sagittal view showing a ZsGreen⁺ axon pathway (arrows) from the OE to the ventrolateral OB (F). C, D, Labeled axons unequivocally innervate glomerular neuropil by 10 d following basal cell division. E, Detail of ZsGreen⁺ axon growth cones (arrow) in the GL. DRAQ-5 is in blue. Dashed circles delineate glomeruli in A–D, and each glomerulus is designated by the letter “G.” Dotted white lines delineate the limit between the GL and the ONL in F. CP, Cribriform plate; GL, glomerular layer. Scale bars: A–D, 25 μm; E, 20 μm; F, 200 μm.