Odorant receptors regulate the final glomerular coalescence of olfactory sensory neuron axons

Abstract

Odorant receptors (OR) are strongly implicated in coalescence of olfactory sensory neuron (OSN) axons and the formation of olfactory bulb (OB) glomeruli. However, when ORs are first expressed relative to basal cell division and OSN axon extension is unknown. We developed an in vivo fate-mapping strategy that enabled us to follow OSN maturation and axon extension beginning at basal cell division. In parallel, we mapped the molecular development of OSNs beginning at basal cell division, including the onset of OR expression. Our data show that ORs are first expressed around 4 d following basal cell division, 24 h after OSN axons have reached the OB. Over the next 6+ days the OSN axons navigate the OB nerve layer and ultimately coalesce in glomeruli. These data provide a previously unidentified perspective on the role of ORs in homophilic OSN axon adhesion and lead us to propose a new model dividing axon extension into two phases. Phase I is OR-independent and accounts for up to 50% of the time during which axons approach the OB and begin navigating the olfactory nerve layer. Phase II is OR-dependent and concludes as OSN axons coalesce in glomeruli.

Keywords: Ascl1; axon guidance; olfactory epithelium; olfactory marker protein; tamoxifen.

Fig. 1.

OSNs migrate radially in the OE. (A) Histogram distributions showing BrdU⁺ cells relative frequency in the OE. The basal lamina is considered as 0 and the surface of the OE as 1 (shown in F). Relative position of BrdU⁺ cells expressed as mean ± SEM (B; *P < 0.001) and migrated distance (C). (D–F) Representative images of BrdU⁺ (green) labeling at 1 (D), 4 (E), and 10 (F) DPI. Nuclear marker Draq-5 is shown in blue. Dotted and dashed lines delineate the surface and the basal lamina of the OE, respectively. (Scale bars, 20 μm.)

Fig. 2.

Timeline of GAP-43, OMP, and AC3 expression. Distribution of GAP-43⁺ (A), OMP⁺ (B), and AC3⁺ (C) OSNs in the OE. (D) Scatter plot for GAP-43, OMP, and AC3 showing their relative position in the OE (Top) and normal distributions associated with histograms (Bottom). Although largely restricted to the lower lamina of the OE, GAP-43 cell bodies exhibit a wider distribution than OMP or AC3. Quantification of cells double-labeled with BrdU and GAP-43 (E), OMP (F), or AC3 (G). Although some OMP⁺ and AC3⁺ cells were also BrdU⁺ at earlier days, these were very few (<0.05%) and inconsistent across subjects. (H–L) BrdU⁺ staining in green and in situ hybridization in red for GAP-43 (H), OMP (I and J), and AC3 (K and L). Arrowheads indicate double-labeled cells. Dashed lines delineate OE basal lamina. Draq-5 is in blue. (Scale bars, 20 μm.)

Fig. 3.

ORs are expressed beginning at 4 DPI. (A) Distribution of MOR31-2, MOR40-14, MOR140-1, and MOR263-5 in the OE. (B) Normal distribution of ORs (solid line) in relation to the three classical markers (dotted lines). (C) OR classification based on class, OE zones of expression, and chromosome location. (D–H) Quantification of cells double-labeled with BrdU and MOR31-2 (D), MOR40-14 (E), MOR140-1 (F), MOR244-1 (G), and MOR263-5 (H) shows OR onset at 4 DPI. (I–M) BrdU staining in green with in situ hybridization in red for MOR31-2 (I), MOR40-14 (J), MOR140-1 (K), MOR244-1 (L), and MOR263-5 (M). Open arrows show cells that are OR⁺/BrdU⁻, and arrowheads identify OSNs double-labeled, OR⁺/BrdU⁺. Quantification of cells double-labeled with BrdU, MOR244-1 protein (N), and MOR171-3 GFP (P) shows translation of OR onset at 4 DPI. BrdU staining in green with immunohistochemistry in red for MOR244-1 (O) and MOR171-3 GFP (Q). Dashed lines delineate OE basal lamina. Draq-5 in blue. (Scale bars, 20 μm.)

Fig. 4.

Ascl1^CreERT2 mice track OSNs in the OE following basal cell division. (A) Strategy for labeling a newly generated subpopulation of OSNs. Timeline shows the relation between DPI and PND. (B–D) Experimental Ascl1^CreERT2/+;R26R^ZsGreen/- mice show extensive ZsGreen expression in OE and OB at 8 DPI after 4HO-Tx (B) and sparse labeling (fewer than four cells per section) after oil injection (D). Control Ascl1^+/+;R26R^ZsGreen/- mice show no labeling after 4HO-Tx (C). (E–J) ZsGreen⁺ cells are immediately adjacent to the basal lamina (E). Labeled cell bodies migrate radially toward the lumen as they develop (F–J), suggesting that Ascl1 cells do not or very rarely divide. Apical dendrites and axons could be seen from 2 DPI onward. Dashed lines delineate OE basal lamina. Draq-5 is in magenta (B–D) and blue (E–J). [Scale bars, 200 μm (B–D); 20 μm (E–J).]

Fig. 5.

Tracking OSN axons in Ascl1^CreERT2 mice. Experimental mice were injected with 4OH-Tx at PND 7 and with BrdU at PND 6 (A and C), at PND 7 (B and C), or at PND 8 (C) (n = 4 for each group). Higher levels of double-labeled cells are observed when both injections are done simultaneously (C), suggesting that BrdU and ZsGreen label the same population of OSNs within the narrow time window of basal cell division. The single asterisk indicates statistically significant (P < 0.05). Within 48 h following a postnatal electroporation of a Tdtomato-expressing plasmid, fluorescent axons can be detected branching in the glomerular layer (D). (Inset) A higher magnification of the square. Electroporation of a CreERT2 plasmid in R26R^ZsGreen/- mice, followed by 4OH-Tx 24 h later, shows ZsGreen expression in the outer ONL (oONL, arrowhead in E), the inner ONL (iONL, arrow in E), and the glomeruli (open arrow in E). Higher magnification of the square in E is shown in F. (G–L) ZsGreen⁺ axons are not observed in axon fascicles at 1 DPI (G), and only a few labeled axons are present by 2 DPI (arrows in H). At 3 DPI labeled axons cross the cribriform plate (I) and at 5 DPI (J) remain in the oONL and rarely transition into the iONL. By 8 DPI, ZsGreen⁺ axons enter the ventral glomerular layer (GL, arrowhead in K) and by 10 DPI innervate most ventral glomeruli (L). Draq-5 in blue. (Scale bars, 20 μm.)

Fig. 6.

OSN differentiation with spatial-temporal gene profiles. The spatial distribution of OSN cell bodies expressing GAP-43 (cyan), ORs (light green), AC3 (red), and OMP (purple) within the olfactory epithelium is on the left. The temporal expression profile of these genes is at the top. The morphology of olfactory sensory neurons, in green, is correlated with the corresponding DPI. Arrows indicate the ending of the axon at each developmental stage. Dashed lines demarcate edges of the inner and outer ONL. Dotted circles represent glomeruli.