Article Figures & Data
Figures
- Figure 1.
Slit1, Slit2, and Robo2 are expressed in ORN axons. A, Coronal section of E13 wild-type forehead immunostained for Robo2 and Robo1. Only Robo2 is expressed in the olfactory nerve (arrowhead). B, Sagittal section of E13 Robo2+/− head immunostained with anti-β-galactosidase and anti-OMP. ORN axons express both markers and project rostrally to the OB. C–F, LRR2-hSlit2-AP binding on coronal sections of E13 foreheads. LRR2-hSlit2-AP binds to olfactory nerves (arrowheads) of wild-type (C) and Robo1−/− (D) mice, but not of Robo2−/− (E) or Robo1−/−;Robo2−/− (F) embryos. G–I, Sagittal section of foreheads from E13 Slit1+/− (G, H) or Slit2+/− (I) mice. H, In Slit1+/−, GFP is highly expressed in the basal telencephalon (Te), a region avoided by OMP-positive ORN axons. G, ORN axons also expressed GFP (arrowheads). I, Likewise, in Slit2+/− embryos, ORN axons coexpress OMP and GFP (arrowhead) and avoid the basal telencephalon, where GFP is highly expressed. J–O, Coronal sections of Slit1+/− (J–L) and Slit2+/− (M–O) E18 embryos. J, K, GFP is expressed in the olfactory nerve layer (ONL) in the OB and ORNs in the epithelium. L, GFP-positive cells in the olfactory epithelium also express OMP. M, N, GFP is coexpressed with OMP in the ONL of Slit2+/− embryo. O, In the olfactory epithelium, all cells express GFP, including OMP-positive ORNs and their axons (arrowhead). Scale bars: A, 100 μm; B–F, H (for G, H), I, 200 μm; J (for J–L), M (for M–O), 500 μm.
- Figure 2.
Robo expression in embryonic olfactory system. A–K, Coronal sections of E16 (C, F, G) or E18 (A, B, D, E, H–K) wild-type (A, D, E), Robo1+/− (B, C), Slit1+/− (F), and Robo2+/− (G–K) embryos. A, GAP43+ ORNs are not Robo1+. B, C, Likewise, in Robo1+/− mice, β-galactosidase is not detected in OMP+ ORNs, but is present in O4+ cells (confocal image, 3 μm). D, E, p75-expressing cells are immunoreactive for Robo1 (3 μm confocal image). F, In Slit1+/− mice, GFP+ ORN axons are surrounded by Robo1+ cells. G, In Robo2+/− embryo, β-galactosidase+ ORN axons are surrounded by Robo1+ cells. G–K, In Robo2+/− embryos, β-galactosidase (H, J) is expressed with OMP (I, J) in ORNs. Note that some OMP neurons do not coexpress β-galactosidase (arrowheads) and vice versa (arrow). K, ORNs expressing β-galactosidase in Robo2+/− embryos are also immunoreactive for MOR256-17. Scale bars: A, B, I (H–J), 15 μm; C, E (for D, E), 20 μm; F, 200 μm; G, 75 μm; K, 10 μm.
- Figure 3.
ORN axon pathfinding defects in Slit and Robo mutants. A–F, OMP immunolabeling of E18 coronal (A, C, E, F) and sagittal (B, D) sections of E16 (B, D) and E18 (A, C, E, F) embryos. A, In wild-type brain, ORN axons cover the whole surface of the OB surface and start forming glomeruli. The arrowhead indicates a glomerulus shown at a higher magnification in the inset. Scale bar (in inset), 50 μm. B, In Robo1+/−;Robo2+/− embryos, OMP+ ORN axons project rostrally to cover the entire surface of the OB. C, In Slit1−/−;Slit2−/− embryos, OMP axons also project to the OB and form glomeruli, but the olfactory nerve is slightly defasciculated (arrowheads). D, In Robo1−/−;Robo2−/− embryos, only a few OMP axons reach the OB, but most of them coalesce under the forebrain before taking a caudal route or invading the septum (SE). E, F, In Robo1−/−;Robo2−/− embryos, OMP+ axons are unable to cover the OB and do not all form clear glomeruli. F, The olfactory nerve is fragmented in smaller fascicles that are spread laterally. In addition, many ORN axons project into the septum (E, arrowheads). Scale bars, 500 μm.
- Figure 4.
MOR256-17 glomerulus duplication in Slit/Robo mutants. A–P, E18 (A–L) and adult (M–P) OBs labeled with anti-MOR256-17, anti-OMP, and Hoescht. A–D, In wild type, MOR256-17 axons project to a single lateral glomerulus (arrowheads) and coexpress OMP. E, One MOR256-17 lateral glomerulus (arrowhead) is found on the same OB of a Slit1−/− embryo. F, In a Slit2−/− OB, the MOR256-17 lateral glomerulus is shifted to a more ventral position. G, H, In Robo1−/− (G) and Robo2−/− (H) mice, only one lateral glomerulus is observed at a normal position. I, J, In a Slit1−/−;Slit2−/− OB, three glomeruli (1–3) were detected (arrowheads) along the lateral surface. K, In Robo1−/−;Robo2−/− double mutants, most OMP-positive axons project caudally to the OB, as shown on a coronal section of the telencephalon. Note that some of these axons are also MOR256-17+ (K, arrowheads). L, However, some MOR256-17-positive axons reach the OB and form a small lateral glomerulus (arrowhead). M–P, In adults, one lateral glomerulus is observed in Slit1+/− (O) and Slit1−/− (P) animals, but two lateral glomeruli (1 and 2 arrowheads) are detected in Slit1−/−;Slit2+/− OB (M, N). Glomerulus 1 is located at the dorsolateral part of the OB, whereas glomerulus 2 is 120 μm behind and in the ventrolateral part. Scale bars: A (for A–D), 50 μm; E–H, J (for I, J), L (for K, L), 100 μm; M (for M, N), O, P, 150 μm.
Additional Files
Supplemental Data
Files in this Data Supplement:
- supplemental material - Supplemental Legend
- supplemental material - Supplemental Figure 1
- supplemental material - Supplemental Figure 2
- supplemental material - Supplemental Figure 3
