Low power images of mouse olfactory bulb at all ages investigated stained with Laminin-1/NCAM (A), Perlecan/NCAM (B), CSPG/NCAM (C) and Tnc/NCAM (D). (A) At all ages laminin-1 (green) was found in the basement membranes of blood vessels as well as in the olfactory ensheathing cells surrounding the OSN axons in the olfactory nerves and in the olfactory nerve layer (blue) of the olfactory bulb (see arrows at E17). (B) At all ages perlecan (green) was found in the basement membranes of blood vessels. Expression was detected in the olfactory ensheathing cells surrounding the OSN axons in the olfactory nerves and in the olfactory nerve layer of the olfactory bulb; however onset of expression was varied in different regions of the pathway. Underneath the olfactory epithelium it was detected at E13 (see arrows at E13) while in the ONL it was first seen at E17 (see arrows at E17). (C) CSPGs (green) were initially detected only in the mesenchyme surrounding the olfactory nerves (see asterisk at E13) but were detected in the OB in layers deep to the olfactory nerve layer (blue) at E17. (D) Tnc was detected in the OB in layers deep to the olfactory nerve layer (blue) from E13. Scale bars (A-D) = 50μm. OB, olfactory bulb; onl, olfactory nerve layer; nc, nasal cavity; oe, olfactory epithelium; s, septum.

Axons fascicles crossing the cribriform plate through small fenestrations at E15 (A,C,E) and PND2 (B,D,F) stained with Laminin-1/CSPG/NCAM (A,B), Tnc/Perlecan/NCAM (C,D), p75/NCAM (E,F). (A) At E15, laminin-1 (green) is found surrounding NCAM+ OSN nerve bundles (blue) crossing the cribriform plate (arrowheads). CSPGs (red) are found in the lamina propria external to the laminin-1 ensheathed axon bundles as well as surrounding the cribriform plate. (B) At PND2, laminin-1 staining (green) is similar to that at E15 with expression surrounding NCAM+ OSN axon bundles (blue) crossing the cribriform plate (arrowheads). CSPGs (red) are no longer detected in this region at this age. (C) At E15 Tnc (green; arrow) is found at low levels in the OECs surrounding NCAM+ OSN axons (blue) in an overlapping pattern with perlecan (red; arrowhead). Some colocalization is apparent (yellow). (D) At PND2, Tnc (green) is detected at much higher levels than at E15 surrounding NCAM+ (blue) axon bundles, colocalizing with perlecan (red), hence appearing yellow (arrowheads). Tnc is also found in cells of the connective tissue lining the cribriform plate (open arrowheads). Perlecan expression is also detected at much higher levels than at E15 in the OECs in this region. (E) At E15, p75+ OECs (green) surround NCAM+ OSN nerve bundles (blue) as they cross the cribriform plate (arrowheads) which resembles localization of laminin-1, perlecan and Tnc staining in panels A and C respectively. (F) At PND2, p75 (green), stains OECs surrounding OSN nerve bundles (blue) crossing the cribriform plate which resembles localization of laminin-1, Tnc and perlecan staining in panels B and D respectively. Scale bars (A-D), (E-F) = 50μm. cp, cribriform plate

The developing olfactory bulb (OB) at E15 (A,C,E) and PND2 (B,D,F) stained with Laminin-1/CSPG/NCAM (A,B), Tnc/Perlecan/NCAM (C,D) and p75/NCAM (E,F). (A) At E15, laminin-1 (green) positive puncta are found throughout the ONL, amongst the OSN axon fascicles in a pattern similar to p75+ staining (c.f. panel E). CSPGs (red) are not detected in the OB at this age. (B) At PND2, laminin-1 staining (green) is restricted to the outer ONL where it surrounded fascicles of NCAM+ OSN axons in a pattern indistinguishable from p75+ OECs (c.f. panel F). CSPGs are present in the OB in the layers deep to the ONL, encapsulating NCAM+ glomeruli (blue) and within glomeruli, interdigitating with NCAM+ axonal compartments (arrowheads). (C) At E15 Tnc (green) is expressed in the dendritic zone (DZ), deep to the ONL in a boundary-like pattern. OSN axons (blue) are directly apposed to the Tnc-boundary. Perlecan (red) is not detected within this region at this age. (D) At PND2, Tnc is found both encapsulating NCAM+ (blue) glomeruli (arrows) and also within glomeruli, interdigitating with NCAM+ axonal compartments within glomeruli (arrowheads), in a simlar distribution to CSPGs (c.f. panel B). Perlecan (red) is detected in the outer ONL where it surrounded fascicles of NCAM+ OSN axons in pattern indistinguishable from laminin-1 (c.f. panel B) and p75 (c.f. panel F). Note red staining in the GL and EPL is perlecan present in the basement membranes of blood vessels (asterisks). (E) At E15, p75+ OECs (green) surround NCAM+ OSN nerve bundles (blue) in the ONL. (F) At PND2, p75 (green) stains OECs surrounding OSN nerve bundles (blue) entering and coursing within the outer ONL of the OB, which resembles laminin-1 and perlecan staining in panels B and D respectively.
Scale bars (A-D), (E-F) = 100μm. cp, cribriform plate; onl^o, outer olfactory nerve layer; onlⁱ, inner olfactory nerve layer; on, olfactory nerve bundle; epl, external plexiform layer; gl, glomerular layer; dz, dendritic zone; *, blood vessel.

(A) Schematic diagram of coronal section through olfactory system indicating four regions analyzed in more detail. Region 1 - Developing olfactory nerves coursing through the mesenchyme underneath the olfactory epithelium. Region 2 - Bundles of olfactory axons passing through the cribiform plate. Region 3 - Nerve entry zone. Region 4 - Developing glomerular and olfactory nerve layers. (B) Time line of olfactory pathway development. d.p.c. = days of gestation; OE = olfactory epithelium; ON = olfactory nerve; pONL = presumptive olfactory nerve layer of the olfactory bulb; DZ = dendritic zone.

OE and underlying lamina propria at E15 (A,C,E) and PND2 (B,D,F) stained with Laminin-1/CSPG/NCAM (A,B), Tnc/Perlecan/NCAM (C,D), p75/NCAM (E,F). (A) At E15, laminin-1 (green) is localized to the basement membrane (BM) of the olfactory epithelium (OE; see arrows) and surrounding NCAM+ OSN nerve bundles (blue) in the lamina propria (arrowheads). CSPGs (red) are found in the lamina propria external to the laminin-1 ensheathed axon bundles. (B) At PND2, laminin-1 staining (green) is similar to that at E15 with expression in the BM of the OE (see arrows) and surrounding NCAM+ OSN axon bundles (blue) in the lamina propria (arrowheads). CSPGs (red) are no longer detected in the lamina propria at this age. (C) At E15 Tnc (green) is not detected in this region while perlecan (red) is found in the BM of the OE (arrows) and surrounding NCAM+ OSN axon bundles (blue) in the lamina propria (arrowheads) in a pattern similar to laminin-1 (c.f. panel A). (D) At PND2, Tnc is detected at low levels in some OECs surrounding NCAM+ (blue) axon bundles, colocalizing with perlecan (red), hence appearing yellow (open arrows). Tnc is also found in cells of the connective tissue lining the nasal septum (open arrowheads). Perlecan continues to be localized to the BM of the OE (arrows) and surrounding OSN axon bundles in the lamina propria (arrowheads). (E) At E15, p75 (green), a marker of olfactory ensheathing cells (OECs) is localized surrounding NCAM+ OSN nerve bundles (blue) in the lamina propria (arrowheads) which resembles localization of laminin-1 and perlecan staining in panels A and C respectively. (F) At PND2, p75 (green), stains OECs surrounding OSN nerve bundles (blue) in the lamina propria (arrowheads) which resembles localization of laminin-1 and perlecan staining in panels B and D respectively. Scale bars (A-D), (E-F) = 50μm. nc, nasal cavity; oe, olfactory epithelium; lp, lamina propria; s, septum.

The entry zone (EZ) where olfactory nerve (ON) fascicles enter the olfactory bulb (OB) and fuse with the olfactory nerve layer (ONL) at E15 (A,C,E) and PND2 (B,D,F) stained with Laminin-1/CSPG/NCAM (A,B), Tnc/Perlecan/NCAM (C,D) and p75/NCAM (E,F). (A) At E15, laminin-1 (green) is found surrounding NCAM+ OSN nerve bundles (blue) entering the OB as well as surrounding axon fascicles within the EZ in a pattern indistinguishable from p75+ OECs (c.f. panel E). CSPGs (red) are present in mesenchymal cells surrounding the entry zone (arrowheads). (B) At PND2, laminin-1 staining (green) is similar to that at E15 with expression surrounding OSN axon bundles (blue) entering the OB as well as surrounding axon fascicles within the EZ. Expression of CSPGs (red) in mesenchymal cells surrounding the entry zone is down regulated compared to E15, with only a few scattered cells surrounding the entry zone stained (arrowheads). (C) At E15 Tnc (green) is not detected within the entry zone. Perlecan is found surrounding the EZ (arrows) but not surrounding OSN fascicles (blue) within the EZ like laminin-1 (c.f. panel A). The red staining seen within the entry zone is perlecan present in the BMs of blood vessels (arrowheads), and not OECs (c.f. p75, panel E). (D) At PND2, Tnc (green) is still not detected within this region. Perlecan (red) is found within the entry zone, surrounding axons fascicles, in a pattern indistinguishable from laminin-1 and p75 (c.f. panels B and F, respectively). (E) At E15, p75+ OECs (green) surround NCAM+ OSN nerve bundles (blue) as they enter and course within the ONL which resembles localization of laminin-1 in panels A. (F) At PND2, p75 (green), stains OECs surrounding OSN nerve bundles (blue) entering and coursing within the ONL of the OB, which resembles laminin-1 and perlecan staining in panels B and D respectively.
Scale bars (A-D), (E-F) = 100μm. cp, cribriform plate; onl, olfactory nerve layer; on, olfactory nerve fascicles; epl, external plexiform layer; gl, glomerular layer; dz, dendritic zone; s, septum; *, large blood vessel.

Colocalization of either inhibitory cues or growth promoting cues in the developing olfactory bulb (OB) at E15 (A,C) and PND2 (B,D) stained with Tnc/CSPG/NCAM (A,B) and Laminin-1/Perlecan/NCAM (C,D). (A) At E15, only Tnc (green) is expressed in the dendritic zone, deep to the OSN axons in the ONL (blue). CSPGs (red) are not present. (B) At PND2, Both Tnc (green) and CSPGs (red) are present in the GL and EPL, appearing yellow, encapsulating NCAM+ glomeruli (blue) and within glomeruli, interdigitating with NCAM+ axonal compartments (arrowheads). Tnc appears to be present at higher levels within glomeruli as the staining is more green than yellow (c.f. levels of Tnc in Figure 5D with CSPGs in 5B). (C) At E15 only laminin-1 (green) is expressed by OECs in the ONL, perlecan (red) is not expressed at this age. (D) At PND2, both laminin-1 (green) and perlecan (red) are expressed by OECS in the outer ONL, surrounding NCAM+ OSN axons (blue). (E) At E15, RC2+ radial glia (green) extend though the dendritic zone to the ONL, where their end feet arrange along the surface of the ONL in a boundary like pattern. (F) At PND2, GFAP+ astrocytes (green) surround and encapsulate glomeruli (blue) in the glomerular layer of the OB.
Scale bar (A-D),(E)= 100μm, (H) = 50μm. onl, olfactory nerve layer; onl^o, outer olfactory nerve layer; onlⁱ, inner olfactory nerve layer; epl, external plexiform layer; gl, glomerular layer; dz, dendritic zone; *, blood vessel.

Summary diagram of expression pattern of laminin-1 (red), perlecan (green), CSPGs (blue) and Tnc (yellow) expression across all 6 ages examined. Protein expression is color coded, and distribution plotted within the pathway. Co-expression is represented by dual or triple hatching of colors. As is evident from the diagram, these proteins display dynamic spatial and temporal patterns of expression. Inhibitory proteins are largely present outside the pathway with growth promoting proteins within, suggesting a coordinated set of ECM cues directing OSN growth during pathway formation.
OE, olfactory epithelium; ON, olfactory nerve; CP, cribriform plate; EZ, entry zone; ONL, olfactory nerve layer; DZ, dendritic zone; ONLⁱ, inner olfactory nerve layer; ONL^o, outer olfactory nerve layer; GL, glomerular layer; EPL, external plexiform layer; OSN, olfactory sensory neuron; OEC, olfactory ensheathing cell; p, protoglomerulus, g, glomerulus.