Fig. 5. Localisation of Robo protein in the VNO and AOB. The expression of Robo protein was examined at E15.5 (A-F) and P21 (G,H) using a Robo specific antiserum (B,D,F-H) on paraffin wax sections. Neighbouring sections (5 µm apart) were stained for ß-tubulin (A,C,E). The outline of the AOB is highlighted by a broken line in G,H. (A,B) VNO axons (stained for tubulin in A) leave the sensory epithelia of the VNO on both sides and navigate dorsally along the nasal septum. These axons clearly express Robo protein (arrows A,B). Note the absence of Robo protein from the cell bodies of the sensory epithelium. (C,D) Analysis of Robo expression (D) along the mediolateral axis of the AOB. Coronal sections through the AOB reveal a stronger Robo expression on the lateral than on the medial region of the VNO nerve layer (arrows in D). (C) The entire length of the VNO nerve layer is indicated by white arrows. (E,F) Robo expression (F) along the anteroposterior axis of the AOB. On a sagittal section Robo staining is mostly confined to the posterior extent of the nerve layer in the AOB (arrows). Importantly, we find this expression already at E15.5, despite the fact that basal neurones are still intermingled with apical neurones within the VNO sensory epithelium. The entire extent of the nerve layer is shown on a neighbouring section stained for tubulin (see arrows in E). (G,H) At P21, Robo protein is clearly restricted to the nerve layer of the posterior AOB, conclusively demonstrating that only the basal subpopulation of VNO axons innervating the posterior half of the AOB expresses Robo. Axons of the main sensory epithelium terminating in glomeruli of the main olfactory bulb also express Robo (G). Arrows in G indicate the expression of Robo in the lateral olfactory tract. A, anterior; D, dorsal; P, posterior; V, ventral Scale-bar: in G, 1 mm for A,C,E,G; in H, 100 µm for B,D,F,H.

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