QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 3 September 2003
doi: 10.1242/dev.00726

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Knöll, B. Articles by Drescher, U. Search for Related Content PubMed PubMed Citation Articles by Knöll, B. Articles by Drescher, U.

On the topographic targeting of basal vomeronasal axons through Slit-mediated chemorepulsion

MRC Centre for Developmental Neurobiology, King's College London, New Hunt's House, Guy's Hospital Campus, London SE1 1UL, UK

View larger version (23K): [in a new window]   Fig. 7. Summary of the expression patterns of guidance molecules involved in the zone-to-zone targeting of vomeronasal axons. The upper part depicts schematically the zone-to-zone projection in the vomeronasal system. Axons from the apical zone of the VNO terminate in the anterior AOB, whereas basal axons terminate in the posterior AOB. Note that apical and basal axons enter the AOB at its medial margin. The lower part summarises the expression patterns of three families of axon guidance molecules described so far in the vomeronasal projection. The expression patterns provided in this study suggest that Robos and Slits act predominantly on basal axons. Robo2 appears to be the principal guidance receptor for basal axons, whereas Robo1 is uniformly expressed and later downregulated, leaving only faint expression on both apical and basal axons (broken line). This suggests a model in which basal axons expressing Robos more strongly than apical axons navigate to the posterior AOB due to repulsive interactions, with Slit proteins secreted from the anterior AOB. Ephrin A proteins and neuropilin 2 instead operate mainly on the apical subpopulation of VNO axons. Apical axons with higher ephrin A expression levels than basal axons project to the anterior AOB, with express higher concentrations of Epha proteins than the posterior AOB. In this scheme, Epha/ephrin A interactions would guide VNO axons on the basis of an attractive guidance mechanism, consistent with data from the stripe assay (Knöll et al., 2001). In turn, apical axons expressing neuropilin 2 are repelled by class 3 semaphorins, which are uniformly localised in the AOB. Neuropilin 2 in the anterior AOB might sequester semaphorins, thus rendering apical axons sensitive to only the semaphorins in the posterior AOB (Cloutier et al., 2000; Walz et al., 2002).

View larger version (191K): [in a new window]   Fig. 1. Sensory neurones expressing Robo and Slit RNA are intermingled in the VNO at P1. Coronal sections through the VNO outline the sensory epithelia (se, broken line) located underneath the nasal septum (s). (A,B) Cell bodies expressing Robo1 (A) or Robo2 (B) are scattered throughout the sensory epithelia of the VNO in newborn mice. (C) Sections stained with Robo2 sense riboprobes are negative. (D,F) Slit1-(D) and Slit3 (F)-expressing cells are distributed all over the entire area of the sensory epithelia. (E) Slit2 is not expressed on the sensory epithelia, but marks the lateral margins of the VNO sensory epithelia. (G,H) Higher magnifications of B and D, indicating that individual neurones expressing Slit1 (G) and Robo2 (H) RNA are distributed in patches in the VNO sensory epithelium. D, dorsal; l, lumen; se, sensory epithelium; s, nasal septum; V, ventral; vv, vomeronasal vein. Scale-bar: 100 µm in A-F; 50 µm in G,H.

View larger version (109K): [in a new window]   Fig. 2. RNA expression patterns of Robos and Slits in the AOB. In situ hybridisation experiments were performed on sagittal sections of the AOB at P1. The outline of the entire AOB is given by broken lines in A. VNO axons in the nerve layer (n) form synapses with mitral- and tufted cells (m/t) located ventrally in the AOB. (A,B) M/t cells along the entire anteroposterior axis of the AOB express Robo1 (A) and Robo2 (B) RNA. Robo2 is also strongly expressed in m/t cells of the MOB (arrows in B). (C) Probing AOB sections with Robo2 sense riboprobes result in no staining. (D,F) Slit1-(D) and Slit3-(F) expressing cells are concentrated at the very anterior border of the AOB (arrows). (E) By contrast, Slit2 RNA is expressed throughout the AP axis of the AOB. AOB, accessory olfactory bulb; A, anterior; D, dorsal; L, lateral; M, medial; MOB, main olfactory bulb; m/t, mitral and tufted cells; n, nerve layer; P, posterior; V, ventral. Scale bar: 100 µm.

View larger version (169K): [in a new window]   Fig. 4. Segregation of Robo- and Slit-expressing neurones in the VNO at P21. After 2 weeks of postnatal development, sensory neurones are restricted to either the apical (a) or basal zone (b) of the VNO sensory epithelium (B) (see also Fig.7). All pictures depict coronal sections of one half of the VNO. (A) Only residual levels of Robo1 RNA are detectable on both apical and basal cell bodies. (B) By contrast, Robo2 expression is prominent and confined to the basal zone of the VNO sensory epithelium. (C) Higher magnification of B, demonstrating the restriction of Robo2-RNA to basal sensory neurones. Note also the patches of Robo2-expressing neurones in the basal zone. (D,F) Slit1 (D) is more strongly expressed in the apical than basal VNO, while Slit3 RNA (F) is mainly restricted to cell bodies of the apical zone. (E) Slit2 RNA, which was labelling the lateral VNO at P1 (see Fig. 1E), has disappeared at P21. a, apical; b, basal, D, dorsal; V, ventral. Scale bars: in F, 100 µm for A,B,D-F; in C, 50 µm for C.

View larger version (101K): [in a new window]   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.

View larger version (46K): [in a new window]   Fig. 3. Differential expression of Slit proteins in the AOB. The experiments were carried out on sagittal sections of the AOB at P1. (A) Schematic drawing of the probes used. As Ig domains 1 and 2 confer Slit binding, a deletion construct omitting these two domains named (1,2)-Robo1-Fc was used as a control, while (3,4,5)-Robo2-Fc containing Ig domains 1 and 2 was used to detect Slit protein expression. (B) Dorsal view of the AOB. Basal vomeronasal axons (green) invade the AOB from its medial margin and project onto the posterior AOB. Approximate location of sagittal sections shown in C,D,E are indicated by broken lines i, ii and iii, respectively. (C,D,E) Detection of Slit proteins using (3,4,5) Robo1-Fc on 150 µm sagittal vibratome sections. Their approximate positions along the mediolateral axis (i,ii,iii) are shown in B. Bound (3,4,5) Robo1-Fc was visualised using an alkaline phosphatase conjugated anti-Fc specific antibody. (G-I) Corresponding DAPI stainings to C,D,E. (F,J) Staining with (1,2) Robo2-Fc (F) serving as a negative control and the corresponding DAPI staining (J). (K-N) Quantification of staining patterns shown in C-F using the Phoretix ID Quantifier V4.0 program. Here, the staining intensity of the area between the two asterisks shown in C-F, which covers the AOB along its entire anteroposterior dimension, was measured. Graphs show average values and standard deviations. K shows the results from the measurement of six comparable sections (n=6) at the level of i, of which C is a typical example (n=10 for L, n=6 for M and n=3 for N). These data indicate a stronger expression of Slits in the anterior than the posterior AOB. AOB, accessory olfactory bulb; A, anterior; D, dorsal; L, lateral; M, medial; MOB, main olfactory bulb; P, posterior; V, ventral. Scale-bar: 100 µm in C-J.

View larger version (90K): [in a new window]   Fig. 6. Slits guide VNO axons by chemorepulsion. E14.5 VNO explants were placed in a collagen-matrix next to COS cell aggregates transfected with Slit1 (B), Slit2 (C), Slit3 (D) or mock transfected (control, A). Lipofection was used to transfect 6 cm2 dishes with a total amount of 0.75 µg Slit2 DNA and 1.5 µg DNA for Slit1 and Slit3. After 3 days in culture, outgrowth of VNO axons was recorded and quantified (E). (A) Axon-outgrowth from VNO explants was radial and not influenced by mock-transfected COS cells (n=33 explants). (B-D) Slit1-Slit3 caused chemorepulsion of VNO axons. (E) The repulsive effect of Slit1-Slit3 on VNO axons was quantified by measuring the length of the neurite front in the proximal (P) and distal quadrants (D) of the VNO explant relative to the cell aggregate. In controls, a P/D ratio of 1 (0.82±0.18) indicates almost no repulsive activity of mock-transfected cell aggregates. By contrast, Slit1-Slit3 shift the P/D ratio to 0.25±0.13, 0.1±0.11 and 0.25±0.18, respectively, indicating a strong Slit-mediated chemorepulsion of VNO axons. Scale bar: 100 µm.