GnRH-1 neuron migration is not altered in Arx-1^null mutants. (A-H) Immunohistochemistry against OMP (brown) and GnRH-1 (blue) on control (A,C,E,G) and Arx-1^null (B,D,F,H) mouse at E15.5 (A-D) and P0 (E-H). In both controls and Arx-1^null, the GnRH-1 ns (dark blue) migrate to the basal forebrain on OMP negative fibers. In Arx-1^null mutants (D) the GnRH-1 ns cross the axonal tangle of the FCM mass and migrate to the basal forebrain (F,H) as in control animals (E,G) (black arrows). (I,J) E15.5, double immunofluorescence against Peripherin and GnRH-1. The GnRH-1 ns migrate to the basal forebrain on Peripherin-positive fibers of the TN on both control and Arx-1^null mice (white arrows). Enlargements illustrate the TN separating from the olfactory fibers that project to the MOB in control (I) or collapsed in the FCM in Arx-1^null mutants (J). (K) At the three analyzed stages a similar percentage of GnRH-1 ns migrated to brain in controls and Arx-1^null mice. (L) Graphs of GnRH-1 ns cell counts at P0 in the nose, OB/FCM, brain and total. No statistical difference was seen in all areas between control and Arx-1^null (data are mean±s.e.m., unpaired student's t-test, P>0.05).

Aberrant formation of the olfactory system does not significantly affect the rostro-caudal distribution of GnRH-1 ns in the brain. (A-B2) Immunostaining against GnRH-1 on parasagittal sections on P0 Arx-1 WT (A-A2) and KO (B-B2) show similar distribution. (A1,B1) GnRH-1 ns cell bodies (arrowheads) in the POA and their (A2,B2) projections (arrows) to the ME (D, dorsal; V, ventral; R, rostral; C, caudal). Mapping of the GnRH-1 ns distribution in Arx-1 WT (n=3) and null (n=3) mice was performed on one series from each animal. (C,D) Scatter plots illustrating GnRH-1 ns distribution in WT (C) (n=3) and KO (n=3) (D), obtained by overlapping the GnRH-1 ns coordinates from one series from each animal in reference to the median eminence (0,0 µm), in along similar migratory paths. (E) Histogram illustrating the average number of GnRH-1 ns on the rostral-caudal axis in 500 µm intervals. The mean cell counts for the WT from distances –500-0 µm: 6.667± 6.667, 0-500 µm: 56±40.067, 1000-1500 µm: 134.667±38.251, 1500-2000 µm: 120±12.20, 2000-2500 µm: 138.667±37.547, 2500-3000 µm: 201.333±67.580, 3000-3500 µm: 78.667±11.2624, 3500-4000 µm: 46.667± 11.624, 4000-4500 µm: 26.667±11.851, 4500-5000 µm: 10.667±3.528. The mean cell counts for the Arx-1 Null from distances –500-0 µm: 0, 0-500 µm: 16±4.619, 1000-1500 µm: 144±43.879, 1500-2000 µm: 90.667±79.644, 2000-2500 µm: 300±84.664, 2500-3000 µm: 256±40.266, 3000-3500 µm: 77.333±12.719, 3500-4000 µm: 25.333±5.812, 4000-4500 µm: 10.667± 5.812, 4500-5000 µm: 0 (data are mean±s.e.m., unpaired student's t-test, P>0.05). FCM, fibrocellular mass; LV, lateral ventricle; ME, median eminence; OB, olfactory bulb; POA, pre-optic area.

Expression of EGFP under the control of a human Peripherin promoter distinguishes the TN from the olfactory/vomeronasal nerve. (A-G) E15.5, parasagittal sections on hPRPH-1G. (A,B) Immunostaining against EGFP showing strong EGFP expression in the AOB and TN invading the basal forebrain (BFB). (C-E) Double immunofluorescence against TAG-1 and EGFP, both EGFP and TAG-1 were found to be strongly expressed in the TN fibers, low TAG-1 expression was detected in putative vomeronasal fibers (VF) projecting to the AOB. (F,G) E15.5, immunofluorescence against EGFP and GnRH-1. GnRH-1 ns (red) access the brain along Peripherin/EGFP-positive TN fibers. (H-O) hPRPH1-G mice reveal that the TN is distinct form OSNs and bypasses the FCM in Arx-1^null mutants. (H,I) Peripherin/GnRH-1 double immunostaining on E15.5 WT (H) and Arx-1^null (I) GnRH-1 ns enter the forebrain along the TN fibers positive for Peripherin (arrows). (I) In the Arx-1^null, GnRH-1 ns access the brain along the Peripherin-positive TN fibers that emerge from the Peripherin-positive FCM. (J,L) hPRPH1-G, E15.5, double immunostaining against OMP and EGFP, OMP+ olfactory sensory neurons projecting to the MOB are mainly negative for EGFP expression while the VSNs projecting to the AOB and the TN invading the basal forebrain (BFB) are positive for EGFP (arrows). (K,M) Double immunostaining against OMP and EGFP on hPRPH1-G /Arx1^null, E15.5. The FCM is composed of OMP+ collapsed axons (red; tangled fibers) mainly negative for EGFP expression while the fibers of the TN, positive for EGFP (arrows), are able to access the brain as in control animals (compare to K,M). Putative VSNs (pVSNs) were found to be tangled as part of the FCM together with the OSNs strongly positive for OMP. (N-N2) E15.5 hPRPH1-G /Arx1^null. Immunostaining against Peripherin and EGFP shows that hPRPH1-G is selective for pVSNs and the TN (arrows).

GPR12-EGFP BAC transgenics show that the TN is distinct from VSNs. (A-C) Postnatal GPR12-EGFP. (A) Coronal section; EGFP expression is limited to the VSNs and to sparse cells in the OE. (B) Whole mount; EGFP is detectable in VSNs projecting to the AOB but not in those projecting to the MOB. (C) Gαi2/EGFP staining on parasagittal section of the AOB, showing that the EGFP-positive fibers project to both the anterior (a) and posterior (p) AOB. (D-F) E15.5 GPR12-EGFP. (D) Double immunostaining against GnRH-1 and EGFP. The GnRH-1 ns access the brain along GPR12-EGFP-negative fibers (arrowheads) while GPR12-EGFP+ axons project from the VNO to the AOB. (E) NRP2 (red) /EGFP (green) double staining showing NRP2 in GPR12-EGFP+ positive axonal bundles projecting to the AOB and in OSNs projecting to the MOB. (F) Peripherin/EGFP double immunofluorescence; EGFP is expressed in the VSNs projecting to the AOB but not in the Peripherin+ TN (arrow). (G,G1) P0, Arx-1^null/GPR12-EGFP immunostaining against OMP (red) and EGFP (green). The GPR12-EGFP-positive vomeronasal fibers (VSNs) project toward the brain and collapse as part of the FCM. (H-K) E15.5 Arx-1^null/GPR12-EGFP. (H) GnRH-1 (red) accessing the brain square (arrowhead in the BFB) on GPR12-EGFP-negative fibers; the EGFP+ VSNs collapse as part of the FCM. (I) NRP2 /EGFP double staining shows NRP2 expression in OSNs and in GPR12-EGFP+ VSNs in the FCM. (J) EGFP expression in the VNO of E15.5 Arx-1^null/GPR12-EGFP (K) Peripherin/EGFP double immunofluorescence; Peripherin highlights the FCM and the TN emerging from the FCM, while EGFP is expressed by the VSNs but not by the TN (arrows).

The TN is positive for Robo3 but not for Robo1 or Robo2. (A1-B4) E15.5 WT animal. (A1-3) Double immunostaining against Robo1 and Peripherin shows that the Peripherin+ TN is negative for Robo1. (B1) ISH against Robo2 combined with immunofluorescence against Peripherin and GnRH-1. Robo2 was detected in the vomeronasal neurons, olfactory epithelium (OE), nasal mesenchyme (NM) and in the olfactory bulb. No Robo2 was detected in GnRH-1 ns. (B2-B4) Immunofluorescence against Robo2 and Peripherin shows lack of immunoreactivity for Robo2 in the TN accessing the brain. (C1) E15.5 WT animal, ISH against Robo3 combined with IF against GnRH-1 and Peripherin. Strong Robo3 mRNA expression was found in cells proximal to the VNO negative for GnRH-1 immunoreactivity (arrow). (C2,C3) E15.5 GPR12-EGFP immunostained for EGFP and Robo3 confirms Robo3 expression in cell bodies and fibers of neurons proximal to the VNO forming bundles with GPR12-EGFP+ VSNs (arrows in C2 and C3). Robo3+ cells proximal to the VNO negative for GnRH-1 and EGFP are indicated as pTN. (D1-D3) E15.5 WT animal. GnRH-1 and Robo3 double immunofluorescence reveals migrating GnRH-1 ns in contact with Robo3+ fibers. (E1-E6) Double immunofluorescence against Robo3 and Peripherin in WT animals (E15.5). The fibers of the pTN accessing the brain are positive for Robo3 and Peripherin immunoreactivity.