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First published online 5 October 2005
doi: 10.1242/dev.02033

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Reelin provides an inhibitory signal in the migration of gonadotropin-releasing hormone neurons

¹ Department of Anatomy and Developmental Biology, University College London, London WC1E 6BT, UK
² Department of Endocrinology, Centre of Excellence on Neurodegenerative Diseases, University of Milan, 20133 Milan, Italy
³ Developmental Neurobiology Unit, University of Louvain Medical School, B1200 Brussels, Belgium

View larger version (86K): [in a new window]   Fig. 1. Expression of reelin during GnRH neuronal migration into the forebrain. GnRH and reelin immunoreactivities in a sagittal section through an E18 rat brain (boxed area in schematic drawing). GnRH immunoreactive cells (green) enter the forebrain (arrow) and migrate caudally to the basal forebrain. Reelin immunoreactivity (red) is present in the olfactory bulb and adjacent region, an area that GnRH neurons tend to avoid. OB, olfactory bulb; BF, basal forebrain; H, hypothalamus. Scale bar: 250 µm.

View larger version (82K): [in a new window]   Fig. 2. Migration of GN11 cells is inhibited by reelin-rich olfactory bulb explants in three-dimensional collagen matrix. Olfactory bulb explants (OB) from E18 rats were cultured with GN11-EGFP cells in collagen gels. (A) GN11-EGFP cells (green) migrated away from the OB (red, axonal staining). (B) The zone surrounding the cell aggregates was divided into quadrants, thus defining a proximal (P) and a distal (D) quadrant in relation to the OB. (C) Migrating cells were counted in 24 co-cultures and expressed as a percentage of cells ± s.e.m. in each of the two quadrants out of the total number of cells in both quadrants. We found that 66.54±1.40% of cells migrated into D and 33.46±1.40% into P. Scale bar: 250 µm. (D,E) Addition of CR-50 antibody resulted in roughly equal proportions of GN11-EGFP cells in P (53.53±2.60%) and D (46.47±2.60%; n=12). Scale bars: 250 µm.

View larger version (17K): [in a new window]   Fig. 3. Reelin inhibits the migration of GN11 cells in Boyden's chamber assay. (A) In a chemotactic assay, reelin-CM did not alter the migration of GN11 cells compared to control-CM. However, the haptotactic response of these cells was significantly inhibited by the presence of reelin-CM (***P<0.001; Student's t-test). On average, 469.1±49.76 cells/mm2 migrated in the presence of control-CM while 183.5±30.90 cells/mm2 migrated in the presence of reelin-CM. Means ± s.e.m. obtained from 12 independent experiments.

View larger version (59K): [in a new window]   Fig. 4. Reelin-blocking antibody alters the direction of migration of basal forebrain neurons. (A,C) Drawings of sagittal sections through E18 rat brains showing placements of fluorescent CMTMR particles at the junction of the olfactory bulb region and the basal forebrain as well as the migratory routes (red dotted lines) taken by labelled cells in control (A) and CR-50-treated slices (C). Vertical lines separate the parts of the sections rostral to the dye placement from those located caudally. (B,D) Streams of labelled cells moving away from the site of CMTMR placement. Note that in the treated slice, a mass of fluorescent cells is directed rostrally. (E) Counts of labelled cells in control and CR-50-treated slices indicated that in the former, cells moved caudal to CMTMR placement but, in the latter, cells took a rostral course (n=12 slices for each case; ***P<0.001; Student's t-test). OB, olfactory bulb; Cx, cerebral cortex; Th, thalamus; BF, basal forebrain; H, hypothalamus. Scale bar: 500 µm.

View larger version (112K): [in a new window]   Fig. 5. Expression of reelin signalling molecules in GN11- and GnRH neurons. (A) RT-PCR analysis, using GN11 cell mRNA, showed the presence of ApoER2 and Vldlr transcripts (313 bp and 253 bp respectively), but absence of Dab1 (predicted amplicon 79 bp). These results were confirmed by nested PCR experiments (B), yielding the predicted 201 bp and 186 bp products for ApoER2 and Vldlr, but not the expected band of 60 bp for Dab1. (C-E) Double immunohistochemistry experiments showed the localization of ApoER2 (red, D and E) in a small proportion (arrow in E) of GnRH neurons (green, C and E). (F-H) Similar experiments failed to identify Dab1 (red, G and H) in GnRH neurons (green, F and H). n=3 brains. Scale bar: 50 µm.

View larger version (83K): [in a new window]   Fig. 6. Reduction in number and aberrant position of GnRH neurons in E18 reeler mice. (A) Cell counts revealed a significant reduction in the total number of GnRH neurons in the brains of reeler mice (reeler, 417.0±31.75; wt, 748.0±4.04; n=3 for each genotype; *P<0.05; Student's t-test), but a relative increase in the number of these cells in the cortex compared to wild type (wt) animals (reeler 41.11±9.77; wt, 9.793±3.97; *P<0.05; Student's t-test). (B) Drawing of a sagittal section depicting the areas (boxes) selected for analysis. (C-H) Images of immunohistochemically stained GnRH neurons in OB, BF and Cx of wild-type and reeler brains. Streams of neurons entering the brain (asterisk in C and D) are directed towards the basal forebrain (large arrows in C and D indicate direction of stream) in both groups of animals, but reeler show an additional stream directed towards the cortex (small arrow in D). Note the overall reduction in GnRH neurons in the BF of reeler mice (arrowheads in F) and the increase of these neurons in the cortex (arrowheads in H). OB, junction of olfactory bulb and forebrain; BF, basal forebrain; H, hypothalamus; Cx, cortex. Scale bar: 50 µm.

View larger version (126K): [in a new window]   Fig. 7. GnRH neuron distribution in the hypothalamus of adult reeler, scrambler and ApoER2/Vldlr mutant mice. (A,B) Sagittal sections taken from adult wild-type and reeler animals, respectively (A) Typical distribution of GnRH neurons in the hypothalamus. (B) In the reeler mouse there are very few, faintly stained GnRH neurons. At this magnification, GnRH neurons are hardly visible in reeler. The inset B', taken from the area indicated by an asterisk in B, illustrates a few of these neurons at a higher magnification. (C) A profuse array of GnRH processes (arrowheads) streaming to the median eminence (me) region from an adult wild-type animal. In reeler mice (D), only very few such processes (arrowheads) converge to the median eminence. (E,F) Sections taken from the brains of ApoER2/Vldlr mutants (E) and scrambler mice (F) illustrate that the density and appearance of GnRH neurons are similar to those in wild-type animals. Scale bars: 100 µm (A,B,E,F); 50 µm (B9,C,D). mpoa, medial preoptic area; db, diagonal band of Broca; me, median eminence; so, supraoptic nucleus.