Figure 4.
Migration of GnRH-1 neurons in PlexinB1−/− mice is delayed. (a and b) Photomicrograph showing GnRH-1 (brown) immunoreactivity in a sagittal section of an E14.5 WT embryo. The areas of analysis for GnRH-1 neuron location along the migratory pathway were the nasal compartment and the brain, respectively. The broken line in panel a indicates the boundary between these regions, and the arrows point to GnRH-1–labeled cells dispersed from the VNO to the basal forebrain (bfb). (b) Analysis of GnRH-1 neurons location revealed a significant accumulation of cells in the nasal region of PlexinB1 KO mice compared with WT. Consistently, fewer GnRH-1 cells were located in the brain of mutant embryos as compared with WT. Quantitative analysis of GnRH-1 neurons was performed as a function of location (nasal region and brain). GnRH-1 cell distribution is presented as the mean percentage of labeled cells located in the nose or in the brain of WT and PlexinB1 KO embryos (E14.5 WT: n = 13; E14.5 PlexinB1−/−: n = 10). Statistical significance was determined using Kruskal-Wallis nonparametric test (P < 0.05). (c) At P3, the GnRH-1 migratory process is over and the great majority of GnRH-1 neurons have reached the ventral forebrain (vfb). The distribution of these cells was analyzed in the nasal region, in the olfactory bulb (ob), and in the vfb at this developmental stage. (d) Approximately 16% of the entire population is accumulated in the ob, whereas fewer cells (∼20% less) are dispersed in the vfb compared with WT littermates. Quantitative analysis of GnRH-1 neurons was performed as a function of location (nose, ob, and vfb). GnRH-1 cell distribution is presented as the mean percentage of labeled cells in each compartment (P3 WT: n = 3; P3 PlexinB1−/−: n = 4). Statistical significance was determined using Kruskall-Wallis nonparametric test (P < 0.05). (e and f) High-power views of sagittal sections of P3 WT and mutants stained for GnRH-1. (e) In WT animals, GnRH-1 neurons are mainly located in the vfb (see arrows and insets, which show enlarged views of the boxed regions), whereas few cells are distributed in the ob area (arrowheads). (f) In PlexinB1−/− mice, abnormal clusters of GnRH-1 cells are located at the level of the cribriform plate (cp; see inset) and of the ob (arrowheads), and fewer cells reached the vfb region (arrows). (g) Photomicrograph showing GnRH-1 immunoreactivity in coronal sections of adult WT and PlexinB1-null mice. The principal fibers' projections of GnRH-1 neurons are to the median eminence (me), and this region showed a dramatic loss of GnRH-1 fibers in mutant mice (g, right) compared with WT littermates (left). The bottom photomicrographs show high-power views of the median eminence in WT and KO brains. (h) Quantitative analysis of GnRH-1 neuronal population in adult brains revealed a 30% reduction of these cells in brains of PlexinB1 mutants as compared with WT (WT: n = 3; PlexinB1−/−: n = 5). The total number of cells was calculated and combined to give group means ± SEM. *, statistical significance was determined using a paired t test (P < 0.0005). cx, cortex. Bars: (a) 500 μm, (c) 600 μm; (e and f) 300 μm, (e and f, insets) 30 μm; (g, top) 125 μm; (g, bottom) 62.5 μm.