Figure 1.
Expression of the Sema3A coreceptor Nrp1 by vomeronasal/terminal nerve fibers and migrating GnRH cells in human and mouse embryos.
(A) Schematic representation of the head of a mouse embryo at E14.5, showing the scaffold of vomeronasal/terminal nerve fibers (in red) along which GnRH cells (in blue) migrate from the nose to the ventral forebrain region. Several areas along this migratory path have been shown to produce Sema3A, including the frontonasal mesenchyme and the olfactory bulb region [21], [33]. Boxes indicate the locations of the sagittal sections shown in (B) and (C). Abbreviations: oe, olfactory epithelium; vno, vomeronasal organ; nm, frontonasal mesenchyme; mob, main olfactory bulb; aob, accessory olfactory bulb; vfb, ventral forebrain; 3V, third ventricle. (B) Sagittal section of the frontonasal region in an E14.5 mouse embryo. In the frontonasal mesenchyme (nm), migrating GnRH-immunoreactive cells (green) are morphologically associated with Nrp1-immunoreactive nerve fibers (red) originating in the vomeronasal organ (vno). Single plane confocal images at higher magnification (insets) show that GnRH cells are Nrp1-immunoreactive (green+red = yellow staining). (C) Sagittal section of the ventral forebrain (vfb) in an E14.5 mouse embryo. The peripherin-immunoreactive (green) fibers of the caudal branch of the vomeronasal nerve (arrows) are also Nrp1-immunoreactive (red), as shown by their yellow staining (green+red). (D) Sagittal section of the olfactory epithelium (oe) and olfactory bulb (ob) regions (left panel) and detail of the frontonasal region (right panel) in a 9 week-old human fetus. Clusters of GnRH-immunoreactive cells (green, arrowheads) are visible in the frontonasal mesenchyme (nm) and the rostral forebrain (fb). In the frontonasal region, these cells migrate in close contact with Nrp1-immunoreactive axons (red). Note that migrating GnRH cells are also Nrp1-immunoreactive, as shown by their yellow staining (green+red) in the right panel (arrows). Scale bars: 100 µm (25 µm in insets).
Figure 2.
Defects in olfactory and vomeronasal axons, and GnRH cell migration in Nrp1sema/sema mutant mice.
(A) Coronal sections of the right olfactory epithelium (oe) and olfactory bulb (ob) regions (left panels), and detail of the olfactory bulb showing the olfactory nerve layer (nl) and glomerular layer (gl) (right panels) in Nrp1+/+ and Nrp1sema/sema newborn (P0) mice. Axons of the olfactory receptor neurons were immunostained (red) using an antibody directed against the olfactory marker protein (OMP). In the Nrp1sema/sema mouse, the immunostaining is both enlarged below the olfactory bulb ventro-medial aspect (asterisks) and markedly reduced in the glomerular layer (arrowheads) compared to wild-type. (B) Sagittal sections of the rostral and ventral forebrain regions (left panels), and detail of the caudal branch of the vomeronasal nerve (right panels) in Nrp1+/+ and Nrp1sema/sema E14.5 mouse embryos. A crystal of the DiI lipophilic fluorescent dye has been placed in the vomeronasal organ lumen to anterogradely label vomeronasal axons. The vomeronasal nerve extends across the medial aspect of the olfactory bulb and projects both dorsally, to the accessory olfactory bulb, and caudally, to the ventral forebrain (vfb). In the mutant mouse, fibers in the caudal branch are scarce compared to wild-type. (C) Sagittal sections of the rostral and ventral forebrain regions at E14.5, immunostained for GnRH (green). Note the abnormal distribution of GnRH-immunoreactive cells in the Nrp1sema/sema mouse (arrows). (D) Coronal sections of the preoptic region (upper panels) showing GnRH neuroendocrine cells (green) and their projections in the median eminence (me, arrows) (lower panels) in Nrp1+/+ and Nrp1sema/sema newborn (P0) mice. The immunostaining is reduced in the Nrp1sema/sema mouse. (E) Quantitative analysis (mean ± s.d.) of GnRH cell distributions in Nrp1+/+ and Nrp1sema/sema mice at E14.5 and P0. * and ** denote statistically significant differences between genotypes in the indicated head regions (two-way ANOVA followed by Tukey's range test) with p<0.05 and p<0.01, respectively. Note that the total numbers of GnRH cells are not statistically different between Nrp1+/+ and Nrp1sema/sema mice at E14.5 or P0 (Student's t-test, p>0.05). Other abbreviations: cx, cerebral cortex; ovlt, organum vasculosum of lamina terminalis; 3v, third ventricle. Scale bars: 100 µm (50 µm in inset).
Figure 3.
Diagram of Sema3A with the mutations found in Kallmann syndrome patients.
Sequence chromatograms of the mutations are shown together with the positions of the corresponding aminoacid residues in the protein domains. Abbreviations: sema, semaphorin; PSI, plexin/semaphorin/integrin; Ig, immunoglobulin-like; C, cysteine residue involved in Sema3A dimerization (interchain disulfide bond) [18].
Figure 4.
Defective secretion or signaling activity of Sema3A proteins harboring the mutations identified in Kallmann syndrome patients.
(A) Western blot analysis of conditioned media from transfected COS-7 cells producing wild-type (WT) or mutated Sema3A proteins. The p.D538fsX31 frameshifting mutation, and the p.V435I and p.R66W missense mutations result in the absence of a secreted protein. (B) Upper panels: Representative western blots for the phosphorylated and total forms of FAK (left panel) and ERK1/2 (right panel) in GN11 cells following a 20 min incubation with serum-free medium (mock, negative control), 100 ng/ml of purified recombinant human Sema3A, or the conditioned media from transfected COS-7 cells producing wild-type or mutated Sema3A proteins. Lower panels: Bar graphs illustrate the mean ratio (± s.d.) of the western blot signal intensity obtained for phosphorylated FAK (P-FAK) or ERK1/2 (P-ERK1/2) to that of total FAK or ERK1/2, respectively. Each experiment was carried out three times independently. * denotes statistically significant difference with wild-type Sema3A (one-way ANOVA followed by Fisher's LSD test, p<0.05). a.u.: arbitrary units (pixel density).
Table 1.
SEMA3A mutations identified in Kallmann syndrome patients.