Fibroblast growth factor 8 signaling through fibroblast growth factor receptor 1 is required for the emergence of gonadotropin-releasing hormone neurons

Abstract

GnRH neurons are essential for the onset and maintenance of reproduction. Mutations in both fibroblast growth factor receptor (Fgfr1) and Fgf8 have been shown to cause Kallmann syndrome, a disease characterized by hypogonadotropic hypogonadism and anosmia, indicating that FGF signaling is indispensable for the formation of a functional GnRH system. Presently it is unclear which stage of GnRH neuronal development is most impacted by FGF signaling deficiency. GnRH neurons express both FGFR1 and -3; thus, it is also unclear whether FGFR1 or FGFR3 contributes directly to GnRH system development. In this study, we examined the developing GnRH system in mice deficient in FGF8, FGFR1, or FGFR3 to elucidate the individual contribution of these FGF signaling components. Our results show that the early emergence of GnRH neurons from the embryonic olfactory placode requires FGF8 signaling, which is mediated through FGFR1, not FGFR3. These data provide compelling evidence that the developing GnRH system is exquisitely sensitive to reduced levels of FGF signaling. Furthermore, Kallmann syndrome stemming from FGF signaling deficiency may be due primarily to defects in early GnRH neuronal development prior to their migration into the forebrain.

Figure 1

Representative bright-field photomicrographs of GnRH ICC in parasagittal sections of Fgf8 hypomorphs. WT (A) and homozygous HOMO (B) E11.5 embryos are shown. Note the presence of GnRH-IR neurons (black arrows) in the invaginated medial-ventral olfactory epithelium (boxed) of WT but not in HOMO embryos. Black asterisk indicates preoptic recess. Black scale bar, 200 μm or 100 μm (inset). C and D, Inverted bright-field photomicrographs of WT and HOMO E14.5 embryos. Note the presence of GnRH-IR neurons in the preoptic area of WT but not in HOMO embryos. White arrow indicates the olfactory bulb. White asterisk indicates the sphenoid cartilage. #, Lateral ventricle. White scale bar, 500 μm or 50 μm (inset). Insets are higher-magnification photomicrographs of the boxed areas.

Figure 2

GnRH neurons in Fgf8 hypomorphs on PN 0. A, Total number of GnRH-IR neurons in the whole head of WT (n = 4), HET (n = 4), and HOMO (n = 4) Fgf8 hypomorphs. Different letters indicate significant differences (P < 0.01). Data are presented as mean ± sem. ND, Not detectable. GnRH mRNA-positive neurons (arrows) are present in coronal sections of the preoptic area in WT (B) but not HOMO animals (C). Asterisk indicates third ventricle. Scale bar, 100 μm.

Figure 3

Representative epifluorescence photomicrographs of Olf-1-IR olfactory neurons (A–C) and peripherin-IR olfactovomeronasal fibers (D–I) in parasagittal sections of Fgf8 hypomorphs. Olf-1-positive staining (red) WT (A), HET (B), and HOMO (C) E11.5 embryos. Note Olf-1-positive staining in the medial-ventral olfactory epithelium (arrows). #, Preoptic recess. Scale bar, 200 μm. D–I, Peripherin-positive staining (red, arrows) in WT (D–F) and HOMO (G) E14.5 embryos. Asterisk indicates the olfactory bulb. Boxed areas are magnified in WT (H) and HOMO (I) E14.5 embryos. Scale bar, 200 μm. oe, Olfactory epithelium; FB, forebrain. Blue is 4′,6′-diamino-2-phenylindole nuclear counterstain.

Figure 4

Representative photomicrographs of E11.5 olfactory epithelium (oe) in cresyl violet-stained parasagittal sections of Fgf8 hypomorphs. A, Apoptotic cells in WT olfactory epithelium (arrows). Scale bar, 20 μm. B–D, Olfactory epithelium in WT, HET, and HOMO embryos. Note the absence of apoptotic cells in the medial-ventral and medial-dorsal olfactory epithelium at higher magnification (insets). The base of the arrows indicate the regions magnified in the insets. Scale bar, 200 μm or 20 μm (insets). Asterisk indicates preoptic recess.

Figure 5

GnRH neurons in Fgfr1 hypomorphs on PN 0. A and B, Representative photomicrographs of GnRH-IR neurons (arrows). C and D, GnRH mRNA positive (arrows) in coronal sections of the preoptic area in WT and HOMO Fgfr1 hypomorphs. Asterisk indicates third ventricle. Dotted line indicates ventral border of preoptic area. Scale bar, 50 μm or 20 μm (inset). E, Total number of GnRH-IR neurons in the whole head of WT (n = 4), HET (n = 4), and HOMO (n = 4) Fgfr1 hypomorphs. Different letters indicate significant differences (P < 0.01). Data are presented as mean ± sem.