THE INVOLVEMENT OF THE NOTCH PATHWAY IN THE GNRH NEURONS DEVELOPMENT

The proper development of the hypothalamic-pituitary-gonadal (HPG) axis is essential for normal reproductive competence. Misfunctions in the axis that impairs GnRH synthesis or function result in GnRH deficiency. Idiopathic congenital hypogonadotropic hypogonadism (CHH) is a rare reproductive disorder, with significant heterogeneity of genetic inheritance, that is primarily caused by gonadotropin-releasing hormone (GnRH) deficiency. Clinically, the disorder is characterized by an absence of puberty and infertility. In approximately 50% of cases, CHH patients also suffer from a reduced or deficient sense of smell (hyposmia or anosmia, respectively). In this case, the disorder is termed Kallmann syndrome (KS) and results from a failure or incomplete embryonic migration of GnRH-producing neurons. The significance to elucidate the genetic causes of CHH is related to the relatively high percentage (about 50%) of patients that are still considered idiopathic. It is known from the literature that Notch signaling has a role in the migration of neurons in the developing cortex and that it is expressed in the olfactory system of many species, so we would like to understand if Notch signaling has a role in the migration of GnRH neurons in the olfactory system. Hence, the aim of my PhD was to understand: 1) if Notch signaling molecules are expressed along the GnRH migratory pathway in human fetal and adult post-mortem brain sections; 2) if Notch plays a role in the establishment of the correct GnRH migratory process or GnRH axonal targeting to the hypothalamic regions, using zebrafish as an in vivo model; 3) to address whether patients affected by CHH present mutations in the Notch signaling pathway; 4) to functionally validate the eventual mutations using an immortalized GnRH cell line (GN11). We explored by multiplex fluorescent in situ hybridization and immunohistochemical assays the expression of both JAG1, NOTCH1 and NOTCH2 in coronal human fetal sections of the nasal compartment and nasal/forebrain junctions in early developmental stages. These experiments revealed that JAG1, NOTCH1 and NOTCH2 are expressed along the GnRH migratory pathway during human fetal development, suggesting a paracrine and/or autocrine mechanism. We then used zebrafish as in vivo model to investigate the involvement of jag1 in GnRH3 neurons development and migration. Firstly, we have demonstrated the expression of jag1a, jag1b, notch1a and GnRH3 in the olfactory placode of zebrafish embryos, revealing a possible collaboration between these factors in the GnRH3 neurons development. Taking advantage of the zebrafish transgenic line tg(GnRH3:EGFP), we demonstrated that downregulation of jag1b, but not jag1a, strongly affects the development of the GnRH3 neurons at 48 and 72hpf. Treatment of tg(GnRH3:EGFP) embryos with the Notch inhibitor, DAPT (γ-secretase inhibitor), phenocopied the morpholino experiments, further supporting a role for notch1/jag1 in the development of GnRH3 neurons. Additionally, we performed migration assay on immortalized GnRH cells, and we observe that JAG1 act as a repellent factor for the motility of these cells. Based on the human and zebrafish data and considering the KS-like phenotype of our jag1b morphants, we next sought in our cohort of CHH/KS patients for possible mutation in JAG1 gene and four rare missense variants in the JAG1 gene were identified (R117G, F206Y, Y931I, 1160N). Overexpression of all JAG1 variants in GN11 cell line coupled with an immunofluorescence assay revealed that only the JAG1 variant D1160N was mislocalized and retained into the cytoplasm. The functionality was evaluated through the Luciferase assay, and D1160N variant did not activate the transcription of the Notch Responsive Element promoter. Combining morphological analysis in vivo (in human and zebrafish), together with genetic and pharmacological manipulation in zebrafish, and human genetic analysis, we provide compelling evidence that Notch1/Jag1 signaling has a role in the development of GnRH neurons/olfactory system and indicate that Notch1/Jag1 signaling insufficiency may contribute to the pathogenesis of CHH in humans.