Impact of wfs1b downregulation on GnRH3 neurons development in the zebrafish model

Congenital hypogonadotropic hypogonadism (CHH) is a rare reproductive disorder characterized by deficiency or action failure of gonadotrophin-releasing hormone and delayed or arrested puberty. CHH shows complex genetic and phenotypic heterogeneity and approximately 50% of patients are still idiopathic, indicating that further regulatory genes remain to be identified. In our cohort of CHH patients, we recently identified two heterozygous missense variants in the WFS1 gene, which encodes for Wolframin, an ubiquitous protein involved in unfolded protein response, regulation of cellular calcium homeostasis and endoplasmic reticulum stress. Homozygous mutations of WFS1 are responsible for “Wolfram Syndrome”, a multisystem disorder characterized by visual dysfunctions, hearing loss, anosmia, diabetes mellitus or insipidus, psychiatric disorders and reduced fertility. The identification of genetic variants in the WFS1 gene in patients with CHH suggests a possible involvement of Wolframin during the various steps underlying the development and function of the hypothalamic-pituitary-gonadal (HPG)-axis. In this work, we took advantage of the zebrafish model to study the impact of wfs1b (orthologues of human WFS1) during the differentiation and migration of GnRH neurons. In vitro experiments were performed to verify the impact of the two allelic variants find by whole exome sequencing on the subcellular localization and expression of the WFS1 protein. Our analyses revealed that the mutated proteins have a lower expression in the ER and tend to form aggregates. In the zebrafish model we generated a knock down model for wfs1b in the tg( GnRH3: EGFP) reporter line to evaluate the role of wfs1b on GnRH3 neurons development. All the wfs1b morphants (wfs1bMOs) embryos showed a global disorganization of GnRH3 neuronal architecture that appeared mislocalized at the level of olfactory bulbs and anterior commissure. To evaluate the potential contribution of wfs1b during GnRH3 neurogenesis we analyzed by whole mount in situ hybridization experiments neuronal progenitors markers expressing the transcription factor pax6 and intermediate neuronal precursors depicted by ngn1 and isl1a expression. We observed a preserved expression of both pax6 and ngn1 in wfs1bMOs but interestingly, morphants embryos displayed differences in isl1a expression suggesting that the reduced expression of this marker may have a role in explaining GnRH3 migration defects. Due to the involvement of WFS1 in UPR mechanism we also performed RT-qPCR experiments in embryos at 48 hpf to investigate the expression of genes involved in the UPR pathways and our analysis revealed that levels of bip, atf6, chop and xbp1 were significantly increased in wfs1bMOs. Our results suggest that the knockdown of wfs1b activates UPR pathway likely affecting isl1a positive neurons, involved in GnRH3 neuronal network development.