P42 - Impact of wfs1b downregulation on GnRH3 neurons development in the zebrafish model

Congenital hypogonadotropic hypogonadism (CHH) is a rare reproductive disorder characterized by impaired secretion or action of gonadotropin-releasing hormone (GnRH), leading to delayed or absent puberty. CHH displays significant genetic and phenotypic heterogeneity, and about 50% of patients remain idiopathic, suggesting that additional regulatory genes are yet to be identified. In our CHH patient cohort, we identified two heterozygous missense variants in the WFS1 gene, which encodes Wolframin, a protein involved in the unfolded protein response (UPR), calcium homeostasis, and endoplasmic reticulum (ER) stress regulation. Homozygous mutations in WFS1 cause Wolfram Syndrome, a multisystem disorder featuring vision and hearing loss, anosmia, diabetes mellitus or insipidus, psychiatric symptoms, and reduced fertility. The discovery of WFS1 variants in CHH patients points to a possible role for Wolframin in the development and function of the hypothalamic-pituitary-gonadal (HPG) axis. To investigate this, we used the zebrafish model, focusing on the role of the WFS1 orthologue wfs1b in the differentiation and migration of GnRH neurons. In vitro assays revealed that the mutant WFS1 proteins had lower ER expression and a tendency to aggregate. We generated a wfs1b knockdown in the Tg(gnrh3:EGFP) zebrafish line to assess its effect on GnRH3 neuron development. All wfs1b morphants displayed disrupted GnRH3 architecture, with neurons mislocalized in the olfactory bulbs and anterior commissure. To explore the underlying mechanism, we analyzed expression of neural progenitor markers (pax6, ngn1) and intermediate precursor marker isl1a using whole- mount in situ hybridization. While pax6 and ngn1 expression was preserved, isl1a was reduced in morphants, suggesting its involvement in the observed GnRH3 migration defects. Given WFS1’s role in UPR, we also examined the expression of UPR-related genes (bip, atf6, chop, xbp1) by RT-qPCR in 48 hpf embryos. These genes were significantly upregulated in wfs1b morphants, indicating activation of the UPR pathway, potentially impacting isl1a-positive neurons critical for GnRH3 network development.