GnRH is essential on reproductive physiology and behaviour. Early in development, GnRH-1 neurons undergo a migratory process from the olfactory placode (OP) to the hypothalamus. Failure of GnRH-1 migration and abnormal olfactory bulb (OB) characterize Kallmann's syndrome (KS) resulting in hypogonadotrophic hypogonadism and anosmia. The X-linked form of KS is due to a dysfunctional KAL-1 gene, which encodes anosmin-1. An autosomal dominant form of KS results from disrupted KAL-2, which encodes FGFR-1. In this study we provide a GnRH-1, KAL-1 and KAL-2 spatio-temporal analysis during human embryogenesis to further understand olfactory system development and elucidate the mechanisms of KAL-1/KAL-2 action. We used GnRH human embryonic primary cells (FNC-B4) to observe in vitro alterations by KAL-1/KAL-2. Human recombinant anosmin-1 (wild type and truncated forms: PIWF4 and PIWF1), correspondent loss of function mutants, specific PIWF1pAb, FGFR-1 mAb and FGFR1 antagonists were used in order to observe these effects.
GnRH-1-immunoreactivity appears in the OP at ED32. Two days later this 'early' GnRH-1 neurons have arrived at the mid-forebrain (before OB morphogenesis proper), whereas another GnRH-1 cell group is accompanied by KAL-1 and KAL-2 cells in the OP, nasal mesenchyme (NM) and rostral forebrain (rF). In vitro assays showed significant neurite outgrowth length and external phenotype changes on FNC-B4 cells: PIWF1, PIWF4 increased length axonal outgrowth and branching which showed a biphasic dose dependent with a 1nM maximum. This effect was not observed when using mutants and blocked by PIWF1pAb, FGFR1 antibodies and FGFR1 antagonists.
The results show 'early' migratory GnRH-1 neurons entering the forebrain before any KAL-1/KAL-2 immunoreactivity in the olfactory system. Soon after, 'late' GnRH-1, KAL-1 and KAL-2 are adjacently localized in OP, NM and rF. Moreover, in vitro studies support the idea that KAL-1 and KAL-2 might interact and affect the migratory phenotype of GnRH-1 neurons.