Role of vascular endothelial growth factor and blood vessels in the development of GnRH neurons

Gonadotropin-releasing hormone (GnRH) neurons, a small number of cells scattered in the hypothalamus, play an essential role in reproductive function. During development, these neurons originate in the olfactory placode and migrate along olfactory/vomeronasal axons in the nasal compartment to gain access into the forebrain and reach their final positions in the hypothalamus. In humans, failure of GnRH neurons to migrate normally results in delayed or absent pubertal maturation and infertility. The movement of these cells through changing molecular environments suggests that numerous factors are involved in their migration. We have recently described that classical neuronal guidance molecules, such neuropilins (NRPs), are expressed by GnRH neurons, and established the importance of NRP2 in their migration in vivo. Using immortalised GnRH cells (GN11), we also found that two NRP ligands modulate their migratory response: the class 3 semaphorins (SEMA3A/3F) and vascular endothelial growth factor A (VEGF). VEGF is a major regulator of vasculogenesis and vascular permeability, interacting with receptor tyrosine kinases Flt-1 and Flk-1 on endothelial cells. Recent evidence indicates that VEGF has additional non-vascular functions. In particular, the identification of NRPs as co-receptors for VEGF, as well as the detection of their receptors on neurons, suggests that VEGF could act directly on neurons to produce effects such survival and migration. In this study, the possible interactions between VEGF, blood vessels and the GnRH-system have been tested. We first visualized the presence of a network of blood vessels along peripherin-positive olfactory axons and migrating GnRH neurons. In embryonic mice, blood vessels, stained with lectin IsoB4, were seen around the olfactory placode, the vomeronasal organ, as well as in the nasal mesenchyme, during the appearance and migration of GnRH neurons. Then, by using RT-PCR and enzymatic staining of VEGF-LacZ reporter mice, we found that VEGF is abundantly expressed in the developing olfactory structures. Moreover, FACS-isolated embryonic GFP-GnRH neurons expressed specific transcripts for VEGF and its receptor Flt-1. We found that GN11 cells-used as a model of migrating GnRH neurons- also express these molecules and respond to VEGF. Functionally, we observed that the latter promotes their survival and stimulates the chemomigration of GN11 cells by activating PI3K and MAPK pathways. Taken together, these results indicate that GnRH neuron migration and development are modulated by VEGF signalling, suggesting the existence of a ‘cross-talk’ between the vascular and GnRH-neuronal system