Gonadotropin-releasing hormone-1 (GnRH-1) neurons (GnRH-1 ns) migrate from the developing olfactory pit into the hypothalamus during embryonic development. Migration of the GnRH-1 neurons is required for mammalian reproduction as these cells control release of gonadotropins from the anterior pituitary gland. Disturbances in GnRH-1 ns migration, GnRH-1 synthesis, secretion or signaling lead to varying degrees of hypogonadotropic hypogonadism (HH), which impairs pubertal onset and fertility. HH associated with congenital olfactory defects is clinically defined as Kallmann Syndrome (KS). The association of olfactory defects with HH in KS suggested a potential direct relationship between defective olfactory axonal routing, lack of olfactory bulbs (OBs) and aberrant GnRH-1 ns migration. However, it has never been experimentally proven that the formation of axonal connections of the olfactory/vomeronasal neurons to their functional targets are necessary for the migration of GnRH-1 ns to the hypothalamus. Loss-of-function of the Arx-1 homeobox gene leads to the lack of proper formation of the OBs with abnormal axonal termination of olfactory sensory neurons ( Yoshihara et al., 2005). Our data prove that correct development of the OBs and axonal connection of the olfactory/vomeronasal sensory neurons to the forebrain are not required for GnRH-1 ns migration, and suggest that the terminal nerve, which forms the GnRH-1 migratory scaffold, follows different guidance cues and differs in gene expression from olfactory/vomeronasal sensory neurons.

The identity of individual neuronal cell types is defined and maintained by the expression of specific combinations of transcriptional regulators that control cell type-specific genetic programs. The epithelium of the vomeronasal organ of mice contains two major types of vomeronasal sensory neurons (VSNs): 1) the apical VSNs which express vomeronasal 1 receptors (V1r) and the G-protein subunit Gαi2 and; 2) the basal VSNs which express vomeronasal 2 receptors (V2r) and the G-protein subunit Gαo. Both cell types originate from a common pool of progenitors and eventually acquire apical or basal identity through largely unknown mechanisms. The transcription factor AP-2ε, encoded by the Tfap2e gene, plays a role in controlling the development of GABAergic interneurons in the main and accessory olfactory bulb (AOB), moreover AP-2ε has been previously described to be expressed in the basal VSNs. Here we show that AP-2ε is expressed in post-mitotic VSNs after they commit to the basal differentiation program. Loss of AP-2ε function resulted in reduced number of basal VSNs and in an increased number of neurons expressing markers of the apical lineage. Our work suggests that AP-2ε, which is expressed in late phases of differentiation, is not needed to initiate the apical-basal differentiation dichotomy but for maintaining the basal VSNs' identity. In AP-2ε mutants we observed a large number of cells that entered the basal program can express apical genes, our data suggest that differentiated VSNs of mice retain a notable level of plasticity.

The ability of terrestrial vertebrates to find food and mating partners, and to avoid predators, relies on the detection of chemosensory information. Semiochemicals responsible for social and sexual behaviors are detected by chemosensory neurons of the vomeronasal organ (VNO), which transmits information to the accessory olfactory bulb. The vomeronasal sensory epithelium of most mammalian species contains a uniform vomeronasal system; however, rodents and marsupials have developed a more complex binary vomeronasal system, containing vomeronasal sensory neurons (VSNs) expressing receptors of either the V1R or V2R family. In rodents, V1R/apical and V2R/basal VSNs originate from a common pool of progenitors. Using single cell RNA-sequencing, we identified differential expression of Notch1 receptor and Dll4 ligand between the neuronal precursors at the VSN differentiation dichotomy. Our experiments show that Notch signaling is required for effective differentiation of V2R/basal VSNs. In fact, Notch1 loss of function in neuronal progenitors diverts them to the V1R/apical fate, whereas Notch1 gain of function redirects precursors to V2R/basal. Our results indicate that Notch signaling plays a pivotal role in triggering the binary differentiation dichotomy in the VNO of rodents.

During mammalian development, gonadotropin-releasing-hormone-1 neurons (GnRH-1ns) migrate from the developing vomeronasal organ (VNO) into the brain asserting control of pubertal onset and fertility. Recent data suggest that correct development of the olfactory ensheathing cells (OEC) is imperative for normal GnRH-1 neuronal migration. However, the full ensemble of molecular pathways that regulate OEC development remains to be fully deciphered. Loss-of-function of the transcription factor Gli3 is known to disrupt olfactory development, however, if Gli3 plays a role in GnRH-1 neuronal development is unclear. By analyzing Gli3 extra-toe mutants (Gli3 Xt/Xt), we found that Gli3 loss-of-function compromises the onset of achaete-scute family bHLH transcription factor 1 (Ascl-1) ϩ vomeronasal progenitors and the formation of OEC in the nasal mucosa. Surprisingly, GnRH-1 neurogenesis was intact in Gli3 Xt/Xt mice but they displayed significant defects in GnRH-1 neuronal migration. In contrast, Ascl-1 null mutants showed reduced neurogenesis for both vomeronasal and GnRH-1ns but less severe defects in OEC development. These observations suggest that Gli3 is critical for OEC development in the nasal mucosa and subsequent GnRH-1 neuronal migration. However, the nonoverlapping phenotypes between Ascl-1 and Gli3 mutants indicate that Ascl-1, while crucial for GnRH-1 neurogenesis, is not required for normal OEC development. Because Kallmann syndrome (KS) is characterized by abnormal GnRH-1ns migration, we examined whole-exome sequencing data from KS subjects. We identified and validated a GLI3 loss-of-function variant in a KS individual. These findings provide new insights into GnRH-1 and OECs development and demonstrate that human GLI3 mutations contribute to KS etiology.