Specific hypothalamic gonadotropin-releasing hormone (GnRH) -secreting cells control the onset and maintenance of the reproductive axis in vertebrates (Gore,2002). These neurons undergo a unique odyssey during embryonic development. They are first detected in association with the olfactory placode, then, by neurophilic migration, they enter the forebrain along olfactory, terminal and veromonasal nerves, and further continue to the preoptic area in the hypothalamus (Schwanzel-Fukuda and Pfaff,1989; Wray et al.,1989). This pathway of developmental migration has been shown in a variety of vertebrate species (Dubois et al.,2002), including zebrafish (Palevitch et al.,2007; Abraham et al.,2008). In mammals, the gene that encodes the hypophysiotropic form is termed GnRH1 (White and Fernald,1998). In the zebrafish genome, which has been almost completely sequenced, the gnrh1 gene is most definitely absent (Palevitch et al.,2007), probably due to selective loss during evolution (Kuo et al.,2005). This gene loss has most likely occurred also in other teleost species belonging to various orders (reviewed in Okubo and Nagahama,2008). Nevertheless, two other genes encoding different forms of GnRH—gnrh2 and gnrh3 (formerly cGnRH-II and sGnRH, respectively)—have been isolated and characterized in zebrafish (Torgersen et al.,2002; Steven et al.,2003). In adult zebrafish, gnrh2 is localized to the midbrain tegmentum, as described in all other investigated jawed vertebrates (Steven et al.,2003). The gnrh3 transcript is located in adults in the terminal nerve ganglion (TNg). A gnrh3-expressing subset group resides in the ventral telencephalon-preoptic area. The GnRH3 axons project to various parts of the brain including the pituitary gland (Abraham et al.,2008). Furthermore, combined high performance liquid chromatography/radioimmunoassay analyses have indicated that the GnRH3 peptide is the predominant GnRH peptide in the adult zebrafish pituitary (Powell et al.,1996). Therefore, GnRH3 is considered the hypophysiotropic form of GnRH in zebrafish. Furthermore, the ontogeny of GnRH3 neurons in zebrafish resembles the development of GnRH1 neurons in mammals: In both cases, the expression of GnRH is initially detected in the olfactory placode, later in association with the olfactory/terminal nerves, and then migrating through the ventral telencephalon to the hypothalamic preoptic area (Schwanzel-Fukuda,1999; Palevitch et al.,2007; Abraham et al.,2008). Of interest, in zebrafish, GnRH3 projections were shown to precede the migration of their cell soma along the terminal nerve, delineating the pathway for the migration of a subset of GnRH3 cells from the olfactory region to the hypothalamus (Palevitch et al.,2007; Abraham et al.,2008). Moreover, projections extended to the pituitary gland even before GnRH3 perikarya entered the forebrain (Abraham et al.,2008).
The importance of an appropriate migration of hypothalamic GnRH neurons for normal reproductive function is well demonstrated by a rare human disease called Kallmann's syndrome (KS; Kallmann et al.,1944). KS is characterized by anosmia coupled with hypogonadotropic hypogonadism that is secondary to deficiency in hypothalamic GnRH (Naftolin et al.,1971). Impaired development of the olfactory nerves in a KS human fetus specimen resulted in failure of GnRH neurons to undergo normal migration (Schwanzel-Fukuda et al.,1989).
The migration of GnRH neurons in vertebrates is modulated by a plethora of factors, such as receptors, secreted molecules, and adhesion molecules (Tobet and Schwarting,2006). However, the exact mechanism that controls these developmental events is poorly understood (Schwarting et al.,2007). One approach that was implemented toward identifying additional regulatory molecules was a cDNA subtraction screen in migrating vs. nonmigrating GnRH neurons in mice (Kramer and Wray,2000). This analysis revealed a new factor, named Nasal Embryonic LHRH factor (NELF), which was shown to be involved in the outgrowth of olfactory axon projections and to affect the migration of GnRH neurons in mice (Kramer and Wray,2000). Consequently, human NELF was considered as a candidate gene for the etiology of idiopathic hypogonadotropic hypogonadism and KS. Indeed, genetic studies in human implicated mutations in the NELF gene in two cases of idiopathic hypogonadotrophic hypogonadism and KS (Miura et al.,2004; Pitteloud et al.,2007). Here, we report on the isolation of the first nonmammalian nelf gene, describe its expression pattern during development, and demonstrate its involvement in the development of the GnRH3 system in zebrafish.