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The positive regulatory (PR) domain containing 13 (PRDM13) putative chromatin modifier and transcriptional regulator functions downstream of the transcription factor PTF1A, which controls GABAergic fate in the spinal cord and neurogenesis in the hypothalamus. Here, we report a recessive syndrome associated with PRDM13 mutation. Patients exhibited intellectual disability, ataxia with cerebellar hypoplasia, scoliosis, and delayed puberty with congenital hypogonadotropic hypogonadism (CHH). Expression studies revealed Prdm13/PRDM13 transcripts in the developing hypothalamus and cerebellum in mouse and human. An analysis of hypothalamus and cerebellum development in mice homozygous for a Prdm13 mutant allele revealed a significant reduction in the number of Kisspeptin (Kiss1) neurons in the hypothalamus and PAX2+ progenitors emerging from the cerebellar ventricular zone. The latter was accompanied by ectopic expression of the glutamatergic lineage marker TLX3. Prdm13-deficient mice displayed cerebellar hypoplasia and normal gonadal structure, but delayed pubertal onset. Together, these findings identify PRDM13 as a critical regulator of GABAergic cell fate in the cerebellum and of hypothalamic kisspeptin neuron development, providing a mechanistic explanation for the cooccurrence of CHH and cerebellar hypoplasia in this syndrome. To our knowledge, this is the first evidence linking disrupted PRDM13-mediated regulation of Kiss1 neurons to CHH in humans.

Gonadotropin releasing hormone (GnRH) neurons are hypothalamic neuroendocrine cells that control sexual reproduction. During embryonic development, GnRH neurons migrate from the nose to the hypothalamus, where they receive inputs from several afferent neurons, following the axonal scaffold patterned by nasal nerves. Each step of GnRH neuron development depends on the orchestrated action of several molecules exerting specific biological functions. Mutations in genes encoding for these essential molecules may cause Congenital Hypogonadotropic Hypogonadism (CHH), a rare disorder characterized by GnRH deficiency, delayed puberty and infertility. Depending on their action in the GnRH neuronal system, CHH causative genes can be divided into neurodevelopmental and neuroendocrine genes. The CHH genetic complexity, combined with multiple inheritance patterns, results in an extreme phenotypic variability of CHH patients. In this review, we aim at providing a comprehensive and updated description of the genes thus far associated with CHH, by dissecting their biological relevance in the GnRH system and their functional relevance underlying CHH pathogenesis.

Idiopathic hypogonadotropic hypogonadism and Kallmann syndrome are rare genetic disorders characterized by isolated gonadotropin-releasing hormone (GnRH) deficiency (IGD) and delayed or absent puberty. Defective GnRH neuron migration during development or secretion of mature GnRH neurons secondary to molecular defects in several key developmental and neuroendocrine pathways are thought to be the primary causes of these disorders. Recent studies have highlighted the importance of semaphorins and their receptors in this system, by showing that these molecules play distinct roles during the development and plasticity of these neurons. Accordingly, mutations in the semaphoring-signaling pathway genes have been found in patients affected by IGD, underlying the importance of semaphorin-mediated signaling pathways in the neuroendocrine axis that control reproduction.

The initiation of puberty is driven by an upsurge in hypothalamic gonadotropin-releasing hormone (GnRH) secretion. In turn, GnRH secretion upsurge depends on the development of a complex GnRH neuroendocrine network during embryonic life. Although delayed puberty (DP) affects up to 2% of the population, is highly heritable, and is associated with adverse health outcomes, the genes underlying DP remain largely unknown. We aimed to discover regulators by whole-exome sequencing of 160 individuals of 67 multigenerational families in our large, accurately phenotyped DP cohort. LGR4 was the only gene remaining after analysis that was significantly enriched for potentially pathogenic, rare variants in 6 probands. Expression analysis identified specific Lgr4 expression at the site of GnRH neuron development. LGR4 mutant proteins showed impaired Wnt/β-catenin signaling, owing to defective protein expression, trafficking, and degradation. Mice deficient in Lgr4 had significantly delayed onset of puberty and fewer GnRH neurons compared with WT, whereas lgr4 knockdown in zebrafish embryos prevented formation and migration of GnRH neurons. Further, genetic lineage tracing showed strong Lgr4-mediated Wnt/β-catenin signaling pathway activation during GnRH neuron development. In conclusion, our results show that LGR4 deficiency impairs Wnt/β-catenin signaling with observed defects in GnRH neuron development, resulting in a DP phenotype., SCOPUS: ar.j, info:eu-repo/semantics/published

In mammals, fertility critically depends on the pulsatile secretion of gonadotropin-releasing hormone (GnRH) by scattered hypothalamic neurons (GnRH neurons). During development, GnRH neurons originate in the nasal placode and migrate first into the nasal compartment and then through the nasal/forebrain junction, before they reach their final position in the hypothalamus. This neurodevelopmental process, which has been extensively studied in mouse models, is regulated by a plethora of factors that might control GnRH neuron migration or survival as well as the fasciculation/targeting of the olfactory/vomeronasal axons along which the GnRH neurons migrate. Defects in GnRH neuron development or release can lead to isolated GnRH deficiency, with the underlying genetic causes still being partially unknown. Recently, semaphorins and their receptors neuropilins and plexins, a large family of molecules implicated in neuronal development and plasticity, are emerging as key regulators of GnRH neuron biology and deficiency. Specifically, semaphorins have been shown to play different roles in GnRH neuron biology by regulating migration and survival during embryonic development as well as secretion in adulthood.

Protein kinase CK2 (CK2) is a highly conserved and ubiquitous kinase is involved in crucial biological processes, including proliferation, migration, and differentiation. CK2 holoenzyme is a tetramer composed by two catalytically active (&alpha, /&alpha, &rsquo, ) and two regulatory (&beta, ) subunits and exerts its function on a broad range of targets. In the brain, it regulates different steps of neurodevelopment, such as neural differentiation, neuritogenesis, and synaptic plasticity. Interestingly, CK2 mutations have been recently linked to neurodevelopmental disorders, however, the functional requirements of the individual CK2 subunits in neurodevelopment have not been yet investigated. Here, we disclose the role of CK2 on the migration and adhesion properties of GN11 cells, an established model of mouse immortalized neurons, by different in vitro experimental approaches. Specifically, the cellular requirement of this kinase has been assessed pharmacologically and genetically by exploiting CK2 inhibitors and by generating subunit-specific CK2 knockout GN11 cells (with a CRISPR/Cas9-based approach). We show that CK2&alpha, subunit has a primary role in increasing cell adhesion and reducing migration properties of GN11 cells by activating the Akt-GSK3&beta, axis, whereas CK2&alpha, subunit is dispensable. Further, the knockout of the CK2&beta, regulatory subunits counteracts cell migration, inducing dramatic alterations in the cytoskeleton not observed in CK2&alpha, knockout cells. Collectively taken, our data support the view that the individual subunits of CK2 play different roles in cell migration and adhesion properties of GN11 cells, supporting independent roles of the different subunits in these processes.

Gonadotropin-releasing hormone (GnRH) neurons regulate puberty onset and sexual reproduction by secreting GnRH to activate and maintain the hypothalamic-pituitary-gonadal axis. During embryonic development, GnRH neurons migrate along olfactory and vomeronasal axons through the nose into the brain, where they project to the median eminence to release GnRH. The secreted glycoprotein SEMA3A binds its receptors neuropilin (NRP) 1 or NRP2 to position these axons for correct GnRH neuron migration, with an additional role for the NRP co-receptor PLXNA1. Accordingly, mutations in SEMA3A, NRP1, NRP2 and PLXNA1 have been linked to defective GnRH neuron development in mice and inherited GnRH deficiency in humans. Here, we show that only the combined loss of PLXNA1 and PLXNA3 phenocopied the full spectrum of nasal axon and GnRH neuron defects of SEMA3A knockout mice. Together with Plxna1, the human orthologue of Plxna3 should therefore be investigated as a candidate gene for inherited GnRH deficiency.

Gonadotropin-releasing hormone (GnRH) neurons are neuroendocrine cells, located in the hypothalamus, that play an essential role in mammalian reproduction. These neurons originate in the nasal placode and migrate during embryonic development, in association with olfactory/vomeronasal nerves, first in the nose, then through the cribriform plate to enter the forebrain, before settling in the hypothalamus. One of the molecules required for their early migration in the nose is the chemokine CXCL12, which is expressed in the embryonic nasal mesenchyme in an increasing ventral to dorsal gradient, presumably guiding GnRH neurons toward the forebrain. Mice lacking CXCR4, the receptor for CXCL12, exhibit defective GnRH cell movement and a significant reduction in their number, suggesting that CXCL12/CXCR4 signaling is important in the migration and survival of these neurons. Here, we investigated the role of the more recently identified second CXCL12 receptor, CXCR7, in GnRH neuron development. We demonstrate that CXCR7 is expressed along the migratory path of GnRH neurons in the nasal cavity and, although not expressed by GnRH neurons, it affects their migration as indicated by the ectopic accumulation of these cells in the nasal compartment inCXCR7−/−mice. Absence of CXCR7 caused abnormal accumulation of CXCL12-RFP at CXCR4-positive sites in the nasal area of CXCL12-RFP-transgenic mice and excessive CXCL12-dependent intracellular clustering of CXCR4 in GnRH neurons, suggesting internalization. These findings imply that CXCR7 regulates CXCL12 availability by acting as a scavenger along the migratory path of GnRH neurons and, thus, influences the migration of these cells in a noncell-autonomous manner.

Neuritin 1 (Nrn1 or cpg15-1) is an activity- dependent protein involved in synaptic plasticity during brain development, a process that relies upon neuronal migration. By analyzing Nrn1 expression, we found that it is highly expressed in a mouse model of migrating immor- talized neurons (GN11 cells), but not in a mouse model of non-migrating neurons (GT1-7 cells). We thus hypothe- sized that Nrn1 might control neuronal migration. By using complementary assays, as Boyden's microchemotaxis, scratch-wounding and live cell imaging, we found that GN11 cell migration is enhanced when Nrn1 is overex- pressed and decreased when Nrn1 is silenced. The effects of Nrn1 in promoting neuronal migration have been then confirmed ex vivo, on rat cortical interneurons, by Boyden chamber assays and focal electroporation of acute embry- onic brain slices. Furthermore, we found that Nrn1 level modulation affects GN11 cell morphology. The process is also paralleled by Nrn1-induced a-tubulin post-translational modifications, a well-recognized marker of microtubule stability. Altogether, the data demonstrate a novel function of Nrn1 in promoting migration of neuronal cells and indicate that Nrn1 levels impact on microtubule stability.

Gonadotropin-releasing hormone (GnRH) neurons are neuroendocrine cells that are born in the nasal placode during embryonic development and migrate through the nose and forebrain to the hypothalamus, where they regulate reproduction. Many molecular pathways that guide their migration have been identified, but little is known about the factors that control the survival of the migrating GnRH neurons as they negotiate different environments. We previously reported that the class 3 semaphorin SEMA3A signals through its neuropilin receptors, NRP1 and NRP2, to organise the axons that guide migrating GnRH neurons from their birthplace into the brain. By combining analysis of genetically altered mice with in vitro models, we show here that the alternative neuropilin ligand VEGF164 promotes the survival of migrating GnRH neurons by co-activating the ERK and AKT signalling pathways through NRP1. We also demonstrate that survival signalling relies on neuronal, but not endothelial, NRP1 expression and that it occurs independently of KDR, the main VEGF receptor in blood vessels. Therefore, VEGF164 provides survival signals directly to developing GnRH neurons, independently of its role in blood vessels. Finally, we show that the VEGF164-mediated neuronal survival and SEMA3A-mediated axon guidance cooperate to ensure that migrating GnRH neurons reach the brain. Thus, the loss of both neuropilin ligands leads to an almost complete failure to establish the GnRH neuron system.

Kallmann syndrome (KS) is a genetic disease characterized by hypogonadotropic hypogonadism and impaired sense of smell. The genetic causes underlying this syndrome are still largely unknown, but are thought to be due to a developmental defect in the migration of gonadotropin-releasing hormone (GnRH) neurons. Understanding the causes of the disease is hampered by lack of appropriate mouse models. GnRH neurons are hypothalamic cells that centrally control reproduction in mammals by secreting the GnRH decapeptide into the portal blood vessels of the pituitary to stimulate the production of gonadotropins. During development, these cells are born in the nasal placode outside the brain and migrate in association with olfactory/vomeronasal axons to reach the forebrain and position themselves in the hypothalamus. By combining the analysis of genetically altered mice with in vitro models, we demonstrate here that a secreted guidance cue of the class 3 semaphorin family, SEMA3A, is essential for the development of the GnRH neuron system: loss of SEMA3A signalling alters the targeting of vomeronasal nerves and the migration of GnRH neurons into the brain, resulting in reduced gonadal size. We found that SEMA3A signals redundantly through both its classical receptors neuropilin (NRP) 1 and, unconventionally, NRP2, while the usual NRP2 ligand SEMA3F is dispensable for this process. Strikingly, mice lacking SEMA3A or semaphorin signalling through both NRP1 and NRP2 recapitulate the anatomical features of a single case of KS analysed so far, and may therefore be used as genetic models to elucidate the pathogenesis of KS.

Gonadotropin-releasing hormone (GnRH) neurons, a small number of cells dispersed in the hypothalamic region of the basal forebrain, play an important role in reproductive function. These neurons originate in the nasal placode and migrate, first in the nasal compartment, then through the cribriform plate and finally through the basal forebrain, before they attain their positions in the hypothalamus. Their movement through changing molecular environments suggests that numerous factors are involved in different phases of their migration. In humans, failure of GnRH neurons to migrate normally results in delayed or absent pubertal maturation and infertility. Advances in genetic and molecular biologic techniques in this decade have allowed us to gain insights into several molecules that affect the migration of GnRH neurons and, consequently, play a role in the establishment and maintenance of reproductive function.

Gonadotropin-releasing hormone (GnRH) neurons in the hypothalamus play an important role in reproductive function. These cells originate in the nasal compartment and migrate into the basal forebrain in association with olfactory/vomeronasal nerves in embryonic life in rodents. Here, we studied the role of neuropilins and their ligands, semaphorins, in the development of the olfactory-GnRH system. We focused onNeuropilin-2knock-out (Npn-2−/−) mice, because they are known to display defasciculation of olfactory nerves and reduced fertility. We found a significant decrease in the number of GnRH neurons in the hypothalamus and a marked reduction in their gonadal size. We then observed an abnormal increase of GnRH neurons in the noses ofNpn-2−/−mice, indicating that these cells failed to migrate into the forebrain. However, because neuropilins and semaphorins are involved in events of neuronal migration in the brain, we asked whether the observed reduction in GnRH neurons was directly attributable to the action of these molecules. Using fluorescence-activated cell sorting and reverse transcription-PCR on mRNA derived from embryonic green fluorescent protein (GFP)–GnRH transgenic mice, we found expression of class 3 semaphorins and their receptors (neuropilin-1/2 and plexin-A1) in GnRH neurons. Furthermore, double-immunofluorescence experiments showed that migrating GnRH neurons, as well as associated olfactory fibers, express Npn-2 in the nasal region. We then used a line of immortalized GnRH neurons (GN11 cells) that display the same expression patterns for semaphorins and their receptors as GFP–GnRH cells and found that class 3 semaphorins and vascular endothelial growth factors modulate their migratory activity. These studies provide support for the direct involvement of neuropilins and their ligands in the establishment of the GnRH neuroendocrine system.

Background Human reproduction depends on an intact hypothalamic-pituitary-gonadal (HPG) axis. Hypothalamic gonadotrophin-releasing hormone (GnRH) has been recognized, since its identification in 1971, as the central regulator of the production and release of the pituitary gonadotrophins that, in turn, regulate the gonadal functions and the production of sex steroids. The characteristic peculiar development, distribution and episodic activity of GnRH-producing neurons have solicited an interdisciplinary interest on the etiopathogenesis of several reproductive diseases. The more recent identification of a GnRH/GnRH receptor (GnRHR) system in both the human endometrium and ovary has widened the spectrum of action of the peptide and of its analogues beyond its hypothalamic function. Methods An analysis of research and review articles published in international journals until June 2015 has been carried out to comprehensively summarize both the well established and the most recent knowledge on the physiopathology of the GnRH system in the central and peripheral control of female reproductive functions and diseases. Results This review focuses on the role of GnRH neurons in the control of the reproductive axis. New knowledge is accumulating on the genetic programme that drives GnRH neuron development to ameliorate the diagnosis and treatment of GnRH deficiency and consequent delayed or absent puberty. Moreover, a better understanding of the mechanisms controlling the episodic release of GnRH during the onset of puberty and the ovulatory cycle has enabled the pharmacological use of GnRH itself or its synthetic analogues (agonists and antagonists) to either stimulate or to block the gonadotrophin secretion and modulate the functions of the reproductive axis in several reproductive diseases and in assisted reproduction technology. Several inputs from other neuronal populations, as well as metabolic, somatic and age-related signals, may greatly affect the functions of the GnRH pulse generator during the female lifespan; their modulation may offer new possible strategies for diagnostic and therapeutic interventions. A GnRH/GnRHR system is also expressed in female reproductive tissues (e.g. endometrium and ovary), both in normal and pathological conditions. The expression of this system in the human endometrium and ovary supports its physiological regulatory role in the processes of trophoblast invasion of the maternal endometrium and embryo implantation as well as of follicular development and corpus luteum functions. The GnRH/GnRHR system that is expressed in diseased tissues of the female reproductive tract (both benign and malignant) is at present considered an effective molecular target for the development of novel therapeutic approaches for these pathologies. GnRH agonists are also considered as a promising therapeutic approach to counteract ovarian failure in young female patients undergoing chemotherapy. Conclusions Increasing knowledge about the regulation of GnRH pulsatile release, as well as the therapeutic use of its analogues, offers interesting new perspectives in the diagnosis, treatment and outcome of female reproductive disorders, including tumoral and iatrogenic diseases.

X-linked Kallmann's syndrome (KS) is a genetic disease characterized by anosmia and hypogonadism due to impairment in the development of olfactory axons and in the migration of gonadotropin-releasing hormone (GnRH)-producing neurons. Deletions or point mutations of a gene located at Xp22.3 (KAL1) are responsible for the disease. This gene encodes for a secreted heparin-binding protein (KAL or anosmin-1) which exhibits similarities with cell-adhesion molecules. In the present study, we show for the first time a direct action of anosmin-1 on the migratory activity of GnRH neurons. Specifically, we exposed immortalized migrating GnRH neurons (GN11 cells) to conditioned media (CM) of COS or CHO cells transiently transfected with human KAL1 gene in microchemotaxis and collagen gel assays. We found that anosmin-1-enriched media produced a cell-specific chemotactic response of GN11 cells. None of the CM enriched on three forms of anosmin-1 carrying different missense mutations (N267K, E514K and F517L) found in patients affected by X-linked KS affected the chemomigration of GN11 cells. Anosmin binds to the GN11 cell surface by interacting with the heparan sulphate proteoglycans, and the chemotactic effect of anosmin-1-enriched CM can be specifically blocked by heparin or by heparitinase pretreatment. These results strongly suggest an involvement of anosmin-1 in the control of the migratory behaviour of GnRH neurons and provide novel information on the pathogenesis of KS.

International audience; Individuals with an inherited deficiency in gonadotropin-releasing hormone (GnRH) have impaired sexual reproduction. Previous genetic linkage studies and sequencing of plausible gene candidates have identified mutations associated with inherited GnRH deficiency, but the small number of affected families and limited success in validating candidates have impeded genetic diagnoses for most patients. Using a combination of exome sequencing and computational modeling, we have identified a shared point mutation in semaphorin 3E (SEMA3E) in 2 brothers with Kallmann syndrome (KS), which causes inherited GnRH deficiency. Recombinant wild-type SEMA3E protected maturing GnRH neurons from cell death by triggering a plexin D1-dependent (PLXND1-dependent) activation of PI3K-mediated survival signaling. In contrast, recombinant SEMA3E carrying the KS-associated mutation did not protect GnRH neurons from death. In murine models, lack of either SEMA3E or PLXND1 increased apoptosis of GnRH neurons in the developing brain, reducing innervation of the adult median eminence by GnRH-positive neurites. GnRH neuron deficiency in male mice was accompanied by impaired testes growth, a characteristic feature of KS. Together, these results identify SEMA3E as an essential gene for GnRH neuron development, uncover a neurotrophic function for SEMA3E in the developing brain, and elucidate SEMA3E/PLXND1/PI3K signaling as a mechanism that prevents GnRH neuron deficiency.

Cajal–Retzius (CR) cells play a crucial role in the formation of the cerebral cortex, yet the molecules that control their development are largely unknown. Here, we show that Ebf transcription factors are expressed in forebrain signalling centres—the septum, cortical hem and the pallial–subpallial boundary—known to generate CR cells. We identified Ebf2, through fate mapping studies, as a novel marker for cortical hem- and septum-derived CR cells. Loss of Ebf2 in vivo causes a transient decrease in CR cell numbers on the cortical surface due to a migratory defect in the cortical hem, and is accompanied by upregulation of Ebf3 in this and other forebrain territories that produce CR cells, without affecting proper cortical lamination. Accordingly, using in vitro preparations, we demonstrated that both Ebf2 and Ebf3, singly or together, control the migration of CR cells arising in the cortical hem. These findings provide evidence that Ebfs directly regulate CR cell development., Highlights ► Ebf2 is a novel marker for hem- and septum-derived Cajal–Retzius cells. ► Ebf2 mutant shows a transient migratory defect of hem-derived Cajal–Retzius cells. ► Ebf3 is expressed in similar territories as Ebf2 and rescues Ebf2 mutant phenotype. ► Both Ebf2 and Ebf3 are required for Cajal–Retzius cell migration.

During development, many neurons display calcium-dependent migration, but the role of this messenger in regulating gene expression leading to this event has not yet been elucidated. Among the decoders of calcium signals is calcineurin, a Ca(2+)/calmodulin serine/threonine phosphatase that has been involved in both short-term and long-term cellular changes. By using immortalized GnRH-secreting neurons, we now show that, in vitro, Ca(2+)-dependent gene expression, proceeding via calcineurin and the transcription factor nuclear factor of activated T cells, is a key player controlling the chemomigratory potential of developing GnRH-secreting neurons. Furthermore, our data highlight the switch nature of this phosphatase, whose activation or inactivation guides cells to proceed from one genetic program to the next.

Leukemia inhibitory factor (LIF) is a pleiotropic cytokine of the IL-6 superfamily. LIF acts through a cell-surface receptor complex formed by two subunits, the specific LIF receptor beta (LIFRbeta) and the glycoprotein 130. Little is known about LIF involvement in modulating the neuroendocrine circuitry governing the reproductive function and, specifically, the development of GnRH-secreting neurons. In the present study, we evaluated the effect of LIF on the in vitro migration of GN11 cells, a model of immature and migratory GnRH neurons, and the signaling pathways involved in this process. GN11 cells expressed both LIFRbeta and glycoprotein 130 subunits. Exposure of GN11 cells to 100 ng/ml LIF resulted in activation of the Janus kinases (Jaks)/signal transducer and activator of transcription 3, MAPK/ERK1/2, and phosphatidylinositol 3-kinase/protein kinase B/Akt pathways. The selective inhibition of Jaks, MAPK kinase, and phosphatidylinositol 3-kinase indicated that these signaling pathways were activated independently by LIF and that Jak2 is not the main kinase involved in LIF signaling. Exposure of GN11 cells to LIF for 3 h induced a concentration-dependent chemotactic response, with a plateau at 100 ng/ml LIF. LIF was also found to induce chemokinesis of GN11 cells. Furthermore, LIF-promoted GN11 migration was the result of the partial and independent contribution of all the three signaling pathways activated by LIF. The present data, together with the observation that LIF and LIFRbeta are expressed prenatally in the mouse nasal compartment, would suggest that LIF might participate in the migration of GnRH neurons.

Infertility and inability to smell are the phenotypical features of Kallmann's syndrome (KS), a genetic disease which affects 1 in 10,000 males and 1 in 50,000 females, the majority of the cases being sporadic. The molecular pathogenesis of KS is complex but mainly referable to the impairment of olfactory axon development and of the migration of gonadotropin-releasing hormone (GnRH) neurons. Only two different genes have been identified so far as responsible for the disease: KAL1 and KAL2, encoding anosmin-1 and fibroblast growth factor receptor 1 (FGFR1), respectively. In this review we focus our attention on insights evoked by recent studies, which propose a new direct role for anosmin-1 in the migration GnRH neurons, and a fascinating hypothesis of interactions between anosmin-1 and FGFR1 systems.

Gonadotropin-releasing hormone (GnRH) neurons, a small number of cells scattered in the hypothalamic region of the basal forebrain, play an important role in reproductive function. These cells originate in the olfactory placode and migrate into the basal forebrain in late embryonic life. Here, we show that reelin, which is expressed along the route of the migrating cells, has an inhibitory role in guiding GnRH neurons to the basal forebrain. Only a small(approximately 5%) subpopulation of these neurons expresses one of the reelin receptors (ApoER2/Lrp8), and all GnRH neurons appear to lack the intracellular adaptor protein Dab1, suggesting that the function of reelin is not mediated by the conventional signal transduction pathway. The importance of reelin in the establishment of GnRH neurons in the hypothalamus was confirmed by our finding that the brains of developing and adult reeler mice of both sexes contained a markedly reduced number of these neuroendocrine neurons. Furthermore, the testes of adult males showed dilation of seminiferous tubules and reduction in their density when compared with controls. Mutants lacking the reelin receptors ApoER2 and Vldlr, and scrambler mice lacking Dab1, showed a normal complement of GnRH neurons in the hypothalamus,confirming that the effect of reelin in their migration is independent of Dab1.

Prenatal exposure of rodents to glucocorticoids (Gc) affects the sexual development of the offspring, possibly interfering with the differentiation of the hypothalamic-pituitary-gonadal axis. Glucocorticoid receptors (GR) are present on gonadotropin-releasing hormone (GnRH) neurons in the rat hypothalamus, suggesting a direct effect of Gc in the control of the synthesis and/or release of the hormone. In this study, we demonstrate the colocalization of immunoreactive GR with GnRH in a subpopulation of mouse hypothalamic GnRH neurons, confirming the possible involvement of Gc in mouse GnRH neuronal physiology. Receptor-binding assay, RT-PCR, immunocytochemistry, and immunoblotting experiments carried out in GN11 immortalized GnRH neurons show the presence of GR even in the more immature mouse GnRH neurons and confirm the expression of GR in GT1-7 mature GnRH cells. In GN11 cells, the activation of GR with dexamethasone produces nuclear translocation, but does not lead to the inhibition of GnRH gene expression already reported in GT1-7 cells. Long-term exposure of GN11 cells to dexamethasone induces an epithelial-like phenotype with a reorganization of F-actin in stress fibers. Finally, we found that Gc treatment significantly decreases the migratory activity in vitro and the levels of phosphorylated focal adhesion kinase of GN11 immature neurons. In conclusion, these data indicate that GR are expressed in mouse hypothalamic GnRH neurons in vivo as well as in the immature GN11 GnRH neurons in vitro. Moreover, the effects of the GR activation in GN11 and in GT1-7 cells may be related to the neuronal maturational stage of the two cell lines, suggesting a differential role of Gc in neuronal development.

In this report we studied and compared the biochemical and the electrophysiological characteristics of two cell lines (GT1-7 and GN11) of immortalized mouse LHRH-expressing neurons and the correlation with their maturational stage and migratory activity. In fact, previous results indicated that GN11, but not GT1-7, cells exhibit an elevated motility in vitro. The results show that the two cell lines differ in terms of immunoreactivity for tyrosine hydroxylase and nestin as well as of production and release of 3,4-dihydroxyphenylalanine (DOPA) and of intracellular distribution and release of the LHRH. Patch-clamp recordings in GN11 cells, reveal the presence of a single inward rectifier K+ current indicative of an immature neuronal phenotype (neither firing nor electrical activity). In contrast, as known from previous studies, GT1-7 cells show the characteristics of mature LHRH neurons with a high electrical activity characterized by spontaneous firing and excitatory postsynaptic potentials. K+-induced depolarization induces in GT1-7 cells, but not in GN11 cells, a strong increase in the release of LHRH in the culture medium. However, depolarization of GN11 cells significantly decreases their chemomigratory response. In conclusion, these results indicate that GT1-7 and GN11 cells show different biochemical and electrophysiological characteristics and are representative of mature and immature LHRH neurons, respectively. The early stage of maturation of GN11 cells, as well as the low electrical activity detected in these cells, appears to correlate with their migratory activity in vitro.

The molecular cues regulating the migratory process of LHRH neurons from the olfactory placode into the brain are not well known, but gradients of chemotropic and chemorepellent factors secreted by the targets are likely to play a key role in guidance mechanisms.Hepatocyte growth factor/scatter factor (HGF/SF) is a pleiotropic cytokine inducing cell migration. It is involved in a variety of developmental processes through interaction with its receptor c-Met. Here we show that c-Met-antibody labels LHRH migrating neurons in the olfactory mesenchyme of E12 mouse and analyze the potential chemotropic effect of HGF/SF on two immortalized LHRH cell lines, GT1-7 and GN11, isolated from tumors developed in the hypothalamus and in the olfactory bulb, respectively.By RT-PCR analysis, Western blotting, and immunocytochemistry, we provide evidence for a high level of c-Met expression in GN11, but not in GT1-7, cells. In addition, HGF/SF treatment promotes specific migratory activity of GN11 cells, as demonstrated by collagen gel assay, time-lapse video microscopy, and Boyden’s chamber experiments. Such promotion is inhibited by the neutralizing antibody.The data reported here represent the first direct evidence of a chemotactic effect of HGF/SF on immortalized LHRH neurons.