Development of the gonadotropin‐releasing hormone system

Abstract This review summarizes the current understanding of the development of the neuroendocrine gonadotropin‐releasing hormone (GnRH) system, including discussion on open questions regarding (1) transcriptional regulation of the Gnrh1 gene; (2) prenatal development of the GnRH1 system in rodents and humans; and (3) paracrine and synaptic communication during migration of the GnRH cells.


| INTRODUC TI ON
Sequencing of the gonadotropin-releasing hormone (GnRH) peptide 1,2 revolutionized GnRH research because antibodies were produced that allowed (1) the anatomical location of GnRH cells to be characterized in a variety of species; (2) secretion studies to be performed; and (3) electrophysiological properties of GnRH neurons to be examined, as verified by post-hoc immunodetection of recorded cells. The majority of these studies concentrated on the GnRH system postnatally as a function of reproductive status. However, between 1984 and 1991, [3][4][5][6][7][8][9] a series of landmark papers transformed our knowledge of the developmental, anatomical, and physiological features of the GnRH. These papers initiated a 'new' look at the neuroendocrine GnRH system and subsequently a wealth of information has been published, 10  This posed an intriguing question: where were the GnRH progenitors and how did the system develop? Thirty-two years ago, two groups independently presented work addressing this question. These studies demonstrated that the GnRH neuroendocrine cells in mouse arise outside the central nervous system (CNS), in the developing olfactory pit, and migrate into the forebrain during embryonic development [4][5][6] ( Figure 1B, C), and that disruption of GnRH migration in humans is associated with Kallmann syndrome. 7 Subsequent to these initial reports, the migration of GnRH cells from the bilateral developing olfactory pits has been documented in all vertebrates, 10 helping to explain the symmetry of the neuroendocrine GnRH cells around the midline brain axis ( Figure 1A), with the final location of (the majority of) neuroendocrine GnRH cells in each species related to guidance cues, as well as the brain development of that particular species.
In addition to disruption of GnRH neuron migration resulting in reproductive dysfunction, the association of GnRH progenitors with

| THE GNRH G ENE: S TRUC TURE AND E XPRE SS ION PAT TERN
The Gnrh (or Gnrh1) gene sequence was first characterized in 1984, encoding the entire 92-amino acid GnRH peptide precursor, prepro-GnRH. 3 This precursor contains a signal peptide located at the Nterminus, GnRH, a cleavage signal, and the GnRH-associated peptide (GAP) located at the C-terminus. Removing the signaling peptide from prepro-GnRH gives rise to the pro-GnRH peptide, which, after being processed by an endopeptidase, produces GnRH and GAP. The final maturation step for GnRH involves removing the basic amino acids at the C-terminus by a carboxypeptidase and conversion of the N-terminal glutamine by a glutaminyl cyclase, resulting in the bioactive GnRH decapeptide. 1,2 In the mouse, these events occur early in development, indicating that the factors needed for initiating GnRH gene expression and peptide processing of GnRH are present within the nasal compartment.
The Gnrh gene contains four exons and three introns. The processing mechanism resulting in Gnrh mRNA and its ultimate translation to the GnRH decapeptide is complex. Exonic splicing enhancers located in exon 3 and 4 of the Gnrh gene are required for splicing of exon 1 from its preceding intron. 11 In the absence of exons 3 and 4, as is the case in the hypogonadal hpg mouse, splicing of exon 1 from the preceding intron is unsuccessful, leading to defective translation and ultimately resulting in the lack of the GnRH peptide. 12,13 Few mutations in the GNRH gene itself, as a primary cause of reproductive dysfunction in human patients, have been found (see below).
Many of the studies on transcriptional regulation of Gnrh dis-   16 with olfactory pits (blue reaction, white arrows), eye (E) and telencephalon (T). Right: E11.5, immunostaining against GnRH detects immunopositive cells (brown, cells in boxed region) in the ventral portion of the anlage of the VNO. (C) Schematic of E12.5 mouse embryonic section through nasal region, olfactory epithelium (OE) and anlage of the VNO are visible. Middle: in situ hybridization histochemistry included in the original 1989 paper 5 showing cells expressing GnRH mRNA in the nasal region, at the nasal forebrain junction (asterisk) and within the forebrain (Tel Later, this group 16 clarified the role of VAX1 by deleting Vax1 specifically in GnRH neurons using an established GnRH-Cre mouse line. 17 The GnRH-Cre/Vax1 KO mouse model had a complete loss of GnRH neuronal immunostaining, which was associated with a hypogonadal, infertile state. 16 However, when crossing this KO mouse with a LacZ reporter strain, they observed that GnRH neurons were still present but not actively expressing Gnrh. To address how Vax1 regulates Gnrh expression, they used luciferase and electrophoretic mobility shift assays in GN11 and GT1-7 cells and determined that Vax1 (which was only highly expressed in GT1-7 cells) acts through the GnRH proximal and enhancer 1 regions to regulate Gnrh.

| TR ANSCRIP TIONAL DIREC TION OF G NRH NEURON -S PECIFI C E XPRE SS I ON
Notably the GnRH gene is also expressed in other reproductive tissues, such as the mammary gland, ovaries, and placenta. 18,19 Although the central function of GnRH neurons in the regulation of reproduction and fertility was established soon after its discovery, the mechanisms underlying the cell-specific expression of the GnRH gene and protein in neuroendocrine GnRH cells, as well as the role of GnRH in the extrahypothalamic tissues, remained a mystery. In addition, during development, GnRH is expressed transiently in other neuronal cell types. In 2002, Wolfe et al. 20 sought to investigate the cis-regulatory elements that specifically target GnRH transcription to hypothalamic neurons. By generating transgenic mice with a luciferase reporter construct bound to the human GnRH promoter (hGnRH), they identified two binding sites for a POU homeodomain F I G U R E 2 Key transcriptional sites and regulatory elements in the mouse and human Gnrh 5′-region. (A) The mouse gonadotropinreleasing hormone (GnRH) gene (mGnRH) contains a GnRH neuron-specific element (NSE) in the proximal promoter, which extends to −1005 bp. In addition, a GnRH neuronal enhancer region is present that also serves as an ovarian GnRH repressor region. The GnRH enhancer contains a kisspeptin-response element (KsRE) with an Otx2 binding site important for GnRH transcriptional activation. Otx2 binding sites are also present in the enhancer, outside of the KsRE, and in the proximal promoter. (B) The human GnRH gene (hGnRH) has a promoter region extending to −551 bp and contains an AP-1 site important for IGF-1 responsiveness. There is also a GnRH NSE upstream of the promoter that contains binding sites for Brn-2, a POU homeodomain factor. ERK, extracellular signal regulated kinase; PI3K, phosphoinositide 3-kinase; pTN, putative terminal nerve. Created with bioRENDER (biorender.com) transcription factor known as Brn-2 ( Figure 2B) as an essential transcriptional target for directing hypothalamic-specific GnRH expression. 20 Brn-2 is characterized by its binding to an octameric DNA sequence (ATGCAAAT), which is common among this family of POU transcription factors. Not only was Brn-2 mRNA determined to be expressed in GnRH neurons in mice, but also Brn-2 was shown to stimulate mouse GnRH (mGnRH) gene expression by transfecting GN11 cells with a Brn-2 expression vector. In another study, the same study group identified specific promoter regions that directed GnRH expression to either the hypothalamus or the ovary ( Figure 2A) using different GnRH promoter deletion fragments fused to a luciferase reporter. These experiments showed that the DNA sequence located −1005 bp from the mGnRH promoter was sufficient to direct both hypothalamic and ovarian GnRH expression. 21 Additionally, an enhancer region between −3446 and −2078 bp was identified that (1) specified neuronal GnRH expression and (2) contained an ovarian repressor element ( Figure 2A). To determine the physiological significance of this GnRH regulatory element, this neuron-specific enhancer region was further characterized by generating a mouse with a deletion of GnRH promoter fragment between −2806 and −2078 bp. In addition to decreased hypothalamic GnRH gene expression and increased ovarian GnRH immunohistochemical staining, this GnRH regulatory element KO mouse also exhibited delayed pubertal onset and abnormal estrous cyclicity. 22 Together, these studies advanced our understanding of the specific elements needed to direct GnRH expression to neurons in critical tissues (i.e., hypothalamus) and repress its expression in other tissues, which may negatively impact the function of the reproductive axis. Clearly, these are interesting regions of the GnRH promoter, for which further research is needed to identify additional proteins that might bind to these sequences, forming complexes that may differentially act on GnRH transcription.

| K ISS PEP TIN A S THE MA JOR NEUROPEP TIDE REG UL ATOR OF G NRH E XPRE SS I ON
The hypothalamic neuropeptide kisspeptin (Kiss1) is now considered to be the major secretagogue for GnRH and activates signaling cascades through its G-protein coupled receptor Kiss1R, which is highly expressed in GnRH neurons. In vitro studies using the Whether other GPCRs can act on Gnrh transcription in a manner similar to that outlined for the signaling through the Kiss1R (i.e., recruit Otx2 and mediate chromatin modifications) remains to be determined.

| G NRH TR AN SCRIP TI ON AND ME TABOLIC/G ROW TH FAC TOR S IG NAL S: INSULIN AND INSULIN -LIKE G ROW TH FAC TO R-1
The hypthalamic-gonadal axis is sensitive to environmental factors such as nutrition, with feedback from peripheral metabolic signals, such as insulin, affecting Gnrh gene expression. Both animal studies and cell culture models have been used to explore the regulatory role of insulin on the reproductive axis, and specifically on GnRH function. Thus, the obstacles presented by the divergence of cis-regulatory elements in the promoter regions of human and mouse GnRH can potentially be overcome by targeting similar signaling pathways such as MAPK signaling, as suggested previously. 30 How such regulatory elements might change in accordance with the species is an interesting question, the answer to which may help unravel unique reproductive strategies used by species to cope with environmental challenges. More recently, miR-375 was shown to indirectly regulate GnRH expression in GT1-7 cells by inhibiting Sp1, resulting in the activation of the GnRH repressor, Cebpb. 32 Another miRNA that was shown to be important in regulating pubertal onset is miR-29. This miRNA inhibits GnRH expression by targeting a transcriptional activator, Tbx21. In studies using GT1-7 cells, it was shown that blocking miR-29 increased GnRH gene expression. In mice, the loss of brain-specific miR-29 resulted in earlier puberty onset and increases in luteinizing hormone secretion. 33  lings with nHH has also been reported. These subjects were found to have an insertion of an adenine (c. 18-19insA) in the N-terminal region of the signaling sequence of the precursor peptide prepro-GnRH. This mutation results in the aberrant production of a peptide that lacks the functional GnRH decapeptide sequence. 35 In these studies, the affected patients had a parent and/or siblings with heterozygous mutations, but no nIHH presentation. However, one heterozygous variant discovered in the study by Chan et al.,34 p.R31C, was directly implicated in GnRH deficiency and predicted to be the primary cause of nIHH in that individual. 34 The R31C

| MUTATI ON S IN THE G NRH G ENE
variant is a missense mutation that changes an arginine in codon 31 to a cysteine in the conserved GnRH decapeptide sequence and results in a mutant GnRH peptide that has a much lower binding affinity for the GnRH receptor (GnRH-R). Subsequent to its discovery in 2009, the R31C variant has been found in other unrelated individuals with nIHH. 36 It is worth noting that the lack of GnRH itself, such as in the

| THE G NRH SYS TEM IN RODENTS AND HUMANS
To understand the beauty and challenges of the neuroendocrine

| Olfactory placode and GnRH ontogenesis
In mice, between embryonic days (E)10.5 and 11.5, the olfactory placode undergoes a first neurogenic wave that gives rise to several migratory neuronal populations, including the GnRH neurons. 40,41 Recent data indicate that the GnRH neurons originate from Achaete-

| The pathway
In mouse, at around E11.5 days of development, GnRH neurons can be immunodetected in the anlage of the prospective VNO. [4][5][6] From this area, the GnRH neurons migrate along TAG-1/ Cntn-2; peripherin; Robo3 positive axonal projections, towards the NFJ [50][51][52] ( Figure 4A-C). In human fetuses, peripherin ( Figure 4E) and Cntn-2/ TAG-1 ( Figure 4F-H) are also expressed by the putative vomeronasal/terminal axons, which form the scaffold for the GnRH migration. 46 In the past, as a result of the high density and often intermingled neuronal fibers in the developing nasal area and the broad immunoreactivity of peripheral nerves against peripherin and Cntn-2/TAG-1, it has been difficult to discriminate between axons of distinct neuronal populations present in the nasal area that GnRH cells associate with. Over the years, researchers proposed that the The terminal nerve, also known as cranial nerve XIII, nervus terminalis or cranial nerve zero "0", is an enigmatic ganglionic structure that projects to the chemosensory mucosa and into the brain, and in some animal species also to the retina. 53 The terminal nerve was first described in 1895 54 as a supernumerary nerve in sharks.
Subsequently, the terminal nerve has been described in multiple animal species, including cetaceans, which do not have an olfactory epithelium or olfactory bulbs, and humans, which do not have a functional VNO in adulthood. [55][56][57][58] In humans, the nervus terminalis has been defined as a microscopic plexus of unmyelinated peripheral nerve fascicles in the subarachnoid space. 59 Moreover, despite many genetic and phenotypical correlations, definitive experimental evidence demonstrating connectivity of the olfactory and/or vomeronasal sensory neurons to the brain is necessary for GnRH neuronal migration into the brain.
If the terminal nerve is distinct from olfactory sensory neurons, can we dissociate the terminal nerve and olfactory development?
Arx-1 is an X-linked homeobox gene and the null animals have defective proliferation and migration of olfactory bulb interneurons. Such developmental impairment translates into absence of protruding olfactory bulbs together with abnormal axonal termination of olfactory sensory neurons. 62 Characterization of the GnRH neuronal migration in Arx-1 deficient mice revealed, quite unexpectedly, that lack of connection of olfactory/vomeronasal sensory neurons to the forebrain, secondary to olfactory agenesis, does not prevent the GnRH neurons from invading the brain. Indeed, in Arx-1 null mutants, the GnRH neurons cross tangles of olfactory fibers and OECs forming the fibrocellular mass and invade the brain along the projections of the putative terminal nerve. In line with this, delayed, but successful, GnRH neuronal migration to the hypothalamus has been previously described in Gli3 pdn hypomorph mice, another mouse mutant with defective olfactory bulb development. 63 These data indicate that the Indeed, the terminal nerve and GnRH neurons invade the brain in areas highly enriched in guidance cues Sema3A and slit guidance ligand 1 (Slit1). Notably Sema3A-and Slit1-mediated repulsion prevent the olfactory and vomeronasal fibers from connecting to wrong parts of the olfactory bulb or invading the brain. 66 However, the terminal nerve does not appear to be repelled by these two guidance cues. 64 GnRH neurons invade the brain in areas rich of the repulsive cue Slit1 and fail to invade the brain in Sem3A null mutants, suggesting that Sema3A could function as an attractive, rather than repulsive, cue for the terminal nerve. 64

| Extra-hypothalamic GnRH cells
As noted above, GnRH cells had been documented in areas outside the 'continuum', such as the cortex and hippocampus in several species, but largely ignored. Recent work in mouse and humans using iDISCO (i.e., immunolabeling-enabled threedimensional imaging of solvent-cleared organs) has revitalized this issue, with the number and distribution of GnRH cells being larger and more diverse than previously assumed. Until recently, data in humans were relatively scarce. Identification of the GnRH population that migrates into the forebrain, as well as their location and function, in a variety of species is needed.

| EMB RYONI C PAR ACRINE AND SYNAP TIC COMMUNIC ATION DURING G NRH NEURONAL DE VELOPMENT ( FI G U RE 6)
In addition to adhesion and guidance molecules that are critical for

| ELEC TRI C AL AC TIVIT Y AND C ALCIUM CONTROL MIG R ATION
Electrical activity of neurons affects their migration directly by affecting cytoskeleton dynamics, or indirectly through modulating gene expression patterns in the cells. In both processes, calcium acts as the prime second messenger, which conveys the information encoded by neuron excitability to the cytoskeleton or to the nucleus. The direct pathway in which calcium affects neuronal motility has been well studied in many types of migrating neurons, 76 as well as in GnRH cells. 77,78 Membrane depolarization causes the opening of voltage-gated calcium channels and the ensuing intracellular calcium rise (from both extracellular sources as well as from intracellular stores) relays the information to the cytoskeleton of cells, thereby affecting their motility. The direct evidence that electrical activity leads to short-lived increases in cytosolic calcium has recently been confirmed in migrating fish GnRH neurons recorded in vivo. 79 In mammalian GnRH neurons, calcium release from intracellular stores was also shown to cause actin flow into the leading process of the migrating neurons, thereby stimulating their forward movement. 78 There is ample evidence for the role of electrical activity on GnRH neuron migration, although there are species differences about the nature of transmitters involved. In zebrafish, glutamatergic inputs through NMDA receptors were shown to be the main excitatory neurotransmitter for GnRH cells during development, 79 although GABAergic inputs were also reported. 80 In mammals, GABA, through its excitatory GABA A receptors, is considered to be the dominant neurotransmitter affecting GnRH migration, [81][82][83] although glutamate probably also plays a role in the control of migration. 84 The electrical activity and migration of GnRH neurons can also be modulated by other signaling molecules, such as cholecystokinin (CCK), anti-Mullerian hormone (AMH) and stromal derived growth factor-1. 81,[85][86][87] In addition to its direct role on cytoskeleton remodeling, elec- are not confined to anatomical boundaries and may produce different responses depending on the concentration that the GnRH cells are exposed to. In addition, the GnRH neurons migrate to the brain along the terminal nerve fibers, and their migration depends on correct olfactory ensheathing cells development. As a community, we should make an effort to elucidate a deeper understanding of the cellular and molecular mechanisms underlying formation and development of both the terminal nerve and olfactory ensheathing cells.
Clearly, to fully comprehend the migration of GnRH cells, we must put all the pieces in their appropriate sequence, and this is what makes this system both challenging and exciting.
With its multifactorial control of migration and the heterogeneity and isolation of its migration environment, the GnRH system provides a particularly attractive opportunity for studying neural migration. In addition to numerous extracellular cues, direct cell-cell interactions add a novel regulatory layer to the complex array of mechanisms that control GnRH neuronal development and migration. These discoveries assign a functional role for OECs that affect GnRH migration and reveal the importance of the dynamic interactions taking place between the neurons of the migratory cell mass. According to this emerging view, GnRH neurons not only follow guidance cues provided by the environment, but also form an inter-hemispheric circuit that serves to relay information and shapes the behavior of the individual units, thus affecting the assembly of the adult circuit.
The journey of the GnRH cells is long in distance and one that is continually expanding as the embryo develops (i.e., in mice, the first GnRH1 cells leave the nasal placode around E11 with cells continuing to migrate through the cribriform at E16.5, at which time the embryo has approximately tripled in size). During migration of the GnRH cells, the milieu through which GnRH cells migrate changes as well. Much is known about the neurogenesis GnRH cells in the forming placode, as well as the pathway, cell interactions, and signals/ cues that they use to cross the nasal region. New data confirms the importance of the NFJ region as a dynamic point for GnRH cell maturation, and indicates that, in mammals, non-neuroendocrine GnRH cells enter the brain, and populate diverse regions.
Once within the brain, independent of final location, the majority of neuroendocrine GnRH axons project to the median eminence where they access (via fenestrated capillaries in the median eminence) the pituitary portal capillary system. Little is known about the molecules used to guide GnRH axons (or other neuroendocrine axons for that matter) to the median eminence. Certainly, however, it is one of the crucial final steps in assuring a functional reproductive axis. Continued research on the development of GnRH neuron is a prerequisite to understanding GnRH neuronal populations postnally, as well as pathophysiologic conditions that disrupt reproductive function. Thus, unraveling the cues/processes underlying the development of the GnRH system, both neuroendocrine as well as non-neuroendocrine, will yield a wealth of information relevant to neuronal development of the brain and reproductive function.

ACK N OWLED G M ENTS
The research reported in this publication was supported by the