Neuropilin‐1 expression in GnRH neurons regulates prepubertal weight gain and sexual attraction
2020; Springer Nature; Volume: 39; Issue: 19 Linguagem: Inglês
10.15252/embj.2020104633
ISSN1460-2075
AutoresCharlotte Vanacker, Sara Trova, Sonal Shruti, Filippo Casoni, Andrea Messina, Sophie Croizier, Samuel A. Malone, Gaëtan Ternier, Naresh K. Hanchate, Sowmyalakshmí Rasika, Sébastien G. Bouret, Philippe Ciofi, Paolo Giacobini, Vincent Prévot,
Tópico(s)Hypothalamic control of reproductive hormones
ResumoArticle5 August 2020free access Transparent process Neuropilin-1 expression in GnRH neurons regulates prepubertal weight gain and sexual attraction Charlotte Vanacker Laboratory of Development and Plasticity of the Neuroendocrine Brain, Univ. Lille, Inserm, CHU Lille, Lille Neuroscience & Cognition, UMR-S 1172, Lille, France FHU, 1000 Days for Health, Lille, France Search for more papers by this author Sara Trova Laboratory of Development and Plasticity of the Neuroendocrine Brain, Univ. Lille, Inserm, CHU Lille, Lille Neuroscience & Cognition, UMR-S 1172, Lille, France FHU, 1000 Days for Health, Lille, France Search for more papers by this author Sonal Shruti Laboratory of Development and Plasticity of the Neuroendocrine Brain, Univ. Lille, Inserm, CHU Lille, Lille Neuroscience & Cognition, UMR-S 1172, Lille, France FHU, 1000 Days for Health, Lille, France Search for more papers by this author Filippo Casoni Laboratory of Development and Plasticity of the Neuroendocrine Brain, Univ. Lille, Inserm, CHU Lille, Lille Neuroscience & Cognition, UMR-S 1172, Lille, France FHU, 1000 Days for Health, Lille, France Search for more papers by this author Andrea Messina Laboratory of Development and Plasticity of the Neuroendocrine Brain, Univ. Lille, Inserm, CHU Lille, Lille Neuroscience & Cognition, UMR-S 1172, Lille, France FHU, 1000 Days for Health, Lille, France Search for more papers by this author Sophie Croizier Center for Integrative Genomics, University of Lausanne, Lausanne, Switzerland Search for more papers by this author Samuel Malone orcid.org/0000-0002-6824-6854 Laboratory of Development and Plasticity of the Neuroendocrine Brain, Univ. Lille, Inserm, CHU Lille, Lille Neuroscience & Cognition, UMR-S 1172, Lille, France FHU, 1000 Days for Health, Lille, France Search for more papers by this author Gaetan Ternier Laboratory of Development and Plasticity of the Neuroendocrine Brain, Univ. Lille, Inserm, CHU Lille, Lille Neuroscience & Cognition, UMR-S 1172, Lille, France FHU, 1000 Days for Health, Lille, France Search for more papers by this author Naresh Kumar Hanchate orcid.org/0000-0003-0849-3367 Laboratory of Development and Plasticity of the Neuroendocrine Brain, Univ. Lille, Inserm, CHU Lille, Lille Neuroscience & Cognition, UMR-S 1172, Lille, France FHU, 1000 Days for Health, Lille, France Search for more papers by this author S Rasika Laboratory of Development and Plasticity of the Neuroendocrine Brain, Univ. Lille, Inserm, CHU Lille, Lille Neuroscience & Cognition, UMR-S 1172, Lille, France FHU, 1000 Days for Health, Lille, France Search for more papers by this author Sebastien G Bouret Laboratory of Development and Plasticity of the Neuroendocrine Brain, Univ. Lille, Inserm, CHU Lille, Lille Neuroscience & Cognition, UMR-S 1172, Lille, France FHU, 1000 Days for Health, Lille, France Search for more papers by this author Philippe Ciofi Inserm U1215, Neurocentre Magendie, Bordeaux, France Université de Bordeaux, Bordeaux, France Search for more papers by this author Paolo Giacobini orcid.org/0000-0002-3075-1441 Laboratory of Development and Plasticity of the Neuroendocrine Brain, Univ. Lille, Inserm, CHU Lille, Lille Neuroscience & Cognition, UMR-S 1172, Lille, France FHU, 1000 Days for Health, Lille, France Search for more papers by this author Vincent Prevot Corresponding Author [email protected] orcid.org/0000-0001-7185-3615 Laboratory of Development and Plasticity of the Neuroendocrine Brain, Univ. Lille, Inserm, CHU Lille, Lille Neuroscience & Cognition, UMR-S 1172, Lille, France FHU, 1000 Days for Health, Lille, France Search for more papers by this author Charlotte Vanacker Laboratory of Development and Plasticity of the Neuroendocrine Brain, Univ. Lille, Inserm, CHU Lille, Lille Neuroscience & Cognition, UMR-S 1172, Lille, France FHU, 1000 Days for Health, Lille, France Search for more papers by this author Sara Trova Laboratory of Development and Plasticity of the Neuroendocrine Brain, Univ. Lille, Inserm, CHU Lille, Lille Neuroscience & Cognition, UMR-S 1172, Lille, France FHU, 1000 Days for Health, Lille, France Search for more papers by this author Sonal Shruti Laboratory of Development and Plasticity of the Neuroendocrine Brain, Univ. Lille, Inserm, CHU Lille, Lille Neuroscience & Cognition, UMR-S 1172, Lille, France FHU, 1000 Days for Health, Lille, France Search for more papers by this author Filippo Casoni Laboratory of Development and Plasticity of the Neuroendocrine Brain, Univ. Lille, Inserm, CHU Lille, Lille Neuroscience & Cognition, UMR-S 1172, Lille, France FHU, 1000 Days for Health, Lille, France Search for more papers by this author Andrea Messina Laboratory of Development and Plasticity of the Neuroendocrine Brain, Univ. Lille, Inserm, CHU Lille, Lille Neuroscience & Cognition, UMR-S 1172, Lille, France FHU, 1000 Days for Health, Lille, France Search for more papers by this author Sophie Croizier Center for Integrative Genomics, University of Lausanne, Lausanne, Switzerland Search for more papers by this author Samuel Malone orcid.org/0000-0002-6824-6854 Laboratory of Development and Plasticity of the Neuroendocrine Brain, Univ. Lille, Inserm, CHU Lille, Lille Neuroscience & Cognition, UMR-S 1172, Lille, France FHU, 1000 Days for Health, Lille, France Search for more papers by this author Gaetan Ternier Laboratory of Development and Plasticity of the Neuroendocrine Brain, Univ. Lille, Inserm, CHU Lille, Lille Neuroscience & Cognition, UMR-S 1172, Lille, France FHU, 1000 Days for Health, Lille, France Search for more papers by this author Naresh Kumar Hanchate orcid.org/0000-0003-0849-3367 Laboratory of Development and Plasticity of the Neuroendocrine Brain, Univ. Lille, Inserm, CHU Lille, Lille Neuroscience & Cognition, UMR-S 1172, Lille, France FHU, 1000 Days for Health, Lille, France Search for more papers by this author S Rasika Laboratory of Development and Plasticity of the Neuroendocrine Brain, Univ. Lille, Inserm, CHU Lille, Lille Neuroscience & Cognition, UMR-S 1172, Lille, France FHU, 1000 Days for Health, Lille, France Search for more papers by this author Sebastien G Bouret Laboratory of Development and Plasticity of the Neuroendocrine Brain, Univ. Lille, Inserm, CHU Lille, Lille Neuroscience & Cognition, UMR-S 1172, Lille, France FHU, 1000 Days for Health, Lille, France Search for more papers by this author Philippe Ciofi Inserm U1215, Neurocentre Magendie, Bordeaux, France Université de Bordeaux, Bordeaux, France Search for more papers by this author Paolo Giacobini orcid.org/0000-0002-3075-1441 Laboratory of Development and Plasticity of the Neuroendocrine Brain, Univ. Lille, Inserm, CHU Lille, Lille Neuroscience & Cognition, UMR-S 1172, Lille, France FHU, 1000 Days for Health, Lille, France Search for more papers by this author Vincent Prevot Corresponding Author [email protected] orcid.org/0000-0001-7185-3615 Laboratory of Development and Plasticity of the Neuroendocrine Brain, Univ. Lille, Inserm, CHU Lille, Lille Neuroscience & Cognition, UMR-S 1172, Lille, France FHU, 1000 Days for Health, Lille, France Search for more papers by this author Author Information Charlotte Vanacker1,2, Sara Trova1,2, Sonal Shruti1,2, Filippo Casoni1,2, Andrea Messina1,2, Sophie Croizier3, Samuel Malone1,2, Gaetan Ternier1,2, Naresh Kumar Hanchate1,2, S Rasika1,2, Sebastien G Bouret1,2, Philippe Ciofi4,5, Paolo Giacobini1,2,‡ and Vincent Prevot *,1,2,‡ 1Laboratory of Development and Plasticity of the Neuroendocrine Brain, Univ. Lille, Inserm, CHU Lille, Lille Neuroscience & Cognition, UMR-S 1172, Lille, France 2FHU, 1000 Days for Health, Lille, France 3Center for Integrative Genomics, University of Lausanne, Lausanne, Switzerland 4Inserm U1215, Neurocentre Magendie, Bordeaux, France 5Université de Bordeaux, Bordeaux, France ‡These authors contributed equally to this work *Corresponding author. Tel: +33 320 62 20 64; Fax: +33 320 53 85 62; E-mail: [email protected] EMBO J (2020)39:e104633https://doi.org/10.15252/embj.2020104633 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Hypothalamic neurons expressing gonadotropin-releasing hormone (GnRH), the "master molecule" regulating reproduction and fertility, migrate from their birthplace in the nose to their destination using a system of guidance cues, which include the semaphorins and their receptors, the neuropilins and plexins, among others. Here, we show that selectively deleting neuropilin-1 in new GnRH neurons enhances their survival and migration, resulting in excess neurons in the hypothalamus and in their unusual accumulation in the accessory olfactory bulb, as well as an acceleration of mature patterns of activity. In female mice, these alterations result in early prepubertal weight gain, premature attraction to male odors, and precocious puberty. Our findings suggest that rather than being influenced by peripheral energy state, GnRH neurons themselves, through neuropilin–semaphorin signaling, might engineer the timing of puberty by regulating peripheral adiposity and behavioral switches, thus acting as a bridge between the reproductive and metabolic axes. Synopsis By regulating GnRH neuronal survival, migration and activity, the expression of Neuropilin 1 in GnRH neurons regulates sexual attraction, prepubertal weight gain and the timing of puberty. Nrp1 expression in GnRH neurons controls their number and distribution during embryogenesis. The lack of Nrp1 expression in GnRH neurons triggers early sexual behavior. GnRH neurons are directly involved in prepubertal weight gain. Disrupting Nrp1 signaling in GnRH neurons leads to central precocious puberty. Introduction Two of the major imperatives of living beings are the maintenance of energy homeostasis and the transmission of genetic material, i.e. the production of young in animals with sexual reproduction. In mammals, both processes are controlled at the level of the brain, albeit by different hypothalamic circuits. Fertility, under the control of the hypothalamic–pituitary–gonadal (HPG) axis, is orchestrated by a small population of neuroendocrine neurons producing gonadotropin-releasing hormone (GnRH). The release of GnRH into the hypothalamic–pituitary portal blood circulation drives gonadotropin secretion by the pituitary. The gonadotropins, luteinizing hormone (LH), and follicle stimulating hormone (FSH), in turn, act on the gonads to regulate sex steroid synthesis, gametogenesis, and the onset of puberty in both sexes (Boehm et al, 2015; Prevot, 2015; Howard & Dunkel, 2019). While puberty consists of the permanent activation of the HPG axis and the commencement of adult reproductive function, the process is controlled by a complex array of genetic and environmental determinants, among which the acceleration of growth during the prepubertal period is thought to be a key permissive factor (Parent et al, 2003; Abreu & Kaiser, 2016; Howard & Dunkel, 2019). In patients with hypogonadotropic hypogonadism, a deficit in GnRH neuronal migration and function leads to an absence of puberty onset (Boehm et al, 2015). However, at the other end of the pathophysiological spectrum, little is known regarding the cellular and molecular mechanisms underlying precocious puberty. GnRH-secreting neurons originate from both the nasal placode and the neural crest during embryonic development and migrate to the forebrain and hypothalamus along olfactory/terminal nerves (Schwanzel-Fukuda & Pfaff, 1989; Wray et al, 1989; Taroc et al, 2017). The complex developmental events leading to correct GnRH neuronal migration and secretion are tightly regulated by the specific spatiotemporal expression patterns of growth factors, adhesion molecules, and diffusible guidance cues that are either attractive or repulsive (Giacobini, 2015). The semaphorins constitute one of the largest families of phylogenetically conserved guidance cues, known to regulate multiple processes crucial for neuronal network formation (Tamagnone & Comoglio, 2004; Van Battum et al, 2015). The repulsive guidance cue semaphorin 3A (Sema3A) and its receptor, neuropilin-1 (Nrp1), are expressed in a complementary manner in olfactory sensory neurons and are involved in the spatial encoding of sensory information in the olfactory bulb (Imai et al, 2009). Olfactory axons expressing Nrp1 also form the scaffold along which GnRH neurons migrate into the brain (Cariboni et al, 2011b; Hanchate et al, 2012). In keeping with their sites of expression, recent studies by us and others have shown that, in humans, mutations in the Sema3A:Nrp1 signaling pathway lead to dysfunctions in olfaction and fertility (Hanchate et al, 2012; Marcos et al, 2017). Here, we studied the role of Nrp1 expression in GnRH neurons in mice. We demonstrate that Nrp1 expression in GnRH neurons is required to control the size of the GnRH neuronal population generated during embryogenesis and their migration to their proper destinations in the brain in the correct proportions. We also show that the knockout of Nrp1 specifically in GnRH neurons results in the accumulation of an abnormally high number of neurons in the accessory olfactory bulb (AOB) and the forebrain/hypothalamus, early attraction to odors of the opposite sex, precocious puberty onset, and, surprisingly, overweight, suggesting an involvement of the GnRH system in energy metabolism. Our data raise the intriguing possibility that, in patients with central precocious puberty, rather than body weight influencing the timing of puberty, GnRH neurons may themselves play a pivotal role in coordinating both peripubertal weight gain and the activation of the HPG axis, in addition to early sexual behavior. Results Selective deletion of Nrp1 expression in GnRH cells in mice in vivo To investigate the role of Nrp1 receptor signaling in GnRH neurons in the mouse, we specifically knocked out Nrp1 expression in GnRH neurons, by crossing Nrp1loxP/loxP mice (Gu et al, 2003) with a Gnrh::cre line (Yoon et al, 2005) expressing the Cre recombinase under the control of the Gnrh promoter. The resulting Gnrh::cre; Nrp1loxP/loxP (hereafter termed "mutant") and Nrp1loxP/loxP (hereafter termed "control") mice were viable and born at Mendelian frequencies. Triple transgenic mice were also produced by crossing them with Gnrh::gfp mice (Spergel et al, 1999), which express green fluorescent protein (GFP) under the control of the Gnrh promoter (Fig 1A), to visualize and isolate these neurons. We first investigated the expression of Nrp1 and the plexins, preferential co-receptors of Nrp1, and the signal-transducing subunits in Sema3A signaling (Takahashi et al, 1999), in GFP-identified GnRH neurons isolated by fluorescence-activated cell sorting (FACS) from the preoptic region of mutant and control mice at P0 (Fig 1B and C). FACS-sorted GFP-positive cells were 1,000–4,000 times enriched in the Gnrh transcript as compared to GFP-negative cells (Mann–Whitney U-test, U = 0, P = 0.03, n = 4; Fig 1C). Real-time quantitative PCR analyses showed a 74.7% decrease in Nrp1 mRNA levels in GnRH neurons from Gnrh::gfp mutant mice as compared to Gnrh::gfp control mice (unpaired t-test, t(9) = 3.851, P = 0.004, n = 5–6), but no significant change in Nrp2 (Mann–Whitney U-test, U = 6, P = 0.13, n = 5–6; Fig 1D). This was associated with a 66.7% decrease in plexin-A1 mRNA levels (unpaired t-test, t(9) = 3.09, P = 0.01, n = 5–6), but no change in plexin-A4 expression (unpaired t-test, t(9) = 0.40, P = 0.70, n = 5–6) (Fig 1D). Nrp1 protein expression was also investigated during embryonic development. GnRH-immunoreactive neurons in control embryos expressed Nrp1 protein (red immunolabeling) by embryonic day 14.5 (E14.5) (Fig 1E). In contrast, littermates with a GnRH neuron-specific Nrp1 knockout did not show any detectable Nrp1 immunolabeling in GnRH neurons, but displayed Nrp1 in GnRH-negative fibers, such as the vomeronasal/terminal nerves that support GnRH neurons in their migration (Fig 1E). These observations, together with the fact that in the specific Gnrh::cre mouse model we used, the expression of the transgene is very limited in ectopic neuronal populations during embryogenesis (Hoffmann et al, 2019), confirm the genetic deletion of Nrp1 in GnRH neurons in mutant mice. In addition, Nrp1 expression remains intact in the non-GnRH hypophysiotropic systems of mutant mice (Giacobini et al, 2014), indicating that any neuroendocrine actions of Nrp1 deletion are mediated by GnRH neurons exclusively. Figure 1. Neuropilin-1 expression is selectively suppressed in GnRH neurons in Gnrh::cre; Nrp1loxP/loxP mice in vivo Genetic strategy to selectively knock out Nrp1 in GnRH-expressing cells, using Nrp1loxP/loxP mice crossed with Gnrh::cre mice. These mice were also crossed with Gnrh::gfp mice expressing green fluorescent protein under the control of the GnRH promoter in order to generate triple transgenic mice. Isolation of GFP-positive cells by FACS: schematic diagram showing the selection of the GFP-positive population (green). Relative mRNA expression from real-time PCR analysis of the GnRH transcript in GFP-positive cells, in comparison with GFP-negative cells. The GnRH transcript appears to be selectively expressed in GFP-positive cells. Mann–Whitney U-test, n = 4 mice from 2 litters. Relative mRNA expression from real-time PCR analysis of neuropilin-1 (Nrp1), Nrp2, plexin-A1 (PlxnA1), and PlxnA4 transcripts in FACS-isolated GFP-positive GnRH neurons from control (Nrp1loxP/loxP; gray) and mutant (Gnrh::cre; Nrp1loxP/loxP; red) P0 mice. Unpaired t-test, n = 5–6 mice per group from 3 litters. Representative immunofluorescence images showing GnRH neurons migrating along Nrp1-immunoreactive vomeronasal/terminal nerve fibers (empty arrowheads) at the nose–brain junction in sagittal slices from E14.5 control and mutant embryos. GnRH neurons (green) themselves express Nrp1 (red, white arrows) in control Nrp1loxP/loxP mice, whereas Nrp1 is undetectable in GnRH neurons from mutant Gnrh::cre; Nrp1loxP/loxP littermates (black arrows). Scale bar: 50 μm. Data information: Bar graphs show individual values and means ± SEM. **P < 0.01, *P < 0.05. Download figure Download PowerPoint Selective deletion of Nrp1 in GnRH neurons alters their number and distribution To investigate the role of Nrp1 expression in GnRH neuron ontogenesis, we used GnRH immunofluorescence to characterize GnRH cell distribution at key stages during embryonic development. In contrast to our previous observations in mutant mice with Nrp1 deletion throughout the body (Hanchate et al, 2012), no alteration of the olfactory sensory or terminal tracts was detected in mutant embryos at any age. GnRH neurons were quantified along the migratory path from the nose to brain at three stages: E12.5, when the majority of GnRH neurons, which become detectable in the vomeronasal epithelium at E11.5, are still located in the nose; E14.5, when around half the GnRH neurons have reached the brain whereas the other half is still located in the nose; and E18.5, when most GnRH neurons have completed their migration and reached their final destination in the ventral forebrain (VFB) (Schwanzel-Fukuda & Pfaff, 1989; Wray et al, 1989). While the total number of GnRH neurons was equivalent in mutant and control embryos at E12.5 (two-way ANOVA, developmental period, F(2,22) = 13.31, P = 0.0002; genotype, F(1,22) = 5.63, P = 0.03; interaction, F(2,22) = 2.18, P = 0.14; Fisher's LSD multiple-comparison test, 1078 ± 201.6 neurons in controls versus 1020 ± 96.72 neurons in mutants, t(22) = 0.36, P = 0.72, n = 4 per group), it was increased by 25.3% in mutant embryos at E14.5 (control: 1353 ± 70 neurons; mutant: 1695 ± 62 neurons, t(22) = 2.51, P = 0.02, n = 5–6) and by 23.8% at E18.5 (control: 1,386 ± 81.35 neurons; mutant: 1,716 ± 103 neurons, t(22) = 2.18, P = 0.04, n = 4–5) (Fig 2A). At E14.5, while the total number of neurons in the nose did not change (two-way ANOVA, anatomical region, F(1,9) = 309.3, P < 0.001; genotype, F(1,9) = 12.7, P = 0.006; interaction, F(1,9) = 7.5, P = 0.02; Fisher's LSD multiple-comparison test, control: 325 ± 36 versus mutant: 368 ± 45, t(18) = 0.63; P = 0.78, n = 6 and 5), the number of neurons reaching the brain markedly increased in mutant embryos at the same developmental stage (control: 1,027 ± 53 versus mutant: 1,327 ± 56, t(18) = 4.46; P < 0.001, n = 6 and 5). These excess cells in the brain were located in the ventral forebrain where their number was found to be significantly increased by 41% (431 ± 50 neurons in controls versus 608.80 ± 69.24 neurons in mutants, Fisher's LSD multiple-comparison test, VFB t(27) = 2.70, P = 0.01; OB; t(27) = 1.87; P = 0.07; two-way ANOVA, anatomical region, F(2,27) = 22.4, P < 0.0001; genotype, F(1,27) = 9.07, P = 0.006; interaction, F(2,27) = 1.07, P = 0.36, n = 6 and 5) (Fig 2B and C), suggesting that a higher proportion of cells had already migrated further toward their destinations at this embryonic stage in mutants than in control littermates. Figure 2. Mice lacking neuropilin-1 expression in GnRH neurons show an increased number of migrating GnRH cells and abnormal GnRH neuronal migration in the accessory olfactory bulb Quantification of total number of GnRH neurons during embryogenesis, at E12.5, E14.5, and E18.5. More GnRH neurons were found in E14.5 and E18.5 mutant embryos. Two-way ANOVA, Fisher's LSD multiple-comparison test, n = 4–6 mouse embryos from at least 3 litters. Schematic representation of the areas containing GnRH migrating cells during embryogenesis at E14.5 and representative immunofluorescence showing migrating GnRH neurons at the level of the nose, the olfactory bulb (OB), and the ventral forebrain (VFB) in control and mutant embryos. Scale bar: 50 μm. VNO, vomeronasal organ; OE, olfactory epithelium; OB, olfactory bulbs; VFB, ventral forebrain. Quantitative analysis showing distribution of GnRH neurons in the nose, OB, and VFB of mutant and control embryos. Two-way ANOVA, Fisher's LSD multiple-comparison test, n = 5 and 6 mouse embryos for mutants and controls, respectively, from at least 3 litters. Representative image of cleared brains and immunolabeling for GnRH at the level of the olfactory bulbs, in a control (left panel) and a mutant (right panel) postnatal brain. Knockout animals show an accumulation of GnRH neurons in the accessory olfactory bulb (circle formed by the GnRH labeling, right panel). AOB, accessory olfactory bulb. Scale bar: 300 μm. Representative image of cleared brains and immunolabeling for GnRH at the level of the organum vasculosum of the lamina terminalis (OVLT) in the median preoptic area, from a control (left) and a mutant (right) female postnatal brain. Scale bar: 500 μm. Oc, optic chiasma. Quantitative analysis of the distribution of GnRH neurons in cleared brains of mutant and control littermates. OB, olfactory bulb; DBB, diagonal band of Broca; 3V, periventricular region of the preoptic area; Tot, total. Two-way ANOVA, Fisher's LSD multiple-comparison test, n = 3 adult mice from 3 litters. Data information: Bar graphs show individual values and means ± SEM. *P < 0.05, ***P < 0.001. Download figure Download PowerPoint We next investigated the distribution of GnRH neurons in the postnatal brain of mutant mice and their control littermates. We used an organic solvent-based clearing methods adapted from iDISCO (Casoni et al, 2016; Belle et al, 2017) coupled with GnRH immunohistofluorescence on brain sections from adult mice to visualize the distribution of GnRH neurons in the brain. Remarkably, while GnRH neurons with abundant fibers were typically localized in the medial and basal parts of the glomerular layer of the olfactory bulbs in controls (Casoni et al, 2016), GnRH neurons in mutant mice clearly accumulated in the accessory olfactory bulb (AOB) (Fig 2D, Movies EV1 and EV2). In parallel with this abnormally high migration of GnRH neurons into the AOB, the number of GnRH neurons that had reached the forebrain/hypothalamus was higher by > 18% in mutant mice, as shown by cell counts both in 3D rendering (Fig 2E and F) and in conventional immunofluorescence (control both sex: 404.10 ± 13.35 neurons, n = 13; mutant both sex: 472.3 ± 23.81 neurons, n = 13, unpaired t-test t(24) = 2.50, P = 0.02) (Fig EV1A). This increase in the GnRH neuronal population was largely restricted to the organum vasculosum of the lamina terminalis (transparentized brain: two-way ANOVA, brain region, F(3,16) = 49.01, P < 0.0001; genotype, F(1,16) = 21.79, P = 0.0003; interaction, F(3,16) = 4.02, P = 0.03; Fisher's LSD multiple-comparison test, OVLT t(16) = 4.03, P = 0.001, n = 3 per group, Fig 2E; conventional immunofluorescence: two-way ANOVA, brain region, F(2,72) = 229.7, P < 0.0001; genotype, F(1,72) = 5.54, P = 0.02; interaction, F(2,72) = 3.15 P = 0.05; Fisher's LSD multiple-comparison test, OVLT, control: 261.4 ± 16.26 neurons; mutant: 316.9 ± 20.89 neurons, t(72) = 3.317, P = 0.001, n = 13 per group, 9 females and 4 males per group, Fig EV1A and B). Similar differences were observed when data from female brains were analyzed alone (two-way ANOVA, brain region, F(2,48) = 158.9, P < 0.0001; genotype, F(1,48) = 4.04 P = 0.05; interaction, F(2,48) = 2.26, P = 0.12; OVLT, control: 263.1 ± 18.87 neurons; mutant: 321.3 ± 26.55 neurons, t(48) = 2.86, P = 0.006, n = 9 per group). The distribution of GnRH neurons in the forebrain/hypothalamus of adult males (n = 4) did not appear to differ markedly from that in females (Fig EV1A). Together, these results show that alterations of GnRH neuronal number and migration during embryonic development in mutant mice have clear repercussions on their distribution in the postnatal brain. Click here to expand this figure. Figure EV1. Migration of supernumerary GnRH neurons in adulthood in the hypothalamus of mice lacking neuropilin-1 expression in GnRH neurons Total number of GnRH neurons in the forebrain and the hypothalamus (Hypoth) and their regional distribution (DBB: diagonal band of Broca; OVLT: organum vasculosum of the lamina terminalis; 3V: periventricular area of the median preoptic) in Nrp1loxP/loxP and Gnrh::cre; Nrp1loxP/loxP littermates using conventional immunofluorescence. Individual males and females used for this analysis are represented by blue and gray/light-red dots, respectively. Two-way ANOVA, Fisher's LSD multiple-comparison test, n = 13 (nine females and four males). Representative immunohistofluorescence for GnRH (green) and Hoechst staining (blue) of sections showing the OVLT region (area framed in the schematic) from control (left) and mutant (right) adult mice. More GnRH cells are observed in OVLT sections of mutant mice (white arrow). Ac, anterior commissure; cc, corpus callosum; Scale bar: 100 μm. Data information: Bar graphs show individual values and means ± SEM. *P < 0.05, **P < 0.01. Download figure Download PowerPoint Knocking out Nrp1 in GnRH neurons increases their migration and survival To understand the origin of these supernumerary GnRH neurons in mutant embryos and adult mice, we first took advantage of immortalized GnRH neurons (the GN11 line) (Radovick et al, 1991), which possess the migratory properties of GnRH neurons (Maggi et al, 2000), in addition to expressing both GnRH and Nrp1 (Cariboni et al, 2007). Semaphorin 3A (Sema3A) is a secreted guidance molecule with known Nrp1-mediated chemorepellent properties, which plays important roles in shaping neural circuits during mammalian embryonic development (Pasterkamp, 2012). In keeping with a chemorepellent action of Sema3A, Transwell assays demonstrated that 100 ng/ml or 250 ng/ml of Sema3A, but not 50 ng/ml, inhibited cell migration by ~50% (Kruskal–Wallis test, SFM versus Sema3A 100 ng/ml, Z = 2.96, P = 0.01, n = 5 and 6; SFM versus Sema3A 250 ng/ml, Z = 2.62, P = 0.035, n = 5 and 6) (Fig 3A and B), an effect that was abolished by pretreatment with neutralizing antibodies to Nrp1 (1/200; SFM versus Sema3A 100 ng/ml + Nrp1Ab, Z = 0.24, P > 0.999, n = 5 and 6 and Sema3A 100 ng/ml versus Sema3A 100 ng/ml + Nrp1Ab, Z = 2.85, P = 0.02, n = 6 per group) (Fig 3A and B). In parallel, two-well removable inserts were used to assess the motility of GN11 cells in response to Sema3A in the presence or absence of Nrp1-neutralizing antibodies (Nrp1Ab) in serum-free culture medium (SFM). Six hours after removing the insert, the area invaded by GN11 cells was significantly lower in the presence of Sema3A at 100 ng/ml (SFM 12.28 ± 0.75 pixels versus Sema3A 100 9.14 ± 0.64 pixels, n = 5 per group; two-way ANOVA, treatment, F(4,66) = 2.78, P = 0.034; time, F(2,66) = 338.60, P < 0.0001; interaction, F(8,66) = 1.49, P = 0.18, Fisher's LSD multiple-comparison test, t(66) = 3.33, P = 0.001) and at 250 ng/ml (9.18 ± 1.23 pixels, n = 6, SFM versus Sema3A 250, t(66) = 3.43, P = 0.001). This effect was abolished by the addition of Nrp1Ab (1/200) to the medium (10.91 ± 0.53 pixels, n = 6, Sema3A 100 ng/ml versus Sema3A 100 ng/ml+Nrp1Ab, t(66) = 1.96, P = 0.05). These in vitro results suggest that Nrp1, acting as the receptor for guidance cues, regulates GnRH cell migration. Figure 3. Neuropilin-1 signaling in GnRH neurons controls their survival and migration Schematic representation of the Transwell assay used to assess the effect of Sema3A, which was placed in the lower chamber, on GN11 cell migration and representative microphotograph of cell nuclei stained with Hoechst (gray) in the lower part of the Transwell membrane after 1
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