Andrea Messina

Fanny Langlet

Konstantina Chachlaki

Juan Roa

S Rasika

Nathalie Jouy

Sarah Gallet

Jyoti Parkash

Manuel Tena-Sempere

Paolo Giacobini

Ph.D Vincent Prevot
email: vincent.prevot@inserm.fr

MicroRNAs flip the switch for the production of hypothalamic GnRH before puberty

A sparse population of a few hundred primarily hypothalamic neurons forms the hub of a complex neuroglial network that controls reproduction in mammals by secreting the "master molecule", gonadotropin-releasing hormone (GnRH). Timely postnatal changes in GnRH expression are essential for puberty and adult fertility. Here, we report that a multilayered microRNA-operated switch with inbuilt feedback controls governs increased GnRH expression during the infantile-juvenile transition, and that impairing microRNA synthesis in GnRH neurons leads to hypogonadotropic hypogonadism and infertility in mice. Two essential components of this switch, miR-200 and miR-155, respectively regulate Zeb1, a repressor of GnRH transcriptional activators and GnRH itself, and Cebpb, an NO-mediated repressor of GnRH that acts both directly and through Zeb1, in GnRH neurons. This alteration in the delicate balance between inductive and repressive signals induces the normal GnRH-fuelled run-up to correct puberty initiation, and interfering with this process disrupts the neuroendocrine control of reproduction.

Introduction

The onset of puberty and the regulation of fertility in mammals are governed by a complex neural network, primarily in the hypothalamus, that converges onto gonadotropin-releasing hormone (GnRH)-producing neurons, the master regulators of gonadotropin secretion and postnatal gonadal growth and function 1 . The proper development of the GnRH system [START_REF] Schwanzel-Fukuda | Luteinizing hormone-releasing hormone (LHRH)-expressing cells do not migrate normally in an inherited hypogonadal (Kallmann) syndrome[END_REF] , including timely changes in GnRH expression and signaling by this sparse population of a few hundred neurons [START_REF] Mason | A deletion truncating the gonadotropin-releasing hormone gene is responsible for hypogonadism in the hpg mouse[END_REF][START_REF] De Roux | A family with hypogonadotropic hypogonadism and mutations in the gonadotropin-releasing hormone receptor[END_REF] , is essential for sexual maturation and the normal functioning of the hypothalamic-pituitary-gonadal axis. However, despite the identification of several factors influencing GnRH production and release [START_REF] De Roux | Hypogonadotropic hypogonadism due to loss of function of the KiSS1derived peptide receptor GPR54[END_REF][START_REF] Ma | Transforming growth factor-alpha gene expression in the hypothalamus is developmentally regulated and linked to sexual maturation[END_REF][START_REF] Prevot | Normal female sexual development requires neuregulin-erbB receptor signaling in hypothalamic astrocytes[END_REF][START_REF] Seminara | The GPR54 gene as a regulator of puberty[END_REF][START_REF] Urbanski | A role for N-methyl-D-aspartate (NMDA) receptors in the control of LH secretion and initiation of female puberty[END_REF] , the mechanisms regulating the clocklike precision of the pubertal activation process remain mysterious.

MicroRNAs (miRNAs) are short (~22-nucleotide) noncoding RNA molecules that silence gene expression posttranscriptionally, principally by binding to the 3' untranslated regions (3'UTR) of target mRNAs (Fig. S1). Mature miRNAs have been shown to be required for the normal differentiation and function of several different cell types [START_REF] Davis | Conditional loss of Dicer disrupts cellular and tissue morphogenesis in the cortex and hippocampus[END_REF][START_REF] Dugas | Dicer1 and miR-219 Are required for normal oligodendrocyte differentiation and myelination[END_REF][START_REF] Harfe | The RNaseIII enzyme Dicer is required for morphogenesis but not patterning of the vertebrate limb[END_REF][START_REF] Lynn | MicroRNA expression is required for pancreatic islet cell genesis in the mouse[END_REF] and, in particular, to regulate somatic growth and fertility at the level of peripheral organs [START_REF] Hasuwa | miR-200b and miR-429 function in mouse ovulation and are essential for female fertility[END_REF][START_REF] Luense | Role of Dicer in female fertility[END_REF][START_REF] Papaioannou | microRNAs in the testis: building up male fertility[END_REF] . We therefore asked whether miRNAs could play a similarly critical role in the central neuroendocrine control of reproduction.

Here we show that a dramatic switch in miRNA expression patterns in infantile GnRH neurons inverts the balance between inductive and repressive signals, triggering an increase in hypothalamic GnRH expression and thereby controlling the crucial transition from the early infantile phase, when its levels are low, to the GnRH-fuelled run-up to puberty. These data raise the intriguing possibility that the miRNA-dependent epigenetic regulation of GnRH secretion could underlie the pathophysiology of human hypogonadotropic hypogonadism when a genetic cause is not evident, and hold therapeutic potential for disorders of puberty and infertility of hypothalamic origin.

Results

Loss of Dicer function in GnRH neurons causes postnatal defects leading to hypogonadotropic hypogonadism and infertility

To determine whether miRNAs play a role in the crucial postnatal increase in GnRH expression, we generated mice in which Dicer, an RNAse III endonuclease essential for miRNA biogenesis [START_REF] Bernstein | Role for a bidentate ribonuclease in the initiation step of RNA interference[END_REF] , was selectively inactivated in GnRH neurons. Animals harboring a floxed Dicer allele [START_REF] Harfe | The RNaseIII enzyme Dicer is required for morphogenesis but not patterning of the vertebrate limb[END_REF] were crossed with those expressing Cre recombinase under the control of the endogenous GnRH promoter [START_REF] Yoon | Olfactory inputs to hypothalamic neurons controlling reproduction and fertility[END_REF] (Fig. 1A), which is expressed in hypothalamic GnRH neurons but not in the gonads (Fig. S2A,B). The resulting Gnrh::Cre; Dicer loxP/loxP mice were viable, born at Mendelian frequencies, and had body weights at adulthood indistinguishable from Dicer loxP/loxP (wild-type) littermates (t (8) =4.35, p=0.390, n=5 per group). All male and female mutant mice, however, exhibited severe hypogonadism (testicular weight: t (13) =24.4, p=0.0001, n=5-10 per group; uterine weight: t (4.47) =10.2, p=0.0005, n=5-6 per group) (Fig. 1B-E) and sterility (males: n=6; females: n= 6) (Fig. 1F). While some female Gnrh::Cre; Dicer loxP/loxP mice underwent vaginal opening (5 of 7) (Fig. 1G), this external sign of sexual maturation occurred significantly later than in Dicer loxP/loxP mice (t (14) =-4.93, p=0.0002), and vaginal smears showed that they never reached puberty (n=7) (Fig. 1H) despite normal somatic growth (two-way ANOVA, F (1.96, 11.96) =0.5741, p=0.54, n=5 per group) (Fig. 1I).

Similarly, male Gnrh::Cre; Dicer loxP/loxP mice (n=5) did not exhibit balanopreputial separation, which occurred at 34.9 ± 1.8 days of age in Dicer loxP/loxP littermates (n=11), or spermatozoa in the seminiferous tubules (Fig. 1C). Additionally, serum levels of the pituitary gonadotropins luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were markedly reduced in adult mutants (Fig. S2C,D). LH levels could readily be increased by systemic GnRH injection, demonstrating that pituitary function in our Gnrh::Cre; Dicer loxP/loxP mice was intact (Fig. S2E).

However, LH levels were unaffected by the intracerebroventricular infusion of two potent stimulators of the GnRH system that trigger LH release in wild-type animals: kisspeptin, which acts through its receptor Gpr54, and NMDA, which bypasses the kisspeptidergic system to act directly on GnRH neurons [START_REF] Urbanski | A role for N-methyl-D-aspartate (NMDA) receptors in the control of LH secretion and initiation of female puberty[END_REF][START_REF] De Tassigny | Kisspeptin signaling is required for peripheral but not central stimulation of gonadotropin-releasing hormone neurons by NMDA[END_REF] (Fig. S2F,G), suggesting that the hypogonadotropic hypogonadism observed was due primarily to a hypothalamic GnRH deficiency.

Following these physiological data, neuroanatomical analyses revealed a striking and complete absence of GnRH immunoreactivity in the hypothalamus of adult mutant mice (Fig. S3A,B), reminiscent of Kallmann syndrome in humans [START_REF] Schwanzel-Fukuda | Luteinizing hormone-releasing hormone (LHRH)-expressing cells do not migrate normally in an inherited hypogonadal (Kallmann) syndrome[END_REF] , in which GnRH neurons, which originate in the olfactory placode, fail to migrate into the brain during development. To determine whether Dicer invalidation in mouse GnRH neurons similarly affected their migration, we examined them on embryonic day (E) 14.5, when half the migrating GnRH neurons are still in the nose, and at birth (postnatal day 0, P0), when GnRH neurons have completed their migration into the hypothalamic preoptic region, their principal site of residence, and sent projections to the median eminence, where they release their neurohormone into portal capillaries for delivery to the anterior pituitary [START_REF] Wierman | Gonadotropin-releasing hormone (GnRH) neuron migration: Initiation, maintenance and cessation as critical steps to ensure normal reproductive function[END_REF][START_REF] Forni | anosmia and hypogonadotropic hypogonadism -where are we?[END_REF] . Surprisingly, and unlike the case in Kallmann patients, GnRH neurons displayed no migratory defects and GnRH immunoreactivity was comparable in Gnrh::Cre; Dicer loxP/loxP and Dicer loxP/loxP littermates at P0 (t (4) =0.27, p=0.8, n=3 per group) (Fig. S4). GnRH deficiency in mutant mice was thus not due to a developmental lack of GnRH neurons, but acquired postnatally.

The GnRH peptide content of the hypothalamus increases progressively between birth and puberty, accelerating markedly during the latter part of the infantile period (P7-P12) [START_REF] Prevot | Normal female sexual development requires neuregulin-erbB receptor signaling in hypothalamic astrocytes[END_REF] .

We thus examined hypothalamic GnRH immunoreactivity at different postnatal ages and found that its disappearance in Gnrh::Cre; Dicer loxP/loxP mice occurred gradually, starting during the infantile period (t (10) =1.96, p=0.08, n=5-7 per group at P12) (Fig. 2A, Fig. S5) and becoming more rapid after weaning, during the juvenile period, i.e. P21 to ~P35 (t (11) =8.53, p<0.0001, n=6-7 per group at P21 and t (6.59) =10.98, p<0.0001, n=7-9 per group at P28) (Fig. 2A, Fig. S5). Unexpectedly, however, the generation of Gnrh::Cre; Dicer loxP/loxP ; tdTomato loxP/STOP trigenic mice, in which the tracer tdTomato continues to be expressed in GnRH neurons even without ongoing GnRH promoter activity, revealed that in the absence of miRNAs, GnRH neurons did not die but simply lost GnRH immunoreactivity (Fig. 2B). Indeed, at P21, ~70% of GnRH neurons had lost GnRH peptide expression in Gnrh::Cre; Dicer loxP/loxP ; tdTomato loxP/STOP mice when compared to Gnrh::Cre; Dicer +/+ ; tdTomato loxP/STOP littermates. Importantly, the concomitant loss of GnRH immunoreactivity and green fluorescent protein (GFP) in Gnrh::Gfp; Gnrh::Cre; Dicer loxP/loxP mice (Fig. S3C), in which GFP is expressed under the GnRH promoter (unlike tdTomato in the previous experiment), indicated that miRNAs probably did not regulate GnRH transcript stability and/or translation directly by binding to its 3'UTR, but rather affected genes necessary for GnRH transcription, such as promoter modulators.

An infantile switch in miRNA species induces GnRH promoter activator expression in

GnRH neurons in vivo

In order to tease apart the mechanism of miRNA action, we started by verifying the expression profile of the GnRH transcript in GnRH neurons isolated by FACS from Gnrh::Gfp mice wild-type for Dicer at two stages: P7, the beginning of the infantile period, and P12, when hypothalamic GnRH peptide content begins to increase in a functionally relevant manner in normal mice [START_REF] Prevot | Normal female sexual development requires neuregulin-erbB receptor signaling in hypothalamic astrocytes[END_REF] , whereas it begins to decrease in mice lacking Dicer even though the vast majority of their GnRH neurons (~80%) still express enough GFP to be detectable (Fig. S3). Real-time PCR analyses of GFP-expressing GnRH neurons isolated by fluorescence-activated cell sorting (FACS) from Gnrh::Gfp; Dicer loxP/loxP mice (Fig. 2C; Fig. S6) revealed that there was a 200% increase in GnRH mRNA levels between P7 and P12 in these Dicer wild-type mice (t (5) =-3.20, p=0.024, n=3-4 per group) (Fig 2D). This increase was accompanied by a significant upregulation of the mRNA levels of several promoter activators known to affect GnRH expression [START_REF] Belsham | Transcription factors Oct-1 and C/EBPbeta (CCAAT/enhancer-binding protein-beta) are involved in the glutamate/nitric oxide/cyclic-guanosine 5'-monophosphate-mediated repression of mediated repression of gonadotropin-releasing hormone gene expression[END_REF][START_REF] Lee | Gonadotropin-releasing hormone: regulation of the GnRH gene[END_REF] (Fig. 2E), while transcripts of three known GnRH repressors [START_REF] Belsham | Transcription factors Oct-1 and C/EBPbeta (CCAAT/enhancer-binding protein-beta) are involved in the glutamate/nitric oxide/cyclic-guanosine 5'-monophosphate-mediated repression of mediated repression of gonadotropin-releasing hormone gene expression[END_REF][START_REF] Lee | Gonadotropin-releasing hormone: regulation of the GnRH gene[END_REF] remained unchanged (Fig. 2F). However, in GnRH neurons lacking Dicer isolated from Gnrh::Gfp; Gnrh::Cre; Dicer loxP/loxP trigenic mice at P12, GnRH mRNA expression was significantly lower than in neurons from Dicer wild-type Gnrh::Gfp; Dicer loxP/loxP mice (t (3.12) =5.88, p=0.0098, n=4 per group) (Fig. 2G). In addition, there was a marked decrease in the expression of most GnRH promoter activators (Fig. 3A) and a striking 15-fold increase in the transcript of the GnRH promoter repressor Cebpb (Fig. 3B) in GnRH neurons in the absence of Dicer (Fig. 3A,B). In silico analysis of the 3'UTR sequence of these promoter modulators revealed the presence of putative binding sites for several miRNAs (p<0.01; Table S1), confirming that miRNAs could regulate the expression of these genes. Concordantly, we uncovered a dramatic inversion of the expression profile of these and other putative regulatory miRNAs in wild-type GnRH neurons between P7 and P12 (Fig. 3C), strongly suggesting that GnRH promoter activity during the infantile period is controlled by a switch operated by miRNAs.

The miR-200/Zeb1 network is involved in controlling hypothalamic GnRH expression during postnatal development

The decrease in the expression of GnRH promoter activators uncovered above suggests that in the absence of miRNAs, repressors of these activators might be reciprocally increased. To identify the putative molecular targets involved in this process, we looked for miRNAs differentially expressed between P7 and P12 (Fig. 3C) that were also specifically enriched in GnRH neurons. The results showed that members of the miRNA-200 family, including miR-141 and miR-429 (Fig. S7A), were among the most highly expressed miRNAs in infantile GnRH neurons when compared to non-GnRH-expressing cells (Fig. 3D and3E). Within this family, the expression of miR-200a and miR-429, known to silence the mRNA for the transcriptional repressor Zeb1 outside the brain in pituitary gonadotropes, where it regulates LH expression [START_REF] Hasuwa | miR-200b and miR-429 function in mouse ovulation and are essential for female fertility[END_REF] , increased significantly between P7 and P12 (miR-200a, t (4) =9.178, p=0.0008; miR-429, t (5) =3.931, p=0.0111, n=3-4 per group) (Fig 3F). To explore the possibility that the miR-200/Zeb1 network [START_REF] Burk | A reciprocal repression between ZEB1 and members of the miR-200 family promotes EMT and invasion in cancer cells[END_REF] (Fig. S7A) also controlled hypothalamic GnRH expression during postnatal development, we analyzed Zeb1 expression in the brain of mice that did or did not express miRNAs in GnRH neurons. Importantly, not only was Zeb1 mRNA expressed in infantile GnRH neurons, its expression decreased significantly between P7 and P12 in Gnrh::Gfp; Dicer +/+ mice (t (6) =4.86, p=0.0028, n=4 per group) (Fig. 4A). RT-PCR experiments in GnRH neurons isolated from Gnrh::Gfp; Gnrh::Cre; Dicer loxP/loxP and Gnrh::Gfp; Dicer loxP/loxP littermates at P12 further showed that Zeb1 mRNA was significantly upregulated in mutant mice (t (5) =-3.38, p=0.0195, n=3-4 per group) (Fig. 4B). In silico sequence analysis revealed varying numbers of putative Zeb1-binding sites in the genes of all GnRH transcriptional activators under scrutiny (Fig. S7B), including Pou2f1, which in addition to activating the GnRH gene directly, could increase levels of miR-200 [START_REF] Bechec | MIR@NT@N: a framework integrating transcription factors, microRNAs and their targets to identify sub-network motifs in a meta-regulation network model[END_REF] , thereby indirectly influencing GnRH activation by repressing Zeb1. In addition, the GnRH gene itself was found to contain putative Zeb1-binding sites, three of which we validated as being functionally active using chromatin immunoprecipitation assays in an immortalized GnRHexpressing mouse cell line (Fig. 4C). Altogether these data support the idea that increased Zeb1 expression in the absence of miRNA biogenesis could mediate the downregulation of these activators and of GnRH in these neurons (Fig. 3A). To test this hypothesis, we infused a target-site blocker, TSB-200, into the brains of Gnrh::Gfp; Dicer +/+ mice to selectively hamper the binding of miR-200b/200c/429 to Zeb1 mRNA (Fig. S7C). TSBs are small, stabilized, freely diffusible locked nucleic acid (LNA) oligonucleotides, complementary to both the miRNA and the binding site being targeted (see for example Fig. S8C,D,E). As shown by fluorescence-tagged sequences (Fig. S8A), TSBs readily target the preoptic region and GnRH neurons 72h after their infusion into the lateral ventricle of Gnrh::Gfp; Dicer +/+ pups. TSB-200 treatment significantly increased Zeb1 mRNA levels in GnRH neurons as compared to a scrambled sequence (t (9) =-3.33, p=0.0087, n=5-6 per group) (Fig. 4D), but did not affect the transcripts of two other miR-200b/200c/429 target genes, Lpin1 26 (t (10) =0.663, p=0.52, n=6 per group) and Maf 27 (t (10) =-0.192, p=0.85, n=6 per group) (Fig. 4D). This increase in Zeb1 expression was accompanied by a significant decrease in the induction of transcripts for two GnRH promoter activators that were upregulated in GnRH neurons at P12 compared to P7 (Fig. 2E), Meis1 (t (8) =2.53, p=0.035, n=5 per group) and Pou2f1 (t (10) =4.15, p=0.002, n=6 per group) (Fig. 4E), while transcripts for other known promoter activators, such as Aes, Otx2, Pbx1 and Pknox1, which did not significantly change between P7 and P12, were also unchanged by TSB-200 infusion. Interestingly, these genes also had fewer putative Zeb1binding sites than those whose expression was strongly modified by altered Zeb1 expression (Fig. S7B). As expected, GnRH transcription was also downregulated by the blockade of miR-200 binding to Zeb1 at P12 (t (9) =5.37, p=0.0004, n=4-7 per group) (Fig. 4F). This repressive effect on GnRH mRNA levels was apparently mediated by the inhibition of GnRH promoter activity, as shown by the dramatic decrease in Gfp reporter mRNA expression (t (10) =14.1, p<0.0001, n=6 per group) (Fig. 4G), suggesting that the miR-200/Zeb1 network is actively involved in the switch in the control of GnRH transcription in the infantile mouse hypothalamus (Fig. 4H).

Hampering selective miR-155 binding to the GnRH repressor Cebpb activates Zeb-1 expression and inhibits infantile GnRH promoter activity in vivo

In contrast to the downregulation of GnRH promoter activators in mice lacking Dicer in GnRH neurons, the mRNA for a GnRH repressor, Cebpb, which codes for the CAAT/enhancer binding protein  (C/EBP), was found to be dramatically upregulated (see Fig. 3B above).

We thus reasoned that the repression of C/EBP expression by miRNAs would increase radically during the infantile period in wild-type mice. Using the miRWalk database, which provides validated information on miRNA-target interactions [START_REF] Dweep | miRWalk--database: prediction of possible miRNA binding sites by "walking" the genes of three genomes[END_REF] , we found that among the miRNAs upregulated in GnRH neurons at P12 (Fig. 3C,3D) was miR-155, whose seed region targets the 3'UTR of the Cebpb transcript (p<0.01, Table S2, Fig. S8B,C) [START_REF] Costinean | Src homology 2 domain-containing inositol-5-phosphatase and CCAAT enhancer-binding protein beta are targeted by miR-155 in B cells of Emicro-MiR-155 transgenic mice[END_REF] . To determine whether the ten-fold increase in miR-155 expression between P7 and P12 (t (4) =-4.85, p=0.0083, n=3 per group) (Fig. 5A) played an actual role in regulating C/EBP expression in GnRH neurons, we used TSB-155 to selectively hamper miR-155 binding to the Cebpb transcript in the hypothalamus of infantile mice (Fig. S8D,S8E). TSB-155 infusion into Gnrh::Gfp; Dicer +/+ mice markedly increased the levels of Cebpb mRNA in GnRH neurons when compared to a scrambled sequence (t (9) =-3.41, p=0.0038, n=5-6 per group) (Fig. 5B), but did not change mRNA levels of Lpin1 (t (10) =0.601, p=0.56, n=6 per group) and Maf (t (10) =0.406, p=0.69, n=6 per group) (Fig. 5B), which are also validated targets for miR-155 [START_REF] Koch | Induction of microRNA-155 is TLRand type IV secretion system-dependent in macrophages and inhibits DNA-damage induced apoptosis[END_REF][START_REF] Rodriguez | Requirement of bic/microRNA-155 for normal immune function[END_REF] . Importantly, this TSB-155-mediated upregulation of the Cebpb transcript was associated with a strong inhibition of both GnRH promoter activity, as indicated by Gfp mRNA levels (t (10) =14, p<0,0001, n=6 per group) (Fig. 5C) and GnRH mRNA levels (t (8) =3.57, p=0.0073, n=4-6 per group) (Fig. 5D). Surprisingly, we found, using ChIPBase, an integrated resource and platform for decoding transcription factor binding maps based on ChipSeq data 32 , that the Zeb1 gene contained binding sites for C/EBP in six different regulatory regions [START_REF] Heinz | Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities[END_REF][START_REF] Lefterova | Cell-specific determinants of peroxisome proliferator-activated receptor gamma function in adipocytes and macrophages[END_REF] (Fig. S9) but no binding site for miR-155, suggesting that C/EBP could regulate GnRH mRNA levels not only directly but indirectly, by influencing Zeb1 expression. In agreement with this hypothesis, TSB-155 treatment, which selectively upregulates the Cebpb transcript, also increased Zeb1 mRNA expression in Dicer +/+ GnRH neurons (t (9) =-2.89, p=0.0177, n=5-6 per group) (Fig. 5E). Furthermore, in silico and ChipSeq analysis revealed several binding sites for both C/EBP-and Zeb1 on the Gpr54 gene (Fig. S7B and Fig. S9), suggesting that in addition to their direct and promoter-activator-mediated effects, C/EBP and Zeb1 could repress GnRH gene transcription indirectly by blocking kisspeptinergic signaling through Gpr54, which has been shown to promote the nuclear translocation of the GnRH promoter activator Otx2 and the subsequent increase in GnRH transcription [START_REF] Novaira | Genetic mechanisms mediating kisspeptin regulation of GnRH gene expression[END_REF] (Fig. 5F). In support of this hypothesis, RT-PCR analysis revealed that in animals lacking Dicer in GnRH neurons, Gpr54 mRNA levels were also strongly reduced (t (6) =4.22, p=0.0056, n=4 per group) (Fig. 5G), to a similar extent as that of other GnRH activators (Fig. 3A).

Blockade of infantile NO release partially rescues GnRH expression in GnRH neurons lacking functional Dicer

C/EBP is also known to mediate the nitric oxide (NO) -induced repression of GnRH gene expression [START_REF] Belsham | Transcription factors Oct-1 and C/EBPbeta (CCAAT/enhancer-binding protein-beta) are involved in the glutamate/nitric oxide/cyclic-guanosine 5'-monophosphate-mediated repression of mediated repression of gonadotropin-releasing hormone gene expression[END_REF] . However, we have recently shown that the activation of neuronal NO synthase (nNOS) in neurons of the preoptic region, itself regulated by the actions of Kisspeptin on its receptor Gpr54 [START_REF] Hanchate | Kisspeptin-GPR54 Signaling in Mouse NO-Synthesizing Neurons Participates in the Hypothalamic Control of Ovulation[END_REF] , is required for sexual maturation [START_REF] Bellefontaine | Leptin-dependent neuronal NO signaling in the preoptic hypothalamus facilitates reproduction[END_REF] . Intriguingly, in wild-type mice in the present study, nNOS activity increased significantly in the preoptic region between P7 and P12, at a time when synaptic connectivity is being established in the GnRH neuronal network 38,39 , as shown by the increase in the phosphorylation of its Ser1412 activation site [START_REF] Rameau | Biphasic coupling of neuronal nitric oxide synthase phosphorylation to the NMDA receptor regulates AMPA receptor trafficking and neuronal cell death[END_REF] (p-nNOS/NOS ratio, t (6) =-5.48, p=0.0015, n=4 per group) (Fig. 5H). To determine whether the marked decrease in GnRH expression in mice lacking Dicer could be related to this infantile increase in NO-C/EBP signaling, we next treated P12 Gnrh::Gfp; Gnrh::Cre; Dicer loxP/loxP mice with the NOS inhibitor N-G-Nitro-L-arginine methyl ester (L-NAME; 50 mg/kg, i.p.) and assessed GnRH mRNA expression in FACS-isolated neurons 12h later. The reduced production of NO due to L-NAME treatment partially but significantly rescued GnRH transcript levels (t(8)=-2.40, p=0.043, n=6-7 per group) (Fig. 5I). In addition to the alleviation of the previously demonstrated repressive effect of C/EBPβ on the GnRH promoter [START_REF] Belsham | Transcription factors Oct-1 and C/EBPbeta (CCAAT/enhancer-binding protein-beta) are involved in the glutamate/nitric oxide/cyclic-guanosine 5'-monophosphate-mediated repression of mediated repression of gonadotropin-releasing hormone gene expression[END_REF] , this increase in GnRH expression could have resulted from the inhibition of the transcriptional activation of Zeb1 by C/EBPβ, as suggested by the 2.4-fold increase in transcripts for Pou2f1 (in L-NAME-injected vs. saline-injected mice; t(8)=-1.40, p=0.017, n=5-6 per group). This suggests that increased NO levels during this period crucial for subsequent GnRH neuronal function might actually participate in the multilayered transcriptional repression of the Gnrh gene in the absence of posttranscriptional silencing of C/EBP by miRNAs (Fig. 5J). In other words, not only do miRNAs control the switch in GnRH promoter activity in infantile GnRH neurons, and thus reproductive capacity, they do so through an intricate mechanism involving crosstalk between inductive and repressive pathways.

miR-200 family members bind to Zeb-1 to control GnRH neuronal function throughout life

To assess the functional involvement of miR-200 family members and miR-155 in GnRH neurons in the onset of puberty, we performed a single bolus injection of the same TSB-200 and/or TSB-155 into the brains of female wild-type mice at P9, and monitored sexual maturation after weaning. Importantly, the use of fluorescently-labeled TSBs demonstrated that they could be detected in the brain up to 2 weeks after injection (Fig. S8A). Surprisingly, while growth (Fig S10 ) was similar in TSB-and scrambled-sequence-injected mice, TSBinjected mice exhibited a precocious onset of puberty when compared to control mice (Fig. 6A). This effect was most pronounced in TSB-200-infused mice, with 50% of the animals displaying their first estrus by P32, i.e. more than 10 days before control mice. In line with these results, TSB-200-infused mice showed significantly elevated peripubertal afternoon surges of plasma LH at P38, while changes in LH levels induced by TSB-155 treatment did not reach statistical significance when compared to control mice, in which plasma LH levels were below the limit of detection (Fig. 6B). The combination of TSB-200 and TSB-155 also increased LH to detectable levels, although this effect was minimal (Fig. 6B non-GnRH hypothalamic cells, which is about 50 fold higher for miR200/429 (Fig. 3E) than for miR-155 (Fig. S11A), Cebpb mRNA levels were significantly increased in non-GnRH cells of the preoptic region after TSB-155 infusion (Fig. S11B), while TSB-200 treatment altered neither Cebpb nor Zeb1 transcripts in these cells (Fig. S11B,S11C). Altogether, these data suggest that, in keeping with the complex feedback mechanisms at work (Fig. 7), selectively applying the brakes to GnRH production in an excessive manner (Fig. 4F,4G) and then releasing them prematurely can trigger precocious puberty, possibly due to a rebound effect.

To determine whether the miR-200/Zeb1 network could control GnRH function outside of this critical infantile time window leading up to puberty, we infused TSB-200 into the hypothalamic preoptic region of cycling adult female mice and followed their estrous cycles (Fig. 6C). While control mice infused with scrambled sequences did not exhibit any marked alteration of their estrous cycles, most mice treated with TSB-200 (5 out of 6) showed a slight prolongation in the number of days spent in diestrus (a basal stage when LH is released at nadir levels) and eventually reached proestrus (when the preovulatory surge of LH occurs), but displayed an incomplete cycle at least once during the first two weeks after injection (Fig. 6C). In line with this observation, LH levels measured during proestrus one week after injection showed a characteristic surge in control animals but not in TSB-200-treated mice (Fig. 6D; F(2, 14)=4.371, p=0.0335, n=5-6 per group; TSB-200 > 1 week, p=0.0262; TSB-200 > 2 weeks, p=0.6976). In 4 of the 6 animals treated with TSB-200, LH levels recovered and displayed normal preovulatory surges by the third week after TSB infusion (Fig. 6D).

Discussion

The balance between inductive and repressive signals is a process essential for successful reproduction, not only in animals but in living organisms in general [START_REF] Lee | Regulation of temperature-responsive flowering by MADS-box transcription factor repressors[END_REF][START_REF] Lomniczi | Epigenetic control of female puberty[END_REF] . Our data provide the first physiological and neuroanatomical evidence that miRNAs, which have been shown to regulate somatic growth, including the development of peripheral sexual organs [START_REF] Hasuwa | miR-200b and miR-429 function in mouse ovulation and are essential for female fertility[END_REF][START_REF] Luense | Role of Dicer in female fertility[END_REF][START_REF] Papaioannou | microRNAs in the testis: building up male fertility[END_REF] , also play an essential role in the postnatal development of the hypothalamic neuroendocrine neurons controlling the onset of puberty and fertility in mammals, by modulating this balance in the GnRH gene network. During this infantile "critical period" lasting just a few days, when the first centrally-driven gonad-independent activation of the hypothalamic-pituitary-gonadal axis occurs, setting in motion, for example, the growth of the first wave of ovarian follicles that will ovulate at puberty in females and the development of the testes in males (see for review [START_REF] Prevot | Puberty in mice and rats[END_REF] ), our findings show that a switch in miRNA expression in turn flips a switch in a multilayered array of GnRH gene activators and repressors, permitting the sustained increase of the neurohormone required for normal sexual maturation. Interfering with the binding and functional effects of two key miRNA species, miR-200/429, which is not only upregulated during this critical period but selectively enhanced in GnRH neurons, and miR-155, which appears to act on other hypothalamic cell types as well, and mediates, for example, the effects of a concomitant release of NO upstream of GnRH neurons, blunts this infantile increase in GnRH expression. In addition, this in vivo alteration of the miR-200/429transcription factor micro-network leads to the disruption of normal puberty onset as well as normal estrous cyclicity in adulthood.

The fact that GnRH neurons form an extremely small population (~800 neurons in a mouse brain) and their scattered distribution in a continuum from the olfactory bulb to the hypothalamus, albeit with a strong concentration in the hypothalamic preoptic area, have made genetic and epigenetic studies in these neurons a veritable technical challenge until now. Our successful separation of GnRH from non-GnRH cells in the preoptic region of GnRH::Gfp mice has allowed us to determine miRNA expression profiles specific to this very limited population of neurons in the postnatal brain, i.e. once the processes of differentiation and the establishment of connectivity, including their projection over several millimeters to the hypothalamic median eminence, are complete. The difficulty of this approach is evident from the fact that other such studies, even those focusing on more abundant neuronal types, have not always succeeded [START_REF] Issler | MicroRNA 135 is essential for chronic stress resiliency, antidepressant efficacy, and intact serotonergic activity[END_REF] . Using this approach, we were not only able to study changes in miRNA expression in GnRH neurons at different stages of postnatal development, but even to analyze the effects of the selective invalidation of miRNA biogenesis and the acute manipulation of specific miRNA networks on the expression of transcription factors, GnRH promoter activity and GnRH expression in the postnatal brain in vivo.

Our findings show that miRNA processing in GnRH neurons is required to promote sexual development, but surprisingly, not GnRH neuronal migration in vivo. The specific role of miRNAs in sustaining the expression of GnRH is shown in Gnrh::Cre; Dicer loxP/loxP mice, in which GnRH expression appears to be normal during the perinatal and the early infantile periods, and the process of sexual maturation commences normally and leads to vaginal opening in 80% of the individuals. However, these mice never achieve puberty because of the progressive decrease in GnRH promoter activity from the second week of postnatal life and its complete extinction during the juvenile period. Our data indicate that miRNA species whose expression is upregulated in GnRH neurons during this critical period of 7 to 12 days of age include miR-155 and members of the miR-200 family. Notably, the striking enrichment of miR-200 family members during this period is restricted to GnRH neurons, indicating a highly specific function for these miRNA species in the regulation of GnRH expression. We have identified several genes targeted directly or indirectly by miR-155 and miR-200/429, whose repression (e.g. Cebpb and Zeb1) or activation (e.g. Pou2f1 or Meis1, which are both targets for Zeb1) is required for normal GnRH expression at 12 days of age. In particular miR-200/429 and its target Zeb1, which can directly repress the GnRH promoter, also seem to form a double-negative loop with the key GnRH promoter activator Pou2f1, while miR-155 counteracts NO/Cebpb-dependent GnRH repression. Nor can we rule out the possibility that some molecules act as both activators and repressors depending upon physiological conditions and cellular context, as we have seen in the case of Cebpb, a GnRH promoter repressor that nevertheless increases Zeb1 levels in GnRH neurons but not in non-GnRH hypothalamic cells (which do not express miR-200/429), or that some of these promoter modulators could exert a reciprocal regulatory effect on the miRNAs that control their expression, as is the case with Zeb1 and miR-200/429 [START_REF] Burk | A reciprocal repression between ZEB1 and members of the miR-200 family promotes EMT and invasion in cancer cells[END_REF] .

Surprisingly, the pattern of expression of various known GnRH promoter modulators is not the same at P12. While activators that are upregulated specifically in GnRH neurons in wildtype mice at P12 show reduced expression with the blockade of miR-200/429 binding (with the exception of Dlx5, which has no Zeb1 binding site and is thus probably regulated by other miRNA-effector pathways), the expression of one putative activator, Dlx1, is increased. Apart from a possible indirect effect, the fact that unlike Pou2f1 or Meis1, the expression of Dlx1 shows a downward trend in wild-type GnRH neurons at P12 could explain this seemingly paradoxical finding. The lack of effect of a miRNA-200/429 blockade on the expression of GnRH activator genes that contain no or few Zeb1-binding sites and that are not differentially expressed during the infantile-juvenile transition in GnRH neurons (but that could nevertheless play a role in baseline GnRH production or its regulation in other physiological contexts) further highlights the importance of the miR-200/Zeb1/Cebpb switch in the prepubertal increase in GnRH expression. Our findings show, in addition, that both miR-155 (by interfering with NO/Cebpb-mediated signaling by nNOS neurons, themselves activated by Kisspeptin [START_REF] Hanchate | Kisspeptin-GPR54 Signaling in Mouse NO-Synthesizing Neurons Participates in the Hypothalamic Control of Ovulation[END_REF] ) and miR-200/429 (through Zeb1) could also play a role in the intricate relationship between different cells of the hypothalamic GnRH network by affecting the expression of the Kisspeptin receptor Gpr54, and thus modulating the Kisspeptin-Gpr54mediated activation of GnRH expression. NO itself, by being both essential for the initiation of puberty [START_REF] Bellefontaine | Leptin-dependent neuronal NO signaling in the preoptic hypothalamus facilitates reproduction[END_REF] and participating in the repression of GnRH expression [START_REF] Belsham | Transcription factors Oct-1 and C/EBPbeta (CCAAT/enhancer-binding protein-beta) are involved in the glutamate/nitric oxide/cyclic-guanosine 5'-monophosphate-mediated repression of mediated repression of gonadotropin-releasing hormone gene expression[END_REF] , could play a more complex role than suspected hithertofore. The Kisspetin/nNOS/GnRH neuronal microcircuit and miRNA-gene network in GnRH neurons (Figure 7) would be ideally poised to ignite rhythmic GnRH gene transcription, which has recently been associated to pulsatile GnRH secretion [START_REF] Choe | Real-Time GnRH Gene Transcription in GnRH Promoter-Driven Luciferase-Expressing Transgenic Mice: Effect of Kisspeptin[END_REF][START_REF] Choe | Synchronous activation of gonadotropin-releasing hormone gene transcription and secretion by pulsatile kisspeptin stimulation[END_REF] in response to developmental and bodily cues during the infantile period in mice [START_REF] Prevot | Puberty in mice and rats[END_REF] , a developmental stage highly reminiscent of minipuberty in humans [START_REF] Kuiri-Hanninen | Activation of the hypothalamic-pituitarygonadal axis in infancy: minipuberty[END_REF] .

Other feedback loops, possibly involving the expression of miR-155 in other cell types, could also exist, as suggested by the fact that simultaneously neutralizing both miR-200/429 binding to Zeb1 and miR-155 binding to Cebpb results in a considerably less striking effect on peripubertal LH levels than the blockade of miR-200/429 alone. Intriguingly, while blocking the binding of miR-200/429 to Zeb1 during this infantile-juvenile critical period dramatically reduces GnRH promoter activity at the cellular level, these animals go on to display precocious, not delayed, puberty. This counterintuitive finding could stem from a rebound effect, where the prolonged inhibition of GnRH expression (TSBs, once injected, remain at the injection site for around 2 weeks) results in a compensatory spike once the inhibition is relieved. Such a phenomenon has been demonstrated previously in human populations, where children whose GnRH system is blunted due to exposure to endocrine disruptors of agricultural origin undergo precocious puberty when they migrate to countries that do not use them [START_REF] Parent | The timing of normal puberty and the age limits of sexual precocity: variations around the world, secular trends, and changes after migration[END_REF] .

To conclude, our data are the first to indicate not only that miRNAs are active in the hypothalamic GnRH network, but that miRNA-200/429 and miRNA-155 are key components of a complex developmental switch that controls the activity of the GnRH promoter, as illustrated in the models presented in Figure 7 and Figure S12, and whose correct functioning is essential for the normal initiation of puberty and reproductive success in adulthood.

Moreover, this dramatic switch in gene expression appears to involve a multilayered and interconnected array of miRNAs and their target genes in GnRH neurons, with its own inbuilt feedback control mechanism, that alters the balance between inductive and repressive signals during the infantile critical period and thus leads to increased GnRH production.

Deciphering the mechanisms through which miRNAs contribute to precocious or delayed puberty through their actions on the functioning of the GnRH neural network may thus not only provide novel insights into the epigenetic regulation of maturational processes, fascinating in itself, but pave the way to a better understanding of idiopathic infertility [START_REF] Pitteloud | Complex genetics in idiopathic hypogonadotropic hypogonadism[END_REF][START_REF] Topaloglu | Molecular causes of hypogonadotropic hypogonadism[END_REF] and the elaboration of new diagnostic and therapeutic options in humans.

Materials and Methods

Animals

All C57Bl/6J mice were housed under specific pathogen-free conditions in a temperaturecontrolled room (21-22°C) with a 12h light/dark cycle and ad libitum access to food and water. Gnrh::Cre (Tg(Gnrh1::Cre)1Dlc), Dicer LoxP/LoxP , and Gnrh::Gfp mice were a generous gift of Dr. Catherine Dulac (Howard Hughes Medical Institute, Cambridge MA) [START_REF] Yoon | Olfactory inputs to hypothalamic neurons controlling reproduction and fertility[END_REF] , Dr. Brian Harfe (University of Florida, FL) [START_REF] Harfe | The RNaseIII enzyme Dicer is required for morphogenesis but not patterning of the vertebrate limb[END_REF] and Dr. Daniel J. Spergel (Section of Endocrinology, Department of Medicine, University of Chicago, IL) [START_REF] Spergel | GABA-and glutamateactivated channels in green fluorescent protein-tagged gonadotropin-releasing hormone neurons in transgenic mice[END_REF] , respectively. tdTomato loxP/STOP mice (B6.Cg-Gt(ROSA)26Sortm9(CAG-tdTomato)Hze/J) were purchased from the Jackson laboratory (Maine, USA). Mice were genotyped by PCR using primers listed in table S3.

Physiological measurements

Fertility index. Male and female fertility indices were calculated from the number of litters per females during a 120-day-long mating.

Puberty onset. Weaned female mice were checked daily for vaginal opening. After vaginal opening, vaginal smears were performed daily and analyzed under an inverted microscope to identify the specific day of the estrous cycle. Weaned males were checked daily for balanopreputial separation.

Hormone level measurements. Protocols and doses for in vivo testing of the LH response to

GnRH, kisspeptin-10 and NMDA were as described in detail elsewhere [START_REF] Garcia-Galiano | Kisspeptin signaling is indispensable for neurokinin B, but not glutamate, stimulation of gonadotropin secretion in mice[END_REF] . Serum LH and FSH levels were measured using radioimmunoassay kits supplied by the National Institutes of Health (Dr. A. F. Parlow, National Hormone and Peptide Program, Torrance, CA). Rat LH-I-10 and FSH-I-9 were labeled with 125 I using Iodo-gen tubes, following the instructions of the manufacturer (Pierce, Rockford, IL). Hormone concentrations were determined using reference preparations of LH-RP-3 and FSH-RP-2 as standards. Intra-and inter-assay coefficients of variation were <8 and 10% for LH and <6 and 9% for FSH, respectively. The sensitivity of the assay was 3.75 pg/tube for LH and 20 pg/tube for FSH. The accuracy of hormone measurements was confirmed by the assessment of rodent serum samples of known concentration (external controls).

Ovarian histology and quantitative analysis

Ovaries and testes were collected from 3-month-old Dicer LoxP/LoxP and Gnrh::Cre; Dicer LoxP/LoxP mice, immersion-fixed in 4% PFA solution and stored at 4°C. Paraffin-embedded ovaries and testes were sectioned at a thickness of 5 μm (histology facility, University of Lille 2, France) and stained with hematoxylin-eosin.

Tissue preparation

For immunohistochemical analysis, embryos were obtained after cervical dislocation from timed-pregnant Gnrh::Cre; Dicer LoxP/+ crossed with Gnrh::Cre; Dicer LoxP/LoxP male mice. Embryos were washed thoroughly in cold 0.1 m PBS, fixed in fixative solution [4% paraformaldehyde (PFA), 0.2% picric acid in 0.1 m PBS, pH 7.4] for 6-8 h at 4°C and cryoprotected in 20% sucrose overnight at 4°C. The following day, embryos were embedded in OCT embedding medium (Tissue-Tek), frozen on dry ice, and stored at -80°C until sectioning. Postnatal (P0 to P28) and adult mice (3-5 months old) were anesthetized with 50-100 mg/kg of Ketamine-HCl and 5-10mg/kg Xylazine-HCl and perfused transcardially with 2-10 ml of saline, followed by 10-100 ml of 4% PFA, pH7.4. Brains were collected, postfixed in the same fixative for 2 h at 4°C, embedded in OCT embedding medium (Tissue-Tek), frozen on dry ice, and stored at -80°C until cryosectioning.

Immunohistochemistry

Tissues were cryosectioned (Leica cryostat) at 16μm for embryos and pre-weaning postnatal mice, and at 35μm (free-floating sections) for post-weaning and adult brains.

Immunohistochemistry was performed as previously reported [START_REF] Hanchate | SEMA3A, a Gene Involved in Axonal Pathfinding, Is Mutated in Patients with Kallmann Syndrome[END_REF][START_REF] Messina | Dysregulation of Semaphorin7A/beta1-integrin signaling leads to defective GnRH-1 cell migration, abnormal gonadal development and altered fertility[END_REF] , using Alexa-Fluor 488-(1:400) and Cy3-conjugated (1:800) secondary antibodies (Invitrogen). Fluorescent specimens were mounted using 1,4-diazabicyclo[2.2.2]octane (Sigma-Aldrich). The primary antisera used were as follows: rabbit anti-GnRH (1:3000), a generous gift from Prof. G.

Tramu (Centre Nationale de la Recherche Scientifique, URA 339, Université Bordeaux I, Talence, France) [START_REF] Beauvillain | Immunocytochemical demonstration of LH-RH, somatostatin, and ACTH-like peptide in osmium-postfixed, resin-embedded median eminence[END_REF] .

Isolation of hypothalamic GnRH neurons using Fluorescence-Activated Cell Sorting

The preoptic regions of Gnrh::Gfp; Dicer LoxP/LoxP and Gnrh::Gfp; Gnrh::Cre; Dicer LoxP/LoxP mice were microdissected and enzymatically dissociated using a Papain Dissociation System (Worthington, Lakewood, NJ) to obtain single-cell suspensions. FACS was performed using an EPICS ALTRA Cell Sorter Cytometer device (BD Bioscience). The sort decision was based on measurements of GFP fluorescence (excitation: 488nm, 50 mW; detection: GFP bandpass 530/30 nm, autofluorescence bandpass 695/40nm) by comparing cell suspensions from Gnrh::Gfp and wild-type animals, as indicated in figure S5. For each animal, 400 to 800

Zeb1 chromatin immunoprecipitation (ChIP) assays

Immortalized GnRH neurons (GN11 cells) were cultured in DMEM + 10% FBS (Life Technology). Prior to experimental assays, cells were washed twice in PBS and incubated overnight in serum-free medium. The next morning, 10% FBS was added for 2h. Chromatin isolation was performed using the ChIP-IT Express kit (Active Motif) following the manufacturer's instructions. After DNA shearing by sonication (9 min total, 20s pulses at 70% power followed by a 40s pause; on ice), protein-bound DNA was immunoprecipitated at 4°C overnight using 5 µg of a polyclonal goat anti-rabbit Zeb1 antibody (E20; Santa Cruz Biotechnology; SC-10572) or control goat IgGs (Santa Cruz Biotechnology; sc-2028). ChIP-DNA fragments were purified using a QIAquick PCR purification kit (Qiagen), and analyzed by qPCR on a CFX96 thermal cycler (Biorad) using GoTaq® qPCR Master Mix (Promega) and the primers indicated in Table S4.

Western blot analyses

The preoptic area of the hypothalamus was dissected from each animal using Wecker scissors (Moria, France) under a binocular magnifying glass, and protein extracted in 200 μl of lysis buffer (25 mM Tris, pH 7.4, β-glycerophosphate, 1.5 mM EGTA, 0.5 mM EDTA, 1 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 10 μg/ml leupeptin and pepstatin A, 10 μg/ml apoprotinin, 100 μg/ml PMSF, and 1% Triton-X100) by trituration of the fragments through 22 and 26 gauge needles in succession. Tissue lysates were cleared by centrifugation at 14000 rpm for 15 min at 4°C. Protein content was determined using the Bradford method (Bio-Rad, Hercules, CA) and equal amounts of protein were mixed with 4X sample buffer (Invitrogen). Samples were boiled for 5 min and stored at -80°C until use. Samples were reboiled for 5 min after thawing and electrophoresed for 75 min at 150 V in 7% Tris-acetate, or for 50 min at 200 V in precast 4-12% MES SDS-polyacrylamide gels according to the protocol supplied with the NuPAGE system (Invitrogen). After size fractionation, the proteins were transferred onto 0.2 μm pore-size polyvinylidene difluoride membranes (LC2002; Invitrogen) in the blot module of the NuPAGE system (Invitrogen) for 75 min at room temperature. Membranes were blocked for 1 h in blocking buffer [TBS with 0.05% Tween 20 (TBST) and 5% nonfat milk] at room temperature, and incubated overnight at 4°C with the appropriate primary antibody diluted in blocking buffer (rabbit polyclonal anti-RFP, 600-401-379, 1:1000, Rockland antibodies & assays; rabbit polyclonal anti-nNOS, sc-8309, 1:500, Santa Cruz technologies; rabbit polyclonal anti-Ser 1412 phospho-nNOS, PA1-032; 1:1000, Affinity BioReagents; goat polyclonal anti-actin, sc-1616, 1:1000). Membranes were washed four times with TBST the following day before being exposed to HRPconjugated secondary antibodies diluted in blocking buffer for 1 h at room temperature.

Immunoreactions were visualized using the ECL detection kit (NEL101; PerkinElmer, Boston, MA). Immunoblots were scanned using a desktop scanner (Epson Expression 1680 PRO) and Adobe Photoshop, and band intensities were determined using ImageJ software (NIH, Bethesda).

Target site blockers (TSBs)

To selectively hamper the silencing activity of miR-155 and miR-200b/200c/429 on Cebpb and Zeb1 respectively in vivo, we used custom-designed target sites blockers with phosphorothioate backbone modifications from Exiqon (miRCURY LNA TM microRNA Target Site Blockers, in vivo ready). TSB sequences are designed with a large arm that covers the miRNA binding site and a short arm outside the miRNA seed to ensure target specificity. In detail, one sequence was generated to protect the unique miR-155-binding site in the Cebpb 3'UTR (TSB-155, Fig. S7). Five sequences were generated to protect the five different miR-200b/200c/429-binding sites in the Zeb1 3'UTR (Fig. S7) and mixed at equimolar ratios (TSB-200).

Stereotactic brain infusions of TSBs

Gnrh::Gfp mice were placed in a stereotaxic frame (Kopf® Instruments, California) under anesthesia (isoflurane), and a burr hole was drilled 1 mm lateral to the bregma, according to a mouse brain atlas (Paxinos and Franklin, 2004). A 10 μl Hamilton syringe was slowly inserted into the bottom of the left lateral ventricle (3.5 mm deep relative to the dura), and 1μl of the different treatment solutions (TSB-200, 50M, n = 6; TSB-155, 50M, n = 6) or vehicle (PBS, pH7.4, n = 6) was injected using an infusion pump (KD Scientific, Holliston, MA) over 4 min. Animals were subjected to intracranial surgery at P9 and killed at P12.

Identification of putative Zeb1-binding sites

To identify putative Zeb1-and C/EBPβ-binding sites in the promoter regions of target genes, 1.5 Kb of the genomic sequence upstream of the transcription initiation site of GnRH, Gpr54 and the GnRH promoter activators under study, were analyzed using ALGEN PROMO 3.0 software (http://alggen.lsi.upc.es/cgi-bin/promo_v3/promo/promoinit.cgi?dirDB=TF_8.3) and jaspar database (http://jaspar.genereg.net/).

Statistics

All analyses were performed using Prism 5 (GraphPad Software) and assessed for normality (Shapiro-Wilk test) and variance, when appropriate. Sample sizes were chosen according to standard practice in the field. Data were compared using an unpaired two-tailed Student's t test, a one-way ANOVA for multiple comparisons or a two-way repeated-measures ANOVA.

  ; F(3, 32)=3.475, p=0.0272, n=8-10 per group; TSB-200, p=0.0361; TSB-155, p=0.7541; TSB-200+TSB-155, p=0.9996) and resulted in earlier puberty onset by about 5 days when compared to control mice. In keeping with its lack of a significant effect on LH levels, treatment with TSB-155 alone failed to significantly change age at puberty (Fig. 6A; F(3, 35)=4.961, p=0.0057, n=9-10 per group; TSB-200, p=0.0019; TSB-155, p=0.073; TSB-200+TSB-155, p=0.0328). To verify whether this discrepancy in the impact of blocking either miR-200/429-Zeb1 binding or miR-155-Cebpb binding on GnRH promoter activity at P12 (Fig. 4F, 4G and Fig. 5C, 5D, respectively) and on subsequent sexual maturation (Fig 6A) was linked to differences in the specific cell types targeted by TSB-155 and TSB-200 in the hypothalamus, we analyzed the effects of TSB injection on the expression of Cebpb and Zeb1 in non-GnRH cells at P12. In agreement with the level of enrichment of miRNA expression in GnRH neurons compared to

  Animal studies were approved by the Institutional Ethics Committees for the Care and Use of Experimental Animals of the Universities of Lille and Cordoba; all experiments were performed in accordance with the guidelines for animal use specified by the European Union Council Directive of September 22, 2010 (2010/63/EU).

AFiguresFigure 1 .

 1 figure legends.

Figure 2 .

 2 Figure 2. A switch in the control of GnRH gene expression is operated by miRNAs

Figure 3 .

 3 Figure 3. MiRNAs regulate the expression of GnRH promoter modulators in infantile

  ) =-4.79, p=0.0173, n=4 per group (*: p<0.05; **: p<0.01; ***: p<0.001). (C-D) miRNAs whose expression changes between P7 and P12 (C) and that are specifically enriched in GnRH neurons at P12 (D). Pos: GFP-expressing neurons; Neg: GFP-negative cells. (E and F) RT-PCR analysis of the expression of miR-200 family members in FACS-sorted cells from preoptic explants isolated from Gnrh::Gfp mice wild-type for Dicer either at P12 (E; Pos vs. Neg: miR-141, t (5) =3.370, p=0.0199; miR-200a, t (4) =13.57, p=0.0002; miR-200b, t (5) =2.775, p=0.0391; miR-200c, t (5) =4.016, p=0.0102; miR-429, t (5) =4.778, p=0.005; n=3-4 per group) or at P7 and P12 (F; P7 vs. P12: miR-141, t (5) =0.6652, p=0.5354; miR-200a, t (4) =9.178, p=0.0008; miR-200b, t (5) =0.9572, p=0.3824; miR-200c, t (5) =0.2604, p=0.805 ; miR-429, t (5) =3.931, p=0.0111; n= 3-4 per group). Values shown are mean ± SEM.

Figure 4 .

 4 Figure 4. Knocking down the ability of miR-200 to repress Zeb1 expression in the brain

Figure 6 .

 6 Figure 6. miR-200 binding to Zeb1 modulates the function of GnRH neurons both in infantile and adult mice. (A) Knocking down the ability of miR-200 and/or miR-155 to

Figure 7 .

 7 Figure 7. Neuronal microcircuit and miRNA-gene network in hypothalamic GnRH neurons regulating the onset of puberty. Schematic diagram illustrating the potential

  ). Aes, t (5) =9.58, p=0.0002; Dlx1, t (5) =3.78, p=0.032; Meis1, t (3.17) =4.71, p=0.018; Otx2, t (5) =4.34, p=0.0074; Pou2f1, t (5) =10, p=0.0002; n=3-4 per group. Pknox1, t (6) =2.63, p=0.039; Cebpb, t (3.17

Acknowledgments

The supplemental information includes 11 figures and 3 tables. This research was supported by the Fondation pour la Recherche Médicale (FRM, Equipe FRM 2005 and DEQ20130326524, France to V.P.), grant BFI2011-025021 from the Spanish Ministry of Economy and Science (to M.T.-S.) and the ERC COST action BM1015 (to V.P, P.G. and M.T.-S.). A.M. was a postdoctoral fellow supported by the FRM. We are indebted to

GFP-positive cells were sorted directly into 10μl extraction buffer: 0.1% Triton® X-100 (Sigma-Aldrich) and 0.4 U/μl RNaseOUT™ (Life Technologies).

Quantitative RT-PCR analyses

For gene expression analyses, mRNAs obtained from FACS-sorted GnRH neurons were reverse transcribed using SuperScript® III Reverse Transcriptase (Life Technologies) and a linear preamplification step was performed using the TaqMan® PreAmp Master Mix Kit protocol (P/N 4366128, Applied Biosystems). Real-time PCR was carried out on Applied Biosystems 7900HT Fast Real-Time PCR System using exon-boundary-specific TaqMan® Gene Expression Assays (Applied Biosystems): Dr. Domenico Accili (University of Columbia) for his precious help in conducting the ChIP assays. We thank M. Tardivel (Imaging University core facility), Marie-Hélène Gevaert (Histology University core Facility), D. Taillieu and J. Devassine (Animal facility, Inserm and University of Lille) for expert technical assistance. GnRH gene expression during the infantile period of postnatal development and how these events may be intertwined with the integration of postmigratory GnRH neurons into the neural network responsible for regulating the timely onset of puberty (solid lines: demonstrated/published; grey lines: ChipSeq data; dotted lines: predictive in silico analyses).


Fanny Langlet

Barry E Levin

Serge Luquet

Massimiliano Mazzone

Andrea Messina

Ambrose A Dunn-Meynell

Eglantine Balland

Amelie Lacombe

Daniele Mazur

Peter Carmeliet

Sebastien G Bouret

Ph.D Vincent Prevot
email: vincent.prevot@inserm.fr

Bénédicte Dehouck

Glucose and tanycyte-derived VEGF-A promote bloodhypothalamus barrier plasticity and access of blood-borne molecules to the arcuate nucleus in response to fasting

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Appetite, energy balance and metabolism are all controlled by select neurons of the hypothalamic arcuate nucleus (ARH) (see for reviews [START_REF] Elmquist | Identifying hypothalamic pathways controlling food intake, body weight, and glucose homeostasis[END_REF][START_REF] Gao | Neurobiology of feeding and energy expenditure[END_REF][START_REF] Levin | Metabolic sensing and the brain: who, what, where, and how?[END_REF][START_REF] Sawchenko | Toward a new neurobiology of energy balance, appetite, and obesity: the anatomists weigh in[END_REF]). These interoceptive sensory neurons participate in neural networks that sense circulating factors such as glucose and adiposity hormones that signal changes in metabolic state [START_REF] Cowley | Leptin activates anorexigenic POMC neurons through a neural network in the arcuate nucleus[END_REF][START_REF] Cowley | The distribution and mechanism of action of ghrelin in the CNS demonstrates a novel hypothalamic circuit regulating energy homeostasis[END_REF][START_REF] Dunn-Meynell | Glucokinase is the likely mediator of glucosensing in both glucose-excited and glucose-inhibited central neurons[END_REF][START_REF] Elias | Leptin differentially regulates NPY and POMC neurons projecting to the lateral hypothalamic area[END_REF][START_REF] Hill | Direct insulin and leptin action on proopiomelanocortin neurons is required for normal glucose homeostasis and fertility[END_REF][START_REF] Liu | Fasting Activation of AgRP Neurons Requires NMDA Receptors and Involves Spinogenesis and Increased Excitatory Tone[END_REF]. However, the physiological mechanisms that control the access of these factors to ARH circuits and their regulation in response to changes in feeding status remain largely unexplored.

Molecular traffic between the periphery and the central nervous system (CNS), including the hypothalamus, is restricted by regulated interfaces, such as the blood-brain barrier (BBB), composed of tight junctions between endothelial cells lining brain microvessels [START_REF] Neuwelt | Engaging neuroscience to advance translational research in brain barrier biology[END_REF]. The blood-cerebrospinal-fluid (CSF) barrier, a lesser known interface, is composed of tanycytes, specialized hypothalamic glia that line the floor of the 3rd ventricle, and microvessels of the median eminence (ME), a circumventricular organ adjacent to the ARH [START_REF] Mullier | Differential distribution of tight junction proteins suggests a role for tanycytes in blood-hypothalamus barrier regulation in the adult mouse brain[END_REF]. While the endothelial cells of the ME are unique in being fenestrated, and thus highly permeable to blood-borne molecules, tight junction complexes between adjacent tanycytes act as a physical barrier preventing their diffusion to the rest of the brain via the CSF. Transcellular transport across the blood-brain and blood-CSF barriers is usually mediated by saturable carriers [START_REF] Banks | Blood-brain barrier and energy balance[END_REF][START_REF] Hawkins | The blood-brain barrier/neurovascular unit in health and disease[END_REF]. However, whether specific hypothalamic areas that regulate energy balance, such as the ARH, directly access peripheral homeostatic signals through the fenestrated microvessel plexus in the adjacent ME is a matter of debate [START_REF] Ciofi | Brain-endocrine interactions: a microvascular route in the mediobasal hypothalamus[END_REF][START_REF] Flier | Obesity wars: molecular progress confronts an expanding epidemic[END_REF][START_REF] Mullier | Differential distribution of tight junction proteins suggests a role for tanycytes in blood-hypothalamus barrier regulation in the adult mouse brain[END_REF].

Here we show that food deprivation, by inducing both tight junction complex reorganization in tanycytes and the increased fenestration and permeability of ME microvessel loops that reach the ventromedial ARH (vmARH) [START_REF] Ambach | The blood supply of the hypothalamus of the rat[END_REF], leads to such direct access. Refeeding, glucose infusion, the selective inhibition of vascular endothelial growth factor (VEGF)-A expression in tanycytes and the manipulation of VEGF signaling all reverse these structural changes and the resulting ability of molecules to enter the ARH.

Results

Fasting-induced plasticity of the blood-hypothalamus barrier (BHB)

To investigate the role of BHB plasticity in the adaptive response to fasting, we compared the hypothalami of mice deprived of food for 24h and those fed ad libitum using immunofluorescence for two constitutive tight junction proteins, zonula occludens-1 (ZO-1) and occludin, expressed in BBB endothelial cells and tanycytes, and claudin-1, expressed in tanycytes at the blood-CSF barrier [START_REF] Mullier | Differential distribution of tight junction proteins suggests a role for tanycytes in blood-hypothalamus barrier regulation in the adult mouse brain[END_REF]. In parallel, antibodies to MECA-32

were used to selectively label the fenestral diaphragms of ME endothelial cells [START_REF] Ciofi | Brain-endocrine interactions: a microvascular route in the mediobasal hypothalamus[END_REF][START_REF] Mullier | Differential distribution of tight junction proteins suggests a role for tanycytes in blood-hypothalamus barrier regulation in the adult mouse brain[END_REF].

Fasting increased the organization of tanycytic tight junction complexes in both the ME and ARH (Fig. 1A,B; Fig. S1A), but not in standard non-ARH hypothalamic vessels composing the BBB (Fig. S1B). Fasting intensified the honeycomb-like pattern of ZO-1 and occludin around the apical pole of tanycytes overlying the ME (Fig. 1A, insets 2,5; Fig. S1A, insets 3,6), and switched ZO-1 and occludin distribution from a diffuse apical pattern to a honeycomb pattern in tanycytes lining the third ventricle (3V) next to the ARH (Fig. 1A, insets 1,4; Fig. S1A, insets 2,5). Claudin-1 expression in ARH tanycytes also became organized in food-deprived mice (Fig. 1C,D). This reorganization was associated with increased protein levels of ZO-1, but not of occludin or claudin-1, in fasting animals (Fig. S1D). Finally, fooddeprived mice refed for an additional 24h displayed a reappearance of the hypothalamic barrier phenotype of fed mice (Fig. 1B-D).

In fasting mice, ME microvessel loops, some of which extend up to the ARH, also demonstrated a marked increase in fenestration associated with changes in MECA-32 distribution (Fig. 1A, insets 1,4; Fig. 1B) and the appearance of fenestrated diaphragms at the ultrastructural level (Fig. 1E, inset 2; Fig. S1C). Western blotting revealed that this increased fenestration was accompanied by a significant increase in MECA-32 protein levels (p<0.05; Fig. 1F). Together, the fasting-induced structural reorganization of the tight junction complexes of tanycytes lining the ventricular wall adjoining the ARH potentially limits paracellular diffusion between the tissue and the CSF, while ME microvessels simultaneously become more leaky. These reversible morphological alterations at the BHB suggest that nutritional state modulates the access of metabolic signals from the periphery to ARH neurons critical for energy homeostasis.

Glucose deprivation mediates fasting-induced plasticity at the BHB

What nutritional factors underlie these profound morphological changes in fasting mice?

Blood glucose acts as a metabolic signal that can alter the activity of hypothalamic neurons [START_REF] Levin | Metabolic sensing and the brain: who, what, where, and how?[END_REF][START_REF] Thorens | Gut-derived signaling molecules and vagal afferents in the control of glucose and energy homeostasis[END_REF]) and evokes robust signaling in tanycytes, which act as glucosensors [START_REF] Frayling | ATP-mediated glucosensing by hypothalamic tanycytes[END_REF], suggesting that reduced glucose levels could underlie the morphological changes seen during fasting. As expected, blood glucose levels were significantly lower after 24h of food deprivation (Fig. 2A, p<0.001). Levels were normalized and fasting-induced BHB reorganization prevented (Fig. 2B) by intravenous (i.v.)

glucose (30%, 1-3 l/min; n = 6) but not saline infusion (n = 4). On the other hand, central neuroglucopenia induced by the intraperitoneal (i.p.) or intracerebroventricular (i.c.v.)

injection of 2-deoxy-D-glucose (2-DG, 300 mg/kg i.p. or 1 mg/mouse i.c.v.; n = 4 each, Fig. 2B; Fig. S2A), a glucose analog that inhibits glucose metabolism but can act on tanycytes [START_REF] Frayling | ATP-mediated glucosensing by hypothalamic tanycytes[END_REF], caused the reorganization of tanycytic tight junction complexes and associated microvessel loops to resemble those in fasting mice. The central detection of glucose deprivation thus appears to play a key role in BHB reorganization after fasting.

VEGF promotes changes in ME microvessel loops and tanycytic tight junction complexes during fasting

Quantitative RT-PCR analyses showed that fasting induced a constellation of hypothalamic transcripts involved in controlling the structural plasticity of the brain (Fig. 2C). However, only VEGF-A expression was upregulated in both fasting and 2-DG-treated animals (Fig. 2C).

VEGF protein levels increased in the hypothalamus of 24h-fasting mice when compared to fed mice (p<0.05; Fig. 2D), and 2DG-treatment for 12h directly triggered VEGF secretion from ME-ARH explants in vitro (p<0.05; Fig. 2E). We therefore treated mice for 24h with Axitinib (25 mg/kg/12h, i.p. or 70 g/mouse, i.c.v.), a tyrosine kinase inhibitor that selectively inhibits VEGF receptors (VEGFR) 1, 2 and 3 [START_REF] Mancuso | Rapid vascular regrowth in tumors after reversal of VEGF inhibition[END_REF]. Axitinib reversed both fasting-and 2DG-evoked changes in tight junction complex organization in tanycytes and the associated microvessel loops in the ME/ARH region (Fig. 2B,F; Fig. S2A,B). Conversely, VEGF infusion (60 g/kg/12h, i.p. or 100 ng/mouse, i.c.v.) for 24h mimicked the effects of fasting on BHB plasticity in fed mice (Fig. 2F,G; Fig. S2B) without affecting tight junction organization in BBB capillaries (Fig. S1B).

Next, we examined the contribution of VEGFR1, 2 and 3, which are all expressed in the mediobasal hypothalamus (MBH; Fig. S2C), to fasting-induced BHB plasticity, by administering selective neutralizing monoclonal antibodies to each receptor (40 mg/kg, i.p.) [START_REF] Pytowski | Complete and specific inhibition of adult lymphatic regeneration by a novel VEGFR-3 neutralizing antibody[END_REF][START_REF] Wang | Targeting autocrine and paracrine VEGF receptor pathways inhibits human lymphoma xenografts in vivo[END_REF]. While antibodies to VEGFR1 and 3 bound to both hypothalamic BBB microvessels and the ME microvessel plexus, antibodies to VEGFR2 bound only to the latter (Fig. S2D). Importantly, the blockade of VEGFR2 and, to a lesser extent, VEGFR1, inhibited the BHB rearrangement observed after fasting (Fig. S2E,F), while antibodies to VEGFR3 had no effect on this plasticity (Fig. S2F). Together, these data suggest that increased hypothalamic VEGF levels during food deprivation target VEGFR1 and 2 in ME endothelial cells to promote microvessel permeability and tight junction complex reorganization in the ME and ARH.

Fasting-induced BHB plasticity requires tanycytic VEGF-A expression

VEGF-A mRNA expression in the hypothalamic tuberal region is restricted to tanycytes (Allen Brain atlas, http://mouse.brain-map.org/experiment/show/74988747, Fig. S3A), suggesting that these cells play a role in the control of VEGF-mediated BHB plasticity. Cell sorting experiments using tdTomato loxP/+ reporter mice in which the tat-cre fusion protein, whose cellular uptake is enhanced compared to cre recombinase [START_REF] Peitz | Ability of the hydrophobic FGF and basic TAT peptides to promote cellular uptake of recombinant Cre recombinase: a tool for efficient genetic engineering of mammalian genomes[END_REF], was stereotaxically infused into the 3 rd ventricle, where it selectively targeted tanycytes (Fig. S2G), to study fasting-dependent changes in tanycytic gene expression in vivo (Fig. 2H).

Sorted Tomato-positive cells abundantly expressed the tanycytic marker DARPP-32 [START_REF] Hokfelt | DARPP-32 as a marker for D-1 dopaminoceptive cells in the rat brain: prenatal development and presence in glial elements (tanycytes) in the basal hypothalamus[END_REF], which was barely detectable in Tomato-negative cells (Fig. S2I). Purified tanycytes also expressed GLUT1, GLUT2 and glucokinase transcripts, as suggested by others [START_REF] Rodriguez | Hypothalamic tanycytes: a key component of brain-endocrine interaction[END_REF], and low levels of VEGFR1 and 2, although VEGFR3 was undetectable (Fig. S2I). Intriguingly, fasting upregulated VEGF-A but not VEGF-B or VEGF-C mRNA expression in tanycytes (Fig. 2H; Fig. S2J), with a concomitant increase in the transcript for hypoxia-inducible factor 1 alpha (HIF-1) (Fig. S2K), recently shown to be involved in hypothalamic glucosensing [START_REF] Zhang | Hypoxia-inducible factor directs POMC gene to mediate hypothalamic glucose sensing and energy balance regulation[END_REF] and known to promote VEGF expression [START_REF] Carmeliet | Role of HIF-1alpha in hypoxia-mediated apoptosis, cell proliferation and tumour angiogenesis[END_REF]. Finally, Vegfa deletion in tanycytes by tat-cre infusion into the third ventricle of Vegfa loxP/loxP mice (Fig. S3A) abolished fasting-induced BHB reorganization (n = 4; Fig. 2F) but did not affect BHB properties in animals fed ad libitum (n = 4; not shown). Together these data demonstrate that tanycytic VEGF-A expression plays a key role in regulating fasting-induced BHB plasticity.

BHB plasticity modulates the access of blood-borne metabolic factors to the ARH

To establish whether the morphological changes described in the MBH above were associated with altered permeability and access of blood-borne molecules to the ARH, the extravasation of intravenously-injected Evans Blue dye from brain microvessels into the hypothalamus was compared between 24h-fasting and fed mice. In fed mice, Evans Blue diffusion was restricted to the vascular bed of hypothalamic BBB microvessels and the ME (Fig. 3A, top panels), and did not spread to neighboring structures such as the ARH. In striking contrast, in 24h-fasting mice, the dye was observed in the ventromedial ARH (vmARH), where plastic BHB changes were observed in previous experiments (Fig. 3A,B).

As with the morphological changes, refeeding reversed dye diffusion into the vmARH (Fig. 3B). Importantly, fasting did not promote dye extravasation from the intrinsic hypothalamic microvessels that compose the BBB (Fig. S3B). The inhibition of BHB reorganization in fooddeprived mice by Axitinib, VEGFR2-neutralizing antibodies, Vegfa gene targeting or glucose infusion prevented dye diffusion into the ARH, whereas the induction of barrier plasticity with VEGF in fed mice clearly elicited such diffusion (Fig. 3B). These findings strongly suggest that the anatomical changes at the BHB in fasting animals facilitate the access of bloodborne signals to the vmARH due to its increased permeability.

To directly test whether fasting-induced structural rearrangements at the BHB do indeed increase the access of critical metabolic substrates to the ARH, we simultaneously assessed glucose levels in the ARH and the adjacent ventromedial nucleus of the hypothalamus (VMH) by microdialysis in fed or 24h-fasting rats (Fig. 3C). First, rats exhibited similar fasting-induced morphological and functional changes to the BHB as did mice (Fig. S3C-E). While ARH glucose levels in fed rats were comparable to those in the VMH (Fig. 3C), in 24h-fasting rats, ARH glucose levels were 300% higher than VMH levels (Fig. 3C). In keeping with the restricted occurrence of microvessels whose permeability changes in response to feeding status in the vmARH, and unlike what would have been expected if the BBB were completely permeable throughout the ARH, ARH glucose levels during fasting ( 2mM) never reached blood levels ( 5mM). However, these data do suggest that fastinginduced structural changes at the BHB create a privileged route for the access of circulating glucose to glucosensing ARH neurons, bypassing both the BBB and the blood-CSF barrier (Fig. 3F).

We further explored this hypothesis by examining the ability of exogenous leptin, a 16-kDa peptide hormone, to access ARH neurons by quantifying the leptin-stimulated phosphorylation of STAT3 in fasting and fed mice. Leptin (3 mg/kg, i.p.) induced a 30% increase in immunoreactivity for phosphorylated STAT3 (pSTAT3) in food-deprived mice when compared to fed mice (Fig. 3D). This increase was restricted to the vmARH (Fig. S3F).

Axitinib (which inhibits BHB rearrangement; Fig. 2F) prevented the leptin-induced increase in pSTAT3 in fasting animals (Fig. 3D,E). Conversely, the treatment of fed mice (in which endogenous leptin levels are significantly higher than in fasting mice; Fig. S3G), with VEGF, which promotes BHB reorganization (Fig. 2F,G), markedly increased STAT3 activation in the ARH (Fig. 3D,E). This effect was blunted by the i.p. injection of a mutated recombinant leptin antagonist (LAN; 3 mg/kg) that is devoid of biological activity but effectively binds to the leptin receptor [START_REF] Niv-Spector | Identification of the hydrophobic strand in the A-B loop of leptin as major binding site III: implications for largescale preparation of potent recombinant human and ovine leptin antagonists[END_REF] 45 min before death (Fig. 3D,E), suggesting that leptin access to the ARH is facilitated by VEGF treatment in fed mice. Together, these data suggest an important role for BHB plasticity in modulating the access of metabolic factors to the ARH.

BHB plasticity modulates feeding

To evaluate the functional consequences of BHB plasticity to feeding behavior, we measured refeeding after fasting in control and Axitinib-treated mice. Compared to vehicle-infused mice refed after a 24h fast, food intake was significantly lower in Axitinib-treated refed mice (Fig. 4A). This difference, which occurred primarily during the first 30 min of refeeding (Fig. 4A), was associated with decreased body weight gain 24h and 48h after Axitinib administration (Fig. 4B). Importantly, Axitinib alone did not inhibit food intake in animals fed ad libitum (Fig. 4A), suggesting that reduced feeding in fasting mice was not due to food aversion.

Conversely, food intake significantly increased in the afternoon and at lights-off (when endogenous anorectic hormones are thought to stimulate food intake) in fed mice 24h after the initiation of VEGF treatment, when compared to vehicle-treated mice (Fig. 4C).

Subsequently, we found that the anorectic and weight-loss-inducing effects of exogenous leptin were greater in fed mice treated with VEGF than in vehicle-treated controls (Fig. 4D).

Together, these findings suggest that VEGF-mediated structural changes at the BHB, by modulating the access of blood-borne metabolic substrates to the ARH, play an important role in the adaptive response to fasting.

Discussion

Energy homeostasis requires increased food intake when energy stores are depleted, and a means of signaling this depletion to central neurons that control feeding behavior [START_REF] Cowley | Leptin activates anorexigenic POMC neurons through a neural network in the arcuate nucleus[END_REF][START_REF] Cowley | The distribution and mechanism of action of ghrelin in the CNS demonstrates a novel hypothalamic circuit regulating energy homeostasis[END_REF][START_REF] Dunn-Meynell | Glucokinase is the likely mediator of glucosensing in both glucose-excited and glucose-inhibited central neurons[END_REF][START_REF] Elias | Leptin differentially regulates NPY and POMC neurons projecting to the lateral hypothalamic area[END_REF][START_REF] Liu | Fasting Activation of AgRP Neurons Requires NMDA Receptors and Involves Spinogenesis and Increased Excitatory Tone[END_REF], such as ARH neurons [START_REF] Hill | Direct insulin and leptin action on proopiomelanocortin neurons is required for normal glucose homeostasis and fertility[END_REF]. Here, we provide evidence that the BHB [START_REF] Mullier | Differential distribution of tight junction proteins suggests a role for tanycytes in blood-hypothalamus barrier regulation in the adult mouse brain[END_REF] undergoes dynamic and reversible structural changes that modulate its permeability in response to glucose and tanycytic VEGF levels, thereby acting as a checkpoint in the access of peripheral metabolic signals to ARH neurons.

Our data show that fasting-evoked dips in blood glucose levels trigger VEGF-A expression in tanycytes and VEGF accumulation in the hypothalamic ME, which acts via VEGFR to promote endothelial cell fenestration. Tanycytes contacting these newly permeable microvessel loops then reorganize their tight junction complexes to seal the paracellular space between the parenchyma and the CSF. In consequence, some target neurons in the vmARH are no longer insulated by the blood-brain and blood-CSF barriers but become directly exposed to peripheral metabolic signals (Fig. 3F), a situation reversed upon refeeding. This increased accessibility is confirmed both by the leakage of intravenously injected dye into the vmARH and by higher physiological glucose levels in the ARH than in the adjacent VMH in the fasting state. Fasting-induced reorganization is blocked by the VEGFR inhibitor Axitinib, which, consistent with the importance of these changes in the adaptive response to fasting, reduces food intake and weight gain when food-deprived mice are refed. Even though Axitinib alone did not affect food intake in control animals fed ad libitum, chronic Axitinib treatment, which causes a marked reduction in endothelial cell [START_REF] Kamba | VEGF-dependent plasticity of fenestrated capillaries in the normal adult microvasculature[END_REF], is known to affect glucose homeostasis [START_REF] Kamba | VEGF-dependent plasticity of fenestrated capillaries in the normal adult microvasculature[END_REF] and to lead to decreased appetite and weight loss in patients [START_REF] Fruehauf | Multicenter, phase II study of axitinib, a selective second-generation inhibitor of vascular endothelial growth factor receptors 1, 2, and 3, in patients with metastatic melanoma[END_REF][START_REF] Rini | Phase II study of axitinib in sorafenib-refractory metastatic renal cell carcinoma[END_REF].

Conversely, triggering BHB permeability in mice fed ad libitum with exogenous VEGF, which signals food deprivation, significantly increases their food intake and sensitivity to the anorectic effects of leptin. We used leptin as a surrogate for other large metabolic peptides such as ghrelin because we could assess its access to the vmARH by monitoring the activation of STAT3. In fact, leptin levels are decreased during fasting, leading to a marked anabolic state within the hypothalamus, with increased neuropeptide Y and agouti-related peptide (AgRP) and decreased proopiomelanocortin levels. This combination acts as a potent stimulus for the animal to seek and ingest food. Contrarily, ghrelin levels increase during fasting, and the increased permeability of ARH microvessels to this peptide could facilitate its selective activation of anabolic AgRP neurons [START_REF] Elmquist | Identifying hypothalamic pathways controlling food intake, body weight, and glucose homeostasis[END_REF][START_REF] Gao | Neurobiology of feeding and energy expenditure[END_REF][START_REF] Levin | Metabolic sensing and the brain: who, what, where, and how?[END_REF][START_REF] Sawchenko | Toward a new neurobiology of energy balance, appetite, and obesity: the anatomists weigh in[END_REF].

Falling blood glucose levels and decreased glucose metabolism appear to be critical signals for the initiation of the BHB response to starvation. Glucose replacement prevents this rearrangement while 2-DG-induced central glucopenia reproduces it. The molecular pathways that underlie the subsequent accumulation of VEGF in the ME are unknown.

However, our results showing that VEGF-A inactivation in these cells blunts fasting-induced BHB plasticity strongly suggest that they are intrinsic to tanycytes, which directly and rapidly respond to changes in glucose levels [START_REF] Frayling | ATP-mediated glucosensing by hypothalamic tanycytes[END_REF]. This response could involve HIF-1, which is upregulated in tanycytes by fasting (Fig. S2J), and is known to be activated by glucoprivation and to promote VEGF expression [START_REF] Carmeliet | Role of HIF-1alpha in hypoxia-mediated apoptosis, cell proliferation and tumour angiogenesis[END_REF][START_REF] Zhang | Hypoxia-inducible factor directs POMC gene to mediate hypothalamic glucose sensing and energy balance regulation[END_REF].

VEGF has long been associated with increased vascular permeability [START_REF] Esser | Vascular endothelial growth factor induces endothelial fenestrations in vitro[END_REF][START_REF] Ioannidou | An in vitro assay reveals a role for the diaphragm protein PV-1 in endothelial fenestra morphogenesis[END_REF] and is required for ependymal cell function and the maintenance of key brain-periphery interfaces such as the choroid plexus [START_REF] Maharaj | VEGF and TGF-beta are required for the maintenance of the choroid plexus and ependyma[END_REF]. VEGF might also contribute to increased BBB permeability in diseased [START_REF] Argaw | VEGF-mediated disruption of endothelial CLN-5 promotes blood-brain barrier breakdown[END_REF] but not healthy brains [START_REF] Hawkins | Rapid, reversible modulation of bloodbrain barrier P-glycoprotein transport activity by vascular endothelial growth factor[END_REF]) (Fig. S1B; Fig. S3A). Our data suggest that VEGF and its signaling receptor, VEGFR2, are key determinants of fasting-induced structural rearrangements at the BHB. Although VEGF involvement in endothelial cell fenestration and the expression of diaphragm proteins (named PV-1 in rats and MECA-32 in mice) is clearly documented [START_REF] Esser | Vascular endothelial growth factor induces endothelial fenestrations in vitro[END_REF][START_REF] Kamba | VEGF-dependent plasticity of fenestrated capillaries in the normal adult microvasculature[END_REF], the mechanisms underlying tight-junction-complex remodeling in tanycytes, which express low levels of VEGFR2, are unknown. These could also involve VEGF signaling, which induces posttranslational modifications in tight junction proteins under some pathological conditions [START_REF] Murakami | Occludin phosphorylation and ubiquitination regulate tight junction trafficking and vascular endothelial growth factorinduced permeability[END_REF], or other as-yet undiscovered signals released by endothelial cells upon fenestration.

Tight junction proteins such as occludin, whose organization is modified in tanycytes under fasting conditions, could also be involved in brain metabolic sensing and body-weight regulation. Occludin-null mice are leaner than their wild-type littermates [START_REF] Saitou | Complex phenotype of mice lacking occludin, a component of tight junction strands[END_REF], and the i.c.v. infusion of antisense oligodeoxynucleotides to occludin restores leptin sensitivity in an animal model of diet-induced leptin resistance and hyperglycemia [START_REF] Oh | Molecular mechanisms associated with leptin resistance: n-3 polyunsaturated fatty acids induce alterations in the tight junction of the brain[END_REF] in which fasting appears not to promote changes in BHB permeability (unpublished observations). This, together with recent findings showing that a high-fat diet triggers neurogenic activity in ME tanycytes [START_REF] Lee | Tanycytes of the hypothalamic median eminence form a diet-responsive neurogenic niche[END_REF], shows that these cells play a dynamic role in metabolic sensing and hold therapeutic potential in metabolic disorders.

Overall our data unveil a new physiological concept in the maintenance of energy homeostasis, in which blood glucose levels, by regulating tanycytic VEGF-A expression, modulate the organization of their tight junctions as well as the permeability of ME capillary loops in the vmARH, and thereby control the access of circulating homeostatic signals to brain circuits that regulate metabolism.

Experimental procedures

Animals

Male C57Bl/6 mice, 3-4 months old (Charles River, France) and male Sprague Dawley rats (Charles River, USA) were given ad libitum access to water and standard laboratory chow.

tdTomato loxP/+ reporter mice were purchased from the Jackson laboratories (Maine, USA) and Vegfa loxP/loxP mice [START_REF] Gerber | VEGF is required for growth and survival in neonatal mice[END_REF] were a gift from N. Ferrara (Novartis, USA). Animal studies were approved by the Institutional Animal Care and Use Committee of Lille and the East Orange Veterans Affairs Medical Center.

Treatment protocols

Glucose infusion and 2-deoxy-D-glucose (2-DG) injection. Mice fed ad libitum and fasting mice were anesthetized with isoflurane and the jugular vein catheterized. After a 7-day recovery period, fed mice were infused with saline solution (0.9%) for 24h while fasting mice were infused with glucose (30% in saline) or saline. Moreover, mice fed ad libitum were given an i.p. or i.c.v. injection of 2-DG (RDS, France) in saline or an equal volume of saline alone, following procedures described previously [START_REF] Mullier | Differential distribution of tight junction proteins suggests a role for tanycytes in blood-hypothalamus barrier regulation in the adult mouse brain[END_REF].

Anti-VEGF and VEGF treatments. Mice were subjected to an i.p. or i.c.v. infusion of Axitinib (in DMSO, LC Laboratories, France) or an equal volume of DMSO during the 24h fasting period. Finally, mice fed ad libitum were given an i.p. or i.c.v. infusion of recombinant mouse VEGF 164 (RDS, France) in PBS for 24h.

Tat-cre delivery. A tat-cre fusion protein produced as detailed previously [START_REF] Peitz | Ability of the hydrophobic FGF and basic TAT peptides to promote cellular uptake of recombinant Cre recombinase: a tool for efficient genetic engineering of mammalian genomes[END_REF] was stereotaxically infused into the third ventricle (1.5 µl over 5 min at 2.1 mg/ml; AP: -1.7 mm, ML: 0 mm DV: -5.6 mm) of isoflurane-anesthetized floxed mice 24h before experiments.

Fluorescence-activated cell-sorting and real-time PCR analyses

Tomato-positive cells were sorted and collected from ME explants microdissected from fasting and fed mice and processed for quantitative RT-PCR, as described in the Supplemental Experimental Procedures.

Physiological measurements

Food intake. Mice were housed 3 per cage with preweighed amounts of food dispensed through the wire cage tops, and food intake was measured every 30 min for the first 3 hours and every hour for 24 hours. The average and cumulative food intake of 3 mice was used for statistical comparisons (n = 4 cages per group).

In vivo leptin sensitivity test. Mice were housed in individual cages two days before the beginning of the experiment. Mice fed ad libitum and fed mice infused with VEGF were injected i.p. at 18:00 with vehicle (5 mM sodium citrate buffer, pH 4.0) or leptin (3 mg/kg, PeproTech, France). Body weight and food consumption were measured at 08:00 the next day.

Permeability assays, immunohistochemistry and image analysis

Mice were given i.v. injections of sterile 1% Evans Blue dye (Sigma, France) in 0.9% saline (50 µl) into the tail vein and killed by decapitation 20 min later. Brains were processed for immunofluorescence as described previously [START_REF] Mullier | Differential distribution of tight junction proteins suggests a role for tanycytes in blood-hypothalamus barrier regulation in the adult mouse brain[END_REF]. The primary antibodies used were: polyclonal rabbit anti-zonula occludens-1 (ZO-1, 1:500, Zymed, USA), rabbit anti-

  of Metabolic Signals to the Arcuate Nucleus in Response to Fasting Fanny Langlet, Barry E. Levin, Serge Luquet, Massimiliano Mazzone, Andrea Messina, Ambrose A. Dunn-Meynell, Eglantine Balland, Amelie Lacombe, Daniele Mazur, Peter Carmeliet, et al. To cite this version: Fanny Langlet, Barry E. Levin, Serge Luquet, Massimiliano Mazzone, Andrea Messina, et al.. Tanycytic VEGF-A Boosts Blood-Hypothalamus Barrier Plasticity and Access of Metabolic Signals to the Arcuate Nucleus in Response to Fasting. Cell Metabolism, 2013, 17 (4), pp.607-617. 10.1016/j.cmet.2013.03.004. inserm-03204462v2

FiguresFigure 1 .

 1 Figures

  Figure 1. Fasting-induced fenestration of ME microvessel loops reaching the ARH, and tight

Figure 2 .Figure 3 .

 23 Figure 2. Fasting-induced BHB plasticity is mediated by glucose deprivation and tanycytic

Figure 4 .

 4 Figure 4. BHB plasticity modulates feeding behavior. (A) Cumulative and absolute food

Acknowledgments

This research was supported by the NEUROBESE International Associated Laboratory (Inserm, SABAN, University of Lille 2; to V.P. and S.G.B.), the Agence National pour la

quantification as described previously [START_REF] Bouret | Distinct roles for specific leptin receptor signals in the development of hypothalamic feeding circuits[END_REF]. Additional details appear in the Supplemental Experimental Procedures.

Microdialysis of the hypothalamus

Placement of hypothalamic cannulae and the assessment of ARH and VMH glucose levels in male rats (n = 9) was performed as described previously [START_REF] Dunn-Meynell | Relationship among brain and blood glucose levels and spontaneous and glucoprivic feeding[END_REF] and detailed in the Supplemental Experimental Procedures.

Immunoblotting

Frozen microdissected ME and MBH of mice fed ad libitum (n = 3) and those fasting for 24h (n = 3) were immunoblotted as described in the Supplemental Experimental Procedures.

Rabbit anti-claudin-1 (1:1000, Zymed, USA), mouse anti-VEGF (1:500, SantaCruz), rat anti-MECA-32 (1:500, Santa Cruz) and goat anti-actin antibodies (1:500, Santa Cruz, France) were used in these experiments.

Statistical analysis

All values are expressed as means  SEM. Data were analyzed for statistical significance with SigmaPlot software (Version 11.0), using one-way or two-way ANOVA followed by a Tukey post hoc test when appropriate. P-values of less than 0.05 were considered to be statistically significant.

Supplemental information

The supplemental information includes four figures, Supplemental Experimental Procedures, and Supplemental References.