Pascal Coumailleau

Elisabeth Pellegrini

Fátima Adrio

Nicolas Diotel

Joel Cano-Nicolau

Ahmed Nasri

Colette Vaillant

Dr Olivier Kah
email: olivier.kah@univ-rennes1.fr

Aromatase, estrogen receptors and brain development in fish and amphibians

Keywords: estrogen, estrogen receptor, aromatase, cyp19a1, neurogenesis, radial glia, plasticity, zebrafish, teleosts, X. laevis

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

Introduction

In 1943, Edward A. Doisy (1893Doisy ( -1986) ) received the Nobel prize for his discovery of the chemical nature of vitamin K, but Doisy and his colleagues should also be remembered as the discoverers of estriol, estrone and estradiol [START_REF] Doisy | The Oestrus--Producing Hormones[END_REF]. Estradiol is a cholesterol derivative mostly known for its actions on the oestrus cycle and the maintenance of female sexual characters. Estradiol was originally considered as a female hormone [START_REF] Doisy | The Oestrus--Producing Hormones[END_REF], until it was found, unexpectedly at the time, that E2 was present in the urine of the stallion [START_REF] Zondek | Estrogenic Hormone in the Urine of the Stallion[END_REF]. This finding set the stage for the discovery that estrogens in fact derive from androgens thanks to the action of cytochrome P450 aromatase, the only enzyme capable of aromatizing the A ring of C19 androgens to convert them into C18 estrogens. With the exception of suidae, in which there are 5 aromatase genes (also named cyp19a1), most vertebrates have a single cyp19a1 gene whose tissue specific expression is driven by multiple aromatase promoters [START_REF] Bulun | Organization of the human aromatase p450 (CYP19) gene[END_REF].

Apart from the gonads, aromatase is expressed in a large variety of tissues such as the bones, the skin, the adrenals, the adipose tissue, the foetal liver, the placenta and some breast cancers. In addition, aromatase is also well expressed in the central nervous system of all vertebrates where it is supposed to play complex and still poorly understood roles [START_REF] Azcoitia | Estradiol synthesis within the human brain[END_REF][START_REF] Garcia--Segura | Aromatase in the brain: not just for reproduction anymore[END_REF][START_REF] Lephart | A review of brain aromatase cytochrome P450, Brain research[END_REF].

Since the seminal work of Alfred Jost on the hormonal control of sex differentiation in the mammalian foetus [START_REF] Jost | Hormonal factors in the sex differentiation of the mammalian foetus[END_REF] and the development of the aromatization hypothesis [START_REF] Maclusky | Estrogen formation in the developing rat brain: sex differences in aromatase activity during early post--natal life[END_REF], our views on aromatase and estrogen functions in the brain are largely influenced by the mammalian literature. In rodents, there is considerable evidence that the masculinization effects of testosterone on the organization of malespecific circuits are caused by aromatization and are in fact mediated by estradiol.

The molecular and cellular mechanisms underlying those estrogenic effects have been thoroughly studied and involve complex age-, sex-and region-specific actions on cell proliferation, apoptosis and differentiation [START_REF] Simerly | Wired on hormones: endocrine regulation of hypothalamic development[END_REF][START_REF] Waters | Estrogen induces caspase--dependent cell death during hypothalamic development[END_REF][START_REF] Gillies | Estrogen actions in the brain and the basis for differential action in men and women: a case for sex--specific medicines[END_REF]. This provides evidence that, at least in this case, estrogens can modulate neurogenesis to cause irreversible sexual dimorphism of some brain structures. In addition, over the last ten years, a number of studies reported potential effects of estrogens and xeno-estrogens, notably bis-phenol A, in either embryonic neurogenesis and/or in the adult neurogenesis in the hippocampus [START_REF] Bayer | Estrogen and the male hippocampus: genetic variation in the aromatase gene predicting serum estrogen is associated with hippocampal gray matter volume in men[END_REF][START_REF] Bowers | A developmental sex difference in hippocampal neurogenesis is mediated by endogenous oestradiol[END_REF][START_REF] Bredemann | 17beta Estradiol increases resilience and improves hippocampal synaptic function in helpless ovariectomized rats[END_REF][START_REF] Suzuki | Neuroprotective effects of estrogens following ischemic stroke[END_REF][START_REF] Waddell | Dysregulation of neonatal hippocampal cell genesis in the androgen insensitive Tfm rat[END_REF]. There has been already several excellent reviews dealing with the putative functions of aromatase in mammals or birds and the role of estrogens in modulating brain functions [START_REF] Gillies | Estrogen actions in the brain and the basis for differential action in men and women: a case for sex--specific medicines[END_REF][START_REF] Czajka--Oraniec | Aromatase research and its clinical significance[END_REF][START_REF] De Nicola | Neuroprotection and sex steroid hormones: evidence of estradiol--mediated protection in hypertensive encephalopathy[END_REF][START_REF] Filova | The effect of testosterone on the formation of brain structures[END_REF][START_REF] Remage--Healey | Estradiol synthesis and action at the synapse: evidence for "synaptocrine" signaling[END_REF][START_REF] Saldanha | Induced synthesis of oestrogens by glia in the songbird brain[END_REF][START_REF] Saldanha | Neuroprotective actions of brain aromatase[END_REF]. On the other hand, a number of reports have shown that upon mechanical or chemical lesions, there is an up regulation of aromatase expression in either reactive astrocytes surrounding the lesions or in radial glial cells (RGCs) facing the lesions as shown in birds, suggesting that aromatase expression in cells of the astroglial lineage would be part of the mechanisms supporting brain repairs after lesion [START_REF] Gillies | Estrogen actions in the brain and the basis for differential action in men and women: a case for sex--specific medicines[END_REF][START_REF] Saldanha | Induced synthesis of oestrogens by glia in the songbird brain[END_REF][START_REF] Garcia--Segura | Aromatase expression by astrocytes after brain injury: implications for local estrogen formation in brain repair[END_REF][START_REF] Luchetti | Gender differences in multiple sclerosis: induction of estrogen signaling in male and progesterone signaling in female lesions[END_REF][START_REF] Peterson | Aromatase expression and cell proliferation following injury of the adult zebra finch hippocampus[END_REF][START_REF] Peterson | Radial glia express aromatase in the injured zebra finch brain[END_REF][START_REF] Peterson | Rapid upregulation of aromatase mRNA and protein following neural injury in the zebra finch (Taeniopygia guttata)[END_REF][START_REF] Spence | Recovery of motor and cognitive function after cerebellar lesions in a songbird: role of estrogens[END_REF]. Thus aromatase can, under certain circumstances, be expressed in cells of the astrocyte lineage, notably under situation of brain repair, while it is admitted in mammals and birds that aromatase is expressed primarily in neurons [START_REF] Balthazart | Distribution of aromatase--immunoreactive cells in the forebrain of zebra finches (Taeniopygia guttata): implications for the neural action of steroids and nuclear definition in the avian hypothalamus[END_REF][START_REF] Balthazart | New insights into the regulation and function of brain estrogen synthase (aromatase)[END_REF].

In contrast to estradiol, testosterone is usually perceived as a male hormone, but this is not the case in all vertebrates. In fish for example, a wealth of data have demonstrated that testosterone is present in the blood of both males and females [START_REF] Campbell | Identification and quantification of steroids in the serum of rainbow trout during spermiation and oocyte maturation[END_REF][START_REF] Scott | Hormone changes during ovulation in the rainbow trout (Salmo gairdneri Richardson)[END_REF] and that fish have some particular non aromatizable androgens, such as 11-ketotestosterone that is found only in males and triggers male secondary sexual characters and sexual behavior [START_REF] Godwin | Neuroendocrinology of sexual plasticity in teleost fishes[END_REF]. Fish are also unique in that they exhibit an amazing sexual plasticity. They are the only vertebrates capable of total sex change, either naturally or upon hormonal treatment. Thus, the above-mentioned aromatization hypothesis probably does not apply to teleost fishes, but it is possible that this sexual plasticity is linked to some of the remarkable characteristics of aromatase expression and functions in the brain of teleost fish that will be reviewed in this article.

Teleost fishes offer an alternative model in which there is a clear neuroanatomical link between estrogen production in the brain and neurogenesis. Indeed, in teleost fishes, radial glial cells strongly express aromatase and the questions are to understand when and why this feature emerged during evolution.

Whether this particular situation is found only in fishes or is also observed in basal tetrapods is unclear. Recently released information on frogs will also be examined in this review for evolutive and comparative purposes.

1-Estrogen receptors, aromatase and radial glia in the brain of fish

Teleost fish (Teleostei) represent one of three infraclasses of Actinopterygians, corresponding to the so-called ray-finned fishes. This is a highly diversified group that arose 280-250 millions years ago and comprises around 26,000 species in about 40 orders and that include most of the living fishes, including the economically-relevant species that are the topic of intense research. A major characteristic of these animals is that, compared to tetrapods, they experienced an additional whole genome duplication that took place early in the emergence of the group. As a result of this duplication, known as the 3R [START_REF] Steinke | Three rounds (1R/2R/3R) of genome duplications and the evolution of the glycolytic pathway in vertebrates[END_REF], teleost fish have potentially twice as much genes than other vertebrates (2:4:8 rule). Although many of these duplicated genes have been lost during evolution, some of them are conserved and developed new or exaggerated functions.

1.1-Estrogen receptors in teleost fishes

The first estrogen receptor cloned in fish was that of the rainbow trout [START_REF] Pakdel | In vivo estrogen induction of hepatic estrogen receptor mRNA and correlation with vitellogenin mRNA in rainbow trout[END_REF][START_REF] Pakdel | Full--length sequence and in vitro expression of rainbow trout estrogen receptor cDNA[END_REF], soon after the cloning of the human ERα (esr1) [START_REF] Green | Human oestrogen receptor cDNA: sequence, expression and homology to v--erb--A[END_REF]. Compared with the mammalian ERα, this rainbow trout (rt ERα) of 65 kDa, named ERα short, exhibited at the Nterminus a deletion of 45 amino acid residues corresponding to the A domain.

However, subsequently a longer ERα form of 71 kDa was retrieved from an ovarian library [START_REF] Metivier | Function of N--terminal transactivation domain of the estrogen receptor requires a potential alpha--helical structure and is negatively regulated by the A domain[END_REF][START_REF] Pakdel | Two estrogen receptor (ER) isoforms with different estrogen dependencies are generated from the trout ER gene[END_REF]. By S1 nuclease protection assays, it was shown that these two isoforms derived from two classes of mRNA generated by an alternative usage of two promoters. Consequently, these mRNA species differ in their 5'-untranslated region and the presence of an ATG in exon 2a permits adding 45 residues at the N-terminus of rtERα-long. Analysis of the transcriptional activities of these isoforms in a yeast cell system demonstrated that, in contrast with rtERα-long, rtERα-short exhibits a ligand-independent transactivation capacity representing 15-25% of the full-length receptor activity. Structural analysis of the AF1 function showed that as it is the case of the mammalian ERα, in the absence of ligand, the A domain of the rtERα-long interacts with the C-terminal region in the absence of ligand, causing inhibition of the AF1 activity located in the B domain [START_REF] Metivier | Function of N--terminal transactivation domain of the estrogen receptor requires a potential alpha--helical structure and is negatively regulated by the A domain[END_REF][START_REF] Pakdel | Two estrogen receptor (ER) isoforms with different estrogen dependencies are generated from the trout ER gene[END_REF]. Studies in rainbow trout showed that the full-length ERα is expressed in liver, brain, pituitary, and ovary, whereas expression of the ERα short is restricted to the liver, demonstrating a tissue-specific expression of these two ERα isoforms [START_REF] Menuet | Tissue--specific expression of two structurally different estrogen receptor alpha isoforms along the female reproductive axis of an oviparous species, the rainbow trout[END_REF].

Following these pioneer studies and the discovery of two estrogen receptors in mammals [START_REF] Kuiper | Cloning of a novel receptor expressed in rat prostate and ovary[END_REF], it rapidly appeared that teleosts have not only one ERα, but also two ERß resulting from an ancient duplication [START_REF] Hawkins | Identification of a third distinct estrogen receptor and reclassification of estrogen receptors in teleosts[END_REF][START_REF] Menuet | Molecular characterization of three estrogen receptor forms in zebrafish: binding characteristics, transactivation properties, and tissue distributions[END_REF]. Zebrafish, probably the bestdocumented fish, has three ER, two ERß, ERß1 (esr2b), and ERß2 (esr2a) and one ERα (esr1). These three estrogen receptors bind estradiol with a high affinity within the 0.5-0.7nM range, similar to what has been observed in mammals [START_REF] Menuet | Molecular characterization of three estrogen receptor forms in zebrafish: binding characteristics, transactivation properties, and tissue distributions[END_REF]. They also have transactivation capacity on reporter genes bearing estrogen responsive element, with ERß2 exhibiting a little more efficiency than the other two receptors, starting at 10 -12 -10 -11 M. All three ERs are expressed in a wide variety of tissues including the brain [START_REF] Menuet | Molecular characterization of three estrogen receptor forms in zebrafish: binding characteristics, transactivation properties, and tissue distributions[END_REF].

One of the characteristics of ERα, at least in the liver, is its spectacular up regulation by its own ligand. This phenomenon has been first deciphered at the molecular level in the rainbow trout and is the basis for the strong induction of vitellogenin expression in the liver upon estrogenic modulation [START_REF] Pakdel | In vivo estrogen induction of hepatic estrogen receptor mRNA and correlation with vitellogenin mRNA in rainbow trout[END_REF]. Such an up regulation of ERα is also observed in the brain, but the induction is much lower than what is observed in the liver [START_REF] Salbert | Localization of the estradiol receptor mRNA in the forebrain of the rainbow trout[END_REF].

-Estrogen receptors in the brain of teleosts

Estrogen receptor expression has been studied in detail in a limited number of species, in particular the rainbow trout [START_REF] Anglade | Distribution of estrogen receptor--immunoreactive cells in the brain of the rainbow trout (Oncorhynchus mykiss)[END_REF][START_REF] Pakdel | Overexpression of rainbow trout estrogen receptor domains in Escherichia coli: characterization and utilization in the production of antibodies for immunoblotting and immunocytochemistry[END_REF], the zebrafish [START_REF] Menuet | Molecular characterization of three estrogen receptor forms in zebrafish: binding characteristics, transactivation properties, and tissue distributions[END_REF][START_REF] Diotel | Aromatase in the brain of teleost fish: expression, regulation and putative functions[END_REF][START_REF] Pellegrini | Relationships between aromatase and estrogen receptors in the brain of teleost fish[END_REF], the European sea bass [START_REF] Muriach | Distribution of estrogen receptor 2 mRNAs (Esr2a and Esr2b) in the brain and pituitary of the sea bass (Dicentrarchus labrax)[END_REF][START_REF] Muriach | Molecular characterization and central distribution of the estradiol receptor alpha (ERalpha) in the sea bass (Dicentrarchus labrax)[END_REF] and the medaka [START_REF] Kanda | Neuroanatomical evidence that kisspeptin directly regulates isotocin and vasotocin neurons[END_REF][START_REF] Zempo | Anatomical distribution of sex steroid hormone receptors in the brain of female medaka[END_REF]. As already documented in mammals, ERα is well expressed in the classical neuroendocrine regions of the brain such as the preoptic area and the mediobasal and caudal hypothalamus, whereas ERßs have a wider distribution in particular along the brain ventricles of the telencephalon and diencephalon. In the preoptic area and the mediobasal hypothalamus, it is very likely that the three receptors are expressed in the same cells (F. Adrio et al., unpublished). Antibodies against the fish estrogen receptors have been developed only in rainbow trout and they are limited to ERα. Although these antibodies permitted to obtain excellent stainings in line with the distribution of the mRNAs, they were rapidly exhausted due to low titer and a necessary purification enrichment step [START_REF] Anglade | Distribution of estrogen receptor--immunoreactive cells in the brain of the rainbow trout (Oncorhynchus mykiss)[END_REF][START_REF] Pakdel | Overexpression of rainbow trout estrogen receptor domains in Escherichia coli: characterization and utilization in the production of antibodies for immunoblotting and immunocytochemistry[END_REF]. Due to the lack of good antibodies, the identity of cells bearing estrogen receptor expression is not entirely clear. However, there are evidences that ERα is expressed primarily in neurons, in particular in dopaminergic neurons of the preoptic region [START_REF] Linard | Estrogen receptors are expressed in a subset of tyrosine hydroxylase--positive neurons of the anterior preoptic region in the rainbow trout[END_REF], GABA neurons [START_REF] Anglade | Distribution of glutamic acid decarboxylase mRNA in the forebrain of the rainbow trout as studied by in situ hybridization[END_REF], GnRH 3 neurons [START_REF] Zempo | Anatomical distribution of sex steroid hormone receptors in the brain of female medaka[END_REF] and kisspeptin neurons [START_REF] Zempo | Anatomical distribution of sex steroid hormone receptors in the brain of female medaka[END_REF][START_REF] Mitani | Hypothalamic Kiss1 but not Kiss2 neurons are involved in estrogen feedback in medaka (Oryzias latipes)[END_REF][START_REF] Escobar | Expression of kisspeptins in the brain and pituitary of the European sea bass (Dicentrarchus labrax)[END_REF]. Because expression of cyp19a1b, encoding aromatase B in fish, is so strongly regulated by E2 through an ER dependent mechanism (see below), it would be expected to observe ER in the RGC. In fact, by increasing the sensitivity of the in situ hybridization techniques, through either amplification of the signal or using radioactive probes with longer exposure, it appears that ERß is likely expressed along the brain ventricles, notably in the telencephalon, preoptic area and mediobasal hypothalamus, but to much lower levels than in neurons of the preoptic area and hypothalamus (Figure 1).

-Radial glia progenitors express aromatase in teleost fishes

A striking feature of the brain of teleost fish is the fact that brain aromatase is massively expressed in radial progenitors of adult animals. Among vertebrates, teleost fish are unique in having two aromatase genes [START_REF] Tchoudakova | Identification of multiple CYP19 genes encoding different cytochrome P450 aromatase isozymes in brain and ovary[END_REF], which clearly derive from the whole genome duplication that occurred at the emergence of the teleost lineage [START_REF] Van Nes | Molecular characterization and expression of two cyp19 (P450 aromatase) genes in embryos, larvae, and adults of Atlantic halibut (Hippoglossus hippoglossus)[END_REF]. One of these genes, cyp19a1a, encodes aromatase A, which is expressed mainly in the gonads, aromatase B, the product of the cyp19a1b gene, is mostly expressed in the brain, but also in the gonads [START_REF] Tchoudakova | Identification of multiple CYP19 genes encoding different cytochrome P450 aromatase isozymes in brain and ovary[END_REF]. Strikingly, it was found, first in toadfish [START_REF] Forlano | Anatomical distribution and cellular basis for high levels of aromatase activity in the brain of teleost fish: aromatase enzyme and mRNA expression identify glia as source[END_REF] and then in rainbow trout and zebrafish [START_REF] Menuet | Expression and estrogen--dependent regulation of the zebrafish brain aromatase gene[END_REF][START_REF] Menuet | Distribution of aromatase mRNA and protein in the brain and pituitary of female rainbow trout: Comparison with estrogen receptor alpha[END_REF], that the aromatase B protein is only expressed in cells resembling radial glial cells. Such cells are present in the embryonic brain of all vertebrate species, but in mammals, they tend to disappear at birth to become star shaped astrocytes [START_REF] Malatesta | Radial glia --from boring cables to stem cell stars[END_REF][START_REF] Pinto | Radial glial cell heterogeneity----the source of diverse progeny in the CNS[END_REF][START_REF] Kriegstein | The glial nature of embryonic and adult neural stem cells[END_REF]. On the contrary, such cells remain abundant in the brain of adult fish, amphibians, birds and reptiles [START_REF] Menuet | Expression and estrogen--dependent regulation of the zebrafish brain aromatase gene[END_REF][START_REF] D'amico | Proliferation, migration and differentiation in juvenile and adult Xenopus laevis brains[END_REF][START_REF] D'amico | The neurogenic factor NeuroD1 is expressed in post--mitotic cells during juvenile and adult Xenopus neurogenesis and not in progenitor or radial glial cells[END_REF][START_REF] Onteniente | Comparative study of the glial fibrillary acidic protein in vertebrates by PAP immunohistochemistry[END_REF][START_REF] Pellegrini | Identification of aromatase--positive radial glial cells as progenitor cells in the ventricular layer of the forebrain in zebrafish[END_REF].

The finding of aromatase expression in RGCs came as a surprise as no one had ever reported such an expression pattern in any other vertebrate. In contrast, in birds and mammals, it is admitted that aromatase expression is strictly confined to neurons under physiological conditions [START_REF] Balthazart | Distribution of aromatase--immunoreactive cells in the forebrain of zebra finches (Taeniopygia guttata): implications for the neural action of steroids and nuclear definition in the avian hypothalamus[END_REF][START_REF] Balthazart | New insights into the regulation and function of brain estrogen synthase (aromatase)[END_REF]. When it was first reported that aromatase is expressed in RGC, virtually nothing was known on this particular cell type in the brain of any adult non-mammalian vertebrate. Since the pioneer studies performed in the late 70s and early 80s by the group of Gloria Callard it was known that the brain of fish exhibits a very high aromatase compared with other vertebrates [START_REF] Callard | Phylogenetic distribution of aromatase and other androgen--converting enzymes in the central nervous system[END_REF][START_REF] Pasmanik | In vivo steroid regulation of aromatase and 5 alpha--reductase in goldfish brain and pituitary[END_REF]. Such a high aromatase was later reported in other species of fish, but the precise sites of expression were unknown [START_REF] Borg | Aromatase activity in the brain of the three-spined stickleback, Gasterosteus aculeatus. I. Distribution and effects of season and photoperiod[END_REF][START_REF] Timmers | Localization of aromatase in the brain of the male African catfish, Clarias gariepinus (Burchell), by microdissection and biochemical identification[END_REF][START_REF] Borg | Aromatase activity in the brain of the three--spined stickleback, Gasterosteus aculeatus. III. Effects of castration under different conditions and of replacement with different androgens[END_REF][START_REF] Gonzalez | Aromatase activity in the European sea bass (Dicentrarchus labrax L.) brain. Distribution and changes in relation to age, sex, and the annual reproductive cycle[END_REF]. It is only in 2001 that, intriguingly, aromatase mRNA and protein were reported in GFAP-positive cells and not in neurons of the toadfish [START_REF] Forlano | Anatomical distribution and cellular basis for high levels of aromatase activity in the brain of teleost fish: aromatase enzyme and mRNA expression identify glia as source[END_REF][START_REF] Forlano | Seasonal plasticity of brain aromatase mRNA expression in glia: divergence across sex and vocal phenotypes[END_REF]. This finding was later confirmed in several other species such as the rainbow trout, the zebrafish, the pejerrey, the Japanese eel, and the medaka [START_REF] Diotel | Aromatase in the brain of teleost fish: expression, regulation and putative functions[END_REF][START_REF] Menuet | Expression and estrogen--dependent regulation of the zebrafish brain aromatase gene[END_REF][START_REF] Menuet | Distribution of aromatase mRNA and protein in the brain and pituitary of female rainbow trout: Comparison with estrogen receptor alpha[END_REF][START_REF] Pellegrini | Identification of aromatase--positive radial glial cells as progenitor cells in the ventricular layer of the forebrain in zebrafish[END_REF][START_REF] Jeng | Expression of aromatase in radial glial cells in the brain of the Japanese eel provides insight into the evolution of the cyp191a gene in Actinopterygians[END_REF][START_REF] Strobl--Mazzulla | Progenitor radial cells and neurogenesis in pejerrey fish forebrain[END_REF][START_REF] Okubo | Sex differences in aromatase gene expression in the medaka brain[END_REF]. In all of these species, aromatase expression is restricted to RGCs which, according to the original definition of early neuroanatomists, are defined as cells exhibiting a small nucleus near the ventricular layer and long processes running radially through the parenchyma and terminating by endfeet at the periphery of the brain [START_REF] Bentivoglio | The history of radial glia[END_REF][START_REF] Garcia--Marin | Cajal's contributions to glia research[END_REF]. These cells were originally believed to serve as a scaffold for migration of new neurons born in the neuroepithelial region [START_REF] Rakic | Neuronal migration and contact guidance in the primate telencephalon[END_REF]. However, since then, a wealth of studies has documented the fact that these cells belong to the astroglial lineage and are actually pluripotent cells [START_REF] Pinto | Radial glial cell heterogeneity----the source of diverse progeny in the CNS[END_REF][START_REF] Xu | Radial Glia, the Keystone of the Development of the Hippocampal Dentate Gyrus[END_REF][START_REF] Morrens | Glial cells in adult neurogenesis[END_REF].

Again, the zebrafish is probably the best-documented species when it comes to aromatase expression and regulation in the brain of fish. In this species the evidence for aromatase expression in RGCs is based on mRNA in situ hybridization, immunohistochemistry using two different kinds of antibodies to zebrafish aromatase B (Figure 2A) and GFP expression driven by cyp19a1b promoter [START_REF] Diotel | Aromatase in the brain of teleost fish: expression, regulation and putative functions[END_REF][START_REF] Pellegrini | Relationships between aromatase and estrogen receptors in the brain of teleost fish[END_REF][START_REF] Menuet | Expression and estrogen--dependent regulation of the zebrafish brain aromatase gene[END_REF][START_REF] Pellegrini | Identification of aromatase--positive radial glial cells as progenitor cells in the ventricular layer of the forebrain in zebrafish[END_REF][START_REF] Tong | A cyp19a1b--gfp (aromatase B) transgenic zebrafish line that expresses GFP in radial glial cells[END_REF].

All these techniques confirm each other and point to the RGCs as the unique cell type expressing aromatase in the zebrafish brain. Such cells are particularly abundant in all regions of the telencephalon, at all levels of the preoptic region, in the thalamus and the hypothalamus, in the optic tectum and the torus semicircularis.

They are also found around the fourth ventricle and in the spinal cord all the way down to the tail. Very interestingly, in the zebrafish the cyp19a1b messengers are observed in the soma lining the ventricles, but also in the radial processes and in the endfeet lining the meninx, where they seem to accumulate [START_REF] Pellegrini | Relationships between aromatase and estrogen receptors in the brain of teleost fish[END_REF]. Surprisingly, in the brain of the rainbow trout, messengers accumulate around the cell nuclei and did not appear to be massively exported in the radial processes [START_REF] Menuet | Expression and estrogen--dependent regulation of the zebrafish brain aromatase gene[END_REF]. The reason for these species differences are unknown at the moment. While no sexual dimorphism in aromatase expression was found in zebrafish, this is not the case in medaka where, in most regions, females have a greater degree of expression in the ventricular layer compared to males, with the optic tectum exhibiting the most conspicuous predominance in females [START_REF] Okubo | Sex differences in aromatase gene expression in the medaka brain[END_REF]. It must be pointed out that studies in European sea bass did not evidence major sexual dimorphism in aromatase activity [START_REF] Gonzalez | Aromatase activity in the European sea bass (Dicentrarchus labrax L.) brain. Distribution and changes in relation to age, sex, and the annual reproductive cycle[END_REF]. It is possible that sex dimorphism is more obvious in medaka, a species with a strong genetic sex determination, unlike zebrafish and sea bass.

-Aromatase expression in the brain of zebrafish is strongly up-regulated by estrogen receptors

In mammals, at least in rodents, aromatase expression and activity is highest during embryonic development and then decrease in adults [START_REF] George | Changes in aromatase activity in the rat brain during embryonic, neonatal, and infantile development[END_REF][START_REF] Lephart | Brain aromatase cytochrome P--450 messenger RNA levels and enzyme activity during prenatal and perinatal development in the rat, Brain research[END_REF]. It is believed that this peak corresponds to the period of brain sexual differentiation which, at least in rodents, relies on aromatization of testosterone produced by the XY embryos [START_REF] Maclusky | Estrogen formation in the developing rat brain: sex differences in aromatase activity during early post--natal life[END_REF].

In contrast, aromatase activity and expression in the brain of the zebrafish, and many other fish species, remain low during the embryonic period and start increasing during the period of sexual maturation corresponding to high levels of circulating sexual steroids such as estradiol and testosterone [START_REF] Gonzalez | Aromatase activity in the European sea bass (Dicentrarchus labrax L.) brain. Distribution and changes in relation to age, sex, and the annual reproductive cycle[END_REF][START_REF] Borg | Aromatase activity in the brain of the three-spined stickleback, Gasterosteus aculeatus. II. Effects of castration in winter[END_REF][START_REF] Pasmanik | Changes in brain aromatase and 5 alpha--reductase activities correlate significantly with seasonal reproductive cycles in goldfish (Carassius auratus)[END_REF][START_REF] Mayer | Effects of castration and androgen treatment on aromatase activity in the brain of mature male Atlantic salmon (Salmo salar L.) parr[END_REF]. In embryos, aromatase expression can be dramatically increased by exposing the animals to any estrogenic compound, including at very low doses [START_REF] Menuet | Expression and estrogen--dependent regulation of the zebrafish brain aromatase gene[END_REF][START_REF] Tong | A cyp19a1b--gfp (aromatase B) transgenic zebrafish line that expresses GFP in radial glial cells[END_REF][START_REF] Brion | Screening estrogenic activities of chemicals or mixtures in vivo using transgenic (cyp19a1b--GFP) zebrafish embryos[END_REF]. In fact, the cyp19a1bgene is so sensitive to estrogens that it has been proposed as a biomarker for estrogenic exposure [START_REF] Brion | Screening estrogenic activities of chemicals or mixtures in vivo using transgenic (cyp19a1b--GFP) zebrafish embryos[END_REF]. For example, the EC50 calculated from dose-response curves of GFP induction in transgenic cyp19a1b-GFP embryos are 0.48 nM and 0.013 nM for 17β-estradiol and 17α-ethinylestradiol, respectively. This relationship between sex steroids levels and aromatase activity has been established long time ago, but it is only in 2005 that molecular dissections of the cyp19a1b promoter showed that an estrogen-responsive element (ERE) located upstream of the transcription start site is absolutely mandatory for up regulating of aromatase expression by estrogens [START_REF] Menuet | Expression and estrogen--dependent regulation of the zebrafish brain aromatase gene[END_REF]. The presence of functional ER is also mandatory as shown by in vitro and in vivo studies [START_REF] Menuet | Expression and estrogen--dependent regulation of the zebrafish brain aromatase gene[END_REF]. Surprisingly, aromatase expression is not observed at all in the numerous neurons expressing ER in any species of fish [START_REF] Menuet | Tissue--specific expression of two structurally different estrogen receptor alpha isoforms along the female reproductive axis of an oviparous species, the rainbow trout[END_REF][START_REF] Menuet | Molecular characterization of three estrogen receptor forms in zebrafish: binding characteristics, transactivation properties, and tissue distributions[END_REF][START_REF] Anglade | Distribution of estrogen receptor--immunoreactive cells in the brain of the rainbow trout (Oncorhynchus mykiss)[END_REF][START_REF] Menuet | Distribution of aromatase mRNA and protein in the brain and pituitary of female rainbow trout: Comparison with estrogen receptor alpha[END_REF], indicating that there is a strict cell specificity of expression of aromatase B in the brain of fish and that, while ER expression is necessary, it is not sufficient. In fact, it was also shown that a sequence, named GxRE, located just upstream the ERE is equally mandatory for the response of the cyp19a1b gene to estradiol [START_REF] Page | Characterization of a cis--acting element involved in cell--specific expression of the zebrafish brain aromatase gene[END_REF]. The hypothesis, which is supported by several lines of evidences is that the GxRE would bind a glial specific factor present in cells of the astrocyte lineage. Indeed, first experiments aiming at dissecting the molecular mechanisms of the estrogenic regulation of cyp19a1b in zebrafish showed that estradiol promotes expression of luciferase from a cyp19a1b-luciferase reporter construct, transfected together with any estrogen receptor, only in cells of the astrocyte lineage, such as U251-MG human astrocytes [START_REF] Menuet | Expression and estrogen--dependent regulation of the zebrafish brain aromatase gene[END_REF][START_REF] Page | Characterization of a cis--acting element involved in cell--specific expression of the zebrafish brain aromatase gene[END_REF]. Additionally, it was shown that, as the GxRE harbours a perfect half binding-site for nuclear receptors (AGGTCA), it is likely that such transcription factors could cooperate with ERs in the context of the cyp19a1b gene. It was also evidenced by gel shift that the GxRE sequence binds nuclear extracts from cells of the astroglial lineage [START_REF] Page | Characterization of a cis--acting element involved in cell--specific expression of the zebrafish brain aromatase gene[END_REF]. However, the identity of this factor still remains uncovered. The fact that the RGC phenotype is absolutely mandatory for aromatase expression is also demonstrated by the observation that, in the transgenic cyp19a1b-GFP fish, GFP expression is rapidly lost in the new-born neuroblasts generated by RGC proliferation when they start migrating [START_REF] Brion | Screening estrogenic activities of chemicals or mixtures in vivo using transgenic (cyp19a1b--GFP) zebrafish embryos[END_REF].

-Aromatase-expressing radial glial cells proliferate and give birth to neurons

It has been know for some time that, in parallel to its dramatic aromatase expression, the brain of fish keeps growing in adult and exhibits an exceptional neurogenic activity in many brain regions [START_REF] Ekstrom | Ventricular proliferation zones in the brain of an adult teleost fish and their relation to neuromeres and migration (secondary matrix) zones[END_REF][START_REF] Kranz | Autoradiographic studies on the localization of the matrix zones of the diencephalon of young and adult Lebistes reticulatus (Teleostae)[END_REF][START_REF] Kranz | Neurogenesis and regeneration in the brain of teleosts in relation to age. (Autoradiographic studies)[END_REF]. Thus, the zebrafish is now a wellrecognized model for studying adult neurogenesis under constitutive and regenerative conditions [START_REF] Pellegrini | Identification of aromatase--positive radial glial cells as progenitor cells in the ventricular layer of the forebrain in zebrafish[END_REF][START_REF] Adolf | Conserved and acquired features of adult neurogenesis in the zebrafish telencephalon[END_REF][START_REF] Chapouton | Adult neurogenesis in non--mammalian vertebrates[END_REF][START_REF] Chapouton | Notch activity levels control the balance between quiescence and recruitment of adult neural stem cells[END_REF][START_REF] Grandel | Neural stem cells and neurogenesis in the adult zebrafish brain: origin, proliferation dynamics, migration and cell fate[END_REF][START_REF] Kaslin | Stem cells in the adult zebrafish cerebellum: initiation and maintenance of a novel stem cell niche[END_REF][START_REF] Marz | Heterogeneity in progenitor cell subtypes in the ventricular zone of the zebrafish adult telencephalon[END_REF][START_REF] Rothenaigner | Clonal analysis by distinct viral vectors identifies bona fide neural stem cells in the adult zebrafish telencephalon and characterizes their division properties and fate[END_REF][START_REF] Zupanc | Adult neurogenesis and neuronal regeneration in the brain of teleost fish[END_REF]. Using combinations of BrdU treatment, PCNA and aromatase as a marker of RGCs, it was shown that aromatase-positive radial cells actively divide to generate newborn cells in many forebrain regions [START_REF] Pellegrini | Identification of aromatase--positive radial glial cells as progenitor cells in the ventricular layer of the forebrain in zebrafish[END_REF]. Such newborn cells can further divide, as shown by BrdU-PCNA (Proliferating Cell Nuclear Antigen) double staining (Figure 2A-C). It was also demonstrated that, over time, new-born cells move away from the ventricles, and migrate along the radial processes, before differentiating in neurons [START_REF] Pellegrini | Identification of aromatase--positive radial glial cells as progenitor cells in the ventricular layer of the forebrain in zebrafish[END_REF][START_REF] Adolf | Conserved and acquired features of adult neurogenesis in the zebrafish telencephalon[END_REF][START_REF] Grandel | Neural stem cells and neurogenesis in the adult zebrafish brain: origin, proliferation dynamics, migration and cell fate[END_REF][START_REF] Marz | Heterogeneity in progenitor cell subtypes in the ventricular zone of the zebrafish adult telencephalon[END_REF].

Given that the estrogen-synthesizing enzyme aromatase B is only expressed in RGCs, acting as neural precursors, this strongly suggests a role for estrogens in constitutive and reactive neurogenesis.

-Effects of estrogens on neurogenesis in fish

2.1-Adult neurogenesis

Recent studies addressed the question of the potential roles of estrogens on zebrafish adult neurogenesis by manipulating the levels of circulating estradiol. For example, treating animals with the aromatase inhibitor ATD caused total disappearance of gonadal and brain aromatase activities and a strong decrease in cyp19a1b gene expression, as expected. Evaluation of the number of PCNA positive cells in both males and females showed a consistent increase in brain cell proliferation that was significant in a limited number of regions [START_REF] Diotel | Effects of estradiol in adult neurogenesis and brain repair in zebrafish[END_REF]. However, inhibition of estrogen receptors using ICI 182,780, which acts as a high affinity antagonist for nuclear receptor (IC50 = 0.29 nM) but presents GPR30 agonist properties, caused a significant increase in the number of PCNA positive cells in the olfactory bulb/telencephalon junction and the mediobasal hypothalamus [START_REF] Diotel | Effects of estradiol in adult neurogenesis and brain repair in zebrafish[END_REF]. These data, suggesting that estradiol inhibits rather than stimulates cell proliferation, were reinforced by the fact that, on the contrary, 17β-estradiol treatment induced a very significant decrease in PCNA positive cells in the telencephalon, mediobasal hypothalamus, periventricular pretectal nucleus and along the posterior recess of the hypothalamus [START_REF] Diotel | Effects of estradiol in adult neurogenesis and brain repair in zebrafish[END_REF]. Furthermore, fish exposure to 17β-estradiol also results in a decrease of newborn cell migration at the olfactory bulb/telencephalon junction and in the mediobasal hypothalamus. Cell survival was also affected by estradiol treatment resulting in a slight survival decrease at the junction between olfactory bulb and the telencephalon. Concerning cell differentiation, no differences were reported in this work [START_REF] Diotel | Effects of estradiol in adult neurogenesis and brain repair in zebrafish[END_REF]. Although these data need to be reinforced, they all suggest that estrogens inhibit cell proliferation and cell migration in adults.

Recently, cell proliferation pattern in the forebrain of adult zebrafish was reported to be sexually dimorphic [START_REF] Ampatzis | Cell proliferation pattern in adult zebrafish forebrain is sexually dimorphic[END_REF]. Ampatzis and colleagues showed that proliferation is higher in females in the medial zone of the dorsal telencephalic area, the pretectal nucleus and the periventricular nucleus of the posterior tuberculum, whereas it is stronger in the dorsal zone of the periventricular hypothalamus in males [START_REF] Ampatzis | Cell proliferation pattern in adult zebrafish forebrain is sexually dimorphic[END_REF]. Such data could reflect sex differences in the steroid and neurosteroids microenvironment and signalling beaten males and females.

2-2 Reparative neurogenesis

In mammals and birds, a wealth of data relates the role of estradiol in the cellular and molecular mechanisms underlying the neurogenic activity that takes place under physiological conditions [START_REF] Bowers | A developmental sex difference in hippocampal neurogenesis is mediated by endogenous oestradiol[END_REF][START_REF] Brock | Short term treatment with estradiol decreases the rate of newly generated cells in the subventricular zone and main olfactory bulb of adult female mice[END_REF][START_REF] Mazzucco | Both estrogen receptor alpha and estrogen receptor beta agonists enhance cell proliferation in the dentate gyrus of adult female rats[END_REF][START_REF] Ormerod | Estradiol enhances neurogenesis in the dentate gyri of adult male meadow voles by increasing the survival of young granule neurons[END_REF] but also in the context of brain repair. Following mechanical and chemical lesions induced in cerebral parenchyma, de novo expression of aromatase is described in reactive astrocytes around the injury site in mammals [START_REF] Garcia--Segura | Aromatase in the brain: not just for reproduction anymore[END_REF][START_REF] Garcia--Segura | Role of astroglia in estrogen regulation of synaptic plasticity and brain repair[END_REF] and in reactive astrocytes and radial glial cells in birds [START_REF] Saldanha | Neuroprotective actions of brain aromatase[END_REF][START_REF] Peterson | Radial glia express aromatase in the injured zebra finch brain[END_REF][START_REF] Pettersson | Role of estrogen receptor beta in estrogen action[END_REF] while under normal condition, the enzyme is mainly localized in neuronal cells. The induction of aromatase mRNA and protein at the injury site is associated with the onset of a high proliferative activity that is thought to contribute to the brain repair, suggesting a strong link between locally produced estradiol and reparative mechanisms. In contrast to the poor capability of the mammalian brain to regenerate after injury, the fish brain possesses a remarkable capacity to reconstruct entire brain regions [START_REF] Zupanc | Adult neurogenesis and neuronal regeneration in the central nervous system of teleost fish[END_REF]. Different studies in zebrafish have documented the fact that lesion of the telencephalon causes very high proliferative activity in the parenchyma and along the brain ventricles, corresponding mainly to the activation of microglia and oligodendrocytes [START_REF] Baumgart | Stab wound injury of the zebrafish telencephalon: a model for comparative analysis of reactive gliosis[END_REF][START_REF] Marz | Regenerative response following stab injury in the adult zebrafish telencephalon[END_REF] and to a certain extent of radial glia [START_REF] Diotel | Effects of estradiol in adult neurogenesis and brain repair in zebrafish[END_REF].

However, aromatase B does not appear to be up-regulated in the proliferating cells [START_REF] Diotel | Effects of estradiol in adult neurogenesis and brain repair in zebrafish[END_REF]. An interesting observation is that, in contrast to birds and mammals, aromatase B expression was rather inhibited in the vicinity of the damaged area. This is consistent with the observed inhibitory effect of estradiol on cell proliferation under physiological situations [START_REF] Diotel | Effects of estradiol in adult neurogenesis and brain repair in zebrafish[END_REF]. Furthermore, treating zebrafish with estradiol or ICI 182,720 does not appeared to affect cell proliferation after lesion [START_REF] Diotel | Effects of estradiol in adult neurogenesis and brain repair in zebrafish[END_REF]. However, E2 impacts on newborn cells differentiation or migration have not been investigating and cannot therefore be excluded.

Taking advantage of the cyp19a1b-GFP transgenic fish line [START_REF] Tong | A cyp19a1b--gfp (aromatase B) transgenic zebrafish line that expresses GFP in radial glial cells[END_REF], our laboratory also showed that estradiol treatment on brain slices results in a rapid modulation of PGC and neuron activities [START_REF] Pellegrini | Expression, regulation and potential functions of aromatase in radial glial cells of the fish brainIn[END_REF]. Indeed, challenging these preparations with 17β-estradiol leads to a rapid increase of intracellular calcium levels in radial glia along the posterior recess of the hypothalamus and also in other cell-types probably corresponding to neurons according to their localization. Such effect of estrogens could possibly occur through the membrane-associated receptor G protein-coupled estrogen receptor 1 (GPER or GPR30), recently reported to be expressed in the adult zebrafish brain [START_REF] Liu | Identification of a membrane estrogen receptor in zebrafish with homology to mammalian GPER and its high expression in early germ cells of the testis[END_REF]. However, further investigations are necessary in order to confirm the presence of Gper in RGCs and to dissect the mechanisms of the rapid modulation on neural stem cell activity and neurons.

-3 Embryonic neurogenesis

We and other groups have shown, that the three estrogen receptors (esr1, esr2b and esr2a) are expressed during early development in zebrafish [START_REF] Bardet | Characterization of oestrogen receptors in zebrafish (Danio rerio)[END_REF][START_REF] Chandrasekar | Levels of 17beta--estradiol receptors expressed in embryonic and adult zebrafish following in vivo treatment of natural or synthetic ligands[END_REF][START_REF] Mouriec | Early regulation of brain aromatase (cyp19a1b) by estrogen receptors during zebrafish development, Developmental dynamics : an official publication of the[END_REF].

The messengers for the three receptors, in particular esr2a, are present in the eggs where they are maternally inherited. Transcripts levels drop down around 4-6 hpf (hour post-fecundation) and start increasing again when the embryonic genome is activated. All three receptor transcripts show a marked increase between 24 hpf and 48 hpf [START_REF] Mouriec | Early regulation of brain aromatase (cyp19a1b) by estrogen receptors during zebrafish development, Developmental dynamics : an official publication of the[END_REF]. Furthermore, esr2a, esr2b and to a lower extent esr1, are detectable by in toto hybridization in different brain areas, mainly the hypothalamus and the preoptic area between 24 and 48 hpf [START_REF] Mouriec | Early regulation of brain aromatase (cyp19a1b) by estrogen receptors during zebrafish development, Developmental dynamics : an official publication of the[END_REF]. In addition, cyp19a1b mRNA show parallel increase, notably between 24 and 48 hpf indicating that aromatase B expression temporally correlates with that of esr [START_REF] Mouriec | Early regulation of brain aromatase (cyp19a1b) by estrogen receptors during zebrafish development, Developmental dynamics : an official publication of the[END_REF]. Furthermore, using the cyp19a1b-GFP embryos, we could show that GFP expression in RGCs can be induced as early as 24 hpf, an effect that can be blocked by ICI182,780, indicating that esr are already functional at this time [START_REF] Mouriec | Early regulation of brain aromatase (cyp19a1b) by estrogen receptors during zebrafish development, Developmental dynamics : an official publication of the[END_REF].

These results were confirmed recently by use of the morpholino (MO) approach showing that esr are fully functional at early developmental stages and exert regulatory actions on estrogen-target genes. Surprisingly, esr2b was the only MO that could block the induction of cyp19a1b [START_REF] Griffin | Morpholino--mediated knockdown of ERalpha, ERbetaa, and ERbetab mRNAs in zebrafish (Danio rerio) embryos reveals differential regulation of estrogen--inducible genes[END_REF]. The development of the lateral line was also studied using MO to block via esr2a showing that this receptor is essential for hair cell development, an effect that could involve interaction with the Notch signalling pathway [START_REF] Froehlicher | Estrogen receptor subtype beta2 is involved in neuromast development in zebrafish (Danio rerio) larvae[END_REF].

Finally, recent studies also suggest a role of the G-protein-coupled estrogen receptor 1, Gper or GPR30 estrogens in brain development of zebrafish [START_REF] Shi | G--protein--coupled estrogen receptor 1 is involved in brain development during zebrafish (Danio rerio) embryogenesis[END_REF]. Gper seems to be strongly expressed in various regions of the developing brain and Gper knockdown by specific morpholinos leads to defects in brain development, causing decreased proliferation in the brain, increased apoptosis and defects in development of sensory and motor neurons [START_REF] Shi | G--protein--coupled estrogen receptor 1 is involved in brain development during zebrafish (Danio rerio) embryogenesis[END_REF].

-Effects of estrogenic endocrine disrupting chemicals on brain development in fish

Because of the above mentioned data showing that estrogens can influence neurogenesis in adults, the impact of estrogenic endocrine disruptors (EDs) on brain development requires attention. It is now clear that the developmental window is critically influenced by many exogenous factors and because these species are very precociously exposed, the structural and functional alterations induced by Eds may lead to significant changes and developmental failures. In light of the observations made in the natural environment, most studies covering the impact of EDs on fish reproduction have focused on the peripheral effects of these molecules. Thus, many data provide information about alterations of gonadal development, egg quality, sperm quality or vitellogenesis disruption [START_REF] Hotchkiss | Fifteen years after "Wingspread"----environmental endocrine disrupters and human and wildlife health: where we are today and where we need to go[END_REF]. Although the peripheral impacts of EDs are clearly established, their actions at the level of the brain are not sufficiently explored and need further considerations.

Based on the extreme sensitivity of the cyp19a1b gene to estrogens, we have used transgenic zebrafish to screen the potential impact of a large spectrum of natural and synthetic molecules (including estrogens, progestagens, androgens) or industrial chemicals (bisphenol A, pesticides). Approximately half of the 45 compounds investigated in that study stimulate in a dose-dependant manner the cyp19a1b-driven GFP expression in radial glia in 5 days larvae. This indicates that many chemicals present in the water can readily access the brain and modify gene expression in brain stem cells. The cyp19a1b gene is not the only gene to be modulated by estradiol or estrogenic EDs. Estradiol, bisphenol A and diéthylstilbestrol also up-regulate expression of ER [START_REF] Chandrasekar | Levels of 17beta--estradiol receptors expressed in embryonic and adult zebrafish following in vivo treatment of natural or synthetic ligands[END_REF][START_REF] Cotter | Multiple structurally distinct ERalpha mRNA variants in zebrafish are differentially expressed by tissue type, stage of development and estrogen exposure[END_REF] and nuclear progesterone receptors in zebrafish embryos [START_REF] Diotel | Nuclear progesterone receptors are up--regulated by estrogens in neurons and radial glial progenitors in the brain of zebrafish[END_REF]. One can expect that a large set of genes is affected by early exposure to estrogens and xenoestrogens with a wide spectrum of consequences in term of brain development and functioning.

Among the potential EDs affecting brain ontogeny, 17α-ethinylestradiol (EE2) is a potent estrogenic molecule widely used in contraceptive pills production and therefore found in waters released from water treatment units. It has been shown that low doses of EE2 disrupts the development of GnRH neuronal network in zebrafish embryos and larvae by increasing the number of GnRH-immunoreactive neurons and fibers in the forebrain and by modifying the migration pattern of GnRH neurons [START_REF] Vosges | 17alpha--Ethinylestradiol and nonylphenol affect the development of forebrain GnRH neurons through an estrogen receptors--dependent pathway[END_REF][START_REF] Vosges | 17alpha--ethinylestradiol disrupts the ontogeny of the forebrain GnRH system and the expression of brain aromatase during early development of zebrafish[END_REF]. However, the underlying mechanisms are unknown. Recent data in our laboratory indicate that exposure to low concentrations of EE2 (10 -10 and 10 -11 M) causes abnormal development, increase esr and npr transcript levels and reduces expression of pcna in the brain (A. Nasri et al., unpublished). While 4-nonylphenol (4-NP) is not estrogenic, nonyphenol mixtures released in water from manufacturing industries and wastewater treatment units possess weak estrogenic activity [START_REF] Brion | Screening estrogenic activities of chemicals or mixtures in vivo using transgenic (cyp19a1b--GFP) zebrafish embryos[END_REF]. The potential effects of NP mixtures on brain development have not been studies so far, however similar to EE2, NP mixtures affect the development of the GnRH systems in developing zebrafish [115[90].

Bisphenol A (BPA), a plastic monomer, has been identified as a xeno-estrogen that can interfere with endogenous estrogen signalling. Since 1950, BPA is a component of a large variety of consumer products, including food containers. Whole mount in situ hybridization has revealed that BPA (5 to 10 µM) induces a strong expression of aromatase B in the brain of developing zebrafish, an induction that is largely dependant of ER nuclear receptors [START_REF] Tse | Early embryogenesis in zebrafish is affected by bisphenol A exposure[END_REF]. In the EASZY screening assay that makes use of cyp19a1b-GFP transgenic zebrafish, the EC50 of BPA is 3 µM, confirming that BPA behaves as a weak estrogen. At this concentration, found in some locations near BPA manufacturing sites [START_REF] Staples | Bisphenol A concentrations in receiving waters near US manufacturing and processing facilities[END_REF], BPA exposure for 5 days induces expression of GFP in radial glial cells indicating that some disruption occurs with unknown consequences [START_REF] Tse | Early embryogenesis in zebrafish is affected by bisphenol A exposure[END_REF]. Whole mount in situ hybridization has revealed that BPA exposure (10 to 75 µM) impairs the normal development during early embryogenesis leading to cell fate determination disruption and organogenesis defects [START_REF] Tse | Early embryogenesis in zebrafish is affected by bisphenol A exposure[END_REF]. In BPA-exposed zebrafish, there is a clear disturbance of the early brain regionalization processes as revealed by inappropriate and unorganized expression of neuronal organization markers as pax2a, otx2, krox2 and eng2b. As a result of these abnormal expressions of regionalization markers during 24 hours of BPA-exposure, rhombomeres are completely unstructured, the mid-hindbrain boundary minimized in size as the midbrain structure [START_REF] Tse | Early embryogenesis in zebrafish is affected by bisphenol A exposure[END_REF]. It must however be pointed out that the dose of BPA (50µM) employed in this study is rather high. Still in zebrafish embryos, BPA treatment (1-15µM) also had adverse impact on motor behavior. The locomotion changes induced by BPA results from an abnormal axonal growth of spinal motoneurons leading to a strong deficit of spontaneaous and stimulated swimming activity of embryos and larvae [START_REF] Wang | Bisphenol A affects axonal growth, musculature and motor behavior in developing zebrafish[END_REF][START_REF] Lam | Toxicogenomic and phenotypic analyses of bisphenol--A early--life exposure toxicity in zebrafish[END_REF]. The use of transcriptomic analysis of BPA-exposed zebrafish larvae of 5 days reveals that several muscular and neuronal markers (including synaptic long-term potentiation or axonal guidance molecules) may be de-regulated by the xeno-estrogen larva [START_REF] Wang | Bisphenol A affects axonal growth, musculature and motor behavior in developing zebrafish[END_REF][START_REF] Lam | Toxicogenomic and phenotypic analyses of bisphenol--A early--life exposure toxicity in zebrafish[END_REF]. BPA exposure (from 10 -8 M to 10 -4 M) was also shown to cause dose-dependent malformations of the otic vesicle in zebrafish and in Xenopus embryos. However, this effect seems to be independent of estrogen receptors or thyroid-hormone receptors [START_REF] Gibert | Bisphenol A induces otolith malformations during vertebrate embryogenesis[END_REF]. BPA (from 0,01 to 1 µM) probably permanently affects brain and behavior development during early life-stages because transient exposure of zebrafish embryos from 8 hpf to 120 hpf elicits in adults long term learning deficits as evidenced by T-maze testing protocol [START_REF] Saili | Neurodevelopmental low--dose bisphenol A exposure leads to early life--stage hyperactivity and learning deficits in adult zebrafish[END_REF]. Recently, it has been shown that early exposures to halogenated-BPA analogs have permanent effects on lipid metabolism in the larvae but also in juveniles. The accumulation of lipids observed in zebrafish treated with halogenated-BPAs correlates with PPARγ (peroxysome proliferatoractivated receptor gamma), a key nuclear factor in the regulation of adipogenesis.

These results strongly suggest that BPA and its analogs may constitute obesogenic compounds whose role in the onset of obesity is increasingly suspected [START_REF] Riu | Halogenated bisphenol--A analogs act as obesogens in zebrafish larvae (Danio rerio)[END_REF].

-Estrogen receptors and aromatase in the brain of amphibians

Until recently, there was no accurate information on the sites of aromatase expression in amphibians, which share common ancestors with the large synapsid group including all mammals. Therefore, it is relevant here to review the available information regarding the situation in amphibians.

.1-Estrogen receptors in the brain of amphibians

Two (ERα, ERß) or three (ERα1, ERα2, ERβ) isoforms of estrogen receptor has been identified in Xenopus Laevis or Xenopus tropicalis, respectively [START_REF] Iwabuchi | Expression profile of the aromatase enzyme in the Xenopus brain and localization of estradiol and estrogen receptors in each tissue[END_REF][START_REF] Lee | Molecular and functional characterization of a novel aryl hydrocarbon receptor isoform, AHR1beta, in the chicken (Gallus gallus)[END_REF][START_REF] Iwabuchi | Isolation of novel isoforms of estrogen receptor genes from Xenopus gonad and brain[END_REF][START_REF] Fumino | Estrogen receptor expression in anomalous arrangement of the pancreaticobiliary duct[END_REF].

Analyses of the estrogen receptors expression in the central nervous system were restricted to specific developmental stages (NF51; 60 and 66) and based on Q-PCR studies [START_REF] Takase | Molecular cloning of two isoforms of Xenopus (Silurana) tropicalis estrogen receptor mRNA and their expression during development[END_REF][START_REF] Wu | Estrogen receptor expression in laryngeal muscle in relation to estrogen--dependent increases in synapse strength[END_REF]. In Xenopus tropicalis, ERα and ERß genes are expressed at low level in the brain at larval stage 51 (before gonadal sex differentiation which occur between NF54 and NF59 stages), but become extremely abundant in the larva at stage 60 (metamorphic climax). They remain expressed at relatively high levels after metamorphosis, i.e at juvenile stages [START_REF] Takase | Molecular cloning of two isoforms of Xenopus (Silurana) tropicalis estrogen receptor mRNA and their expression during development[END_REF][START_REF] Duarte--Guterman | Developmental profiles and thyroid hormone regulation of brain transcripts in frogs: a species comparison with emphasis on Physalaemus pustulosus[END_REF]. The precise distribution of estrogen receptors mRNA or proteins have not been studied during development in amphibian species. To the best of our knowledge, estrogen receptors have only been localized in adult animals, using in situ hybridization, in the brain of the tungara frogs (Physalaemus pustulosus) and, using immunohistochemistry methods, in the male roughskin newt (Taricha granulosa) [START_REF] Chakraborty | Sexually dimorphic androgen and estrogen receptor mRNA expression in the brain of tungara frogs[END_REF][START_REF] Davis | Neuroanatomical distribution of androgen and estrogen receptor--immunoreactive cells in the brain of the male roughskin newt[END_REF][START_REF] Guerriero | Estrogen receptors and aromatase activity in the hypothalamus of the female frog, Rana esculenta. Fluctuations throughout the reproductive cycle[END_REF].. In Physalaemus pustulosus, ERα and ERß expressions were found mostly in limbic areas (e.g. preoptic areas, hypothalamus, nucleus accumbens, striatum, septum, amygdala), parts of thalamus, in the laminar nucleus of the torus semicircularis and the pallium (medial and dorsal).

Males and females had similar anatomical distributions of ERα and ERß expression in the brain. Importantly, no sex differences in the expression levels of ERs were found in the preoptic area, hypothalamus and septal region. However, females had higher ERß and ERα expression than males in the laminar and principal nuclei of the torus semicircularis [START_REF] Chakraborty | Sexually dimorphic androgen and estrogen receptor mRNA expression in the brain of tungara frogs[END_REF]. ERß expression was many-fold higher than ERα in the preoptic area suggesting that ERß plays an important role in modulating sexual behavior in anurans.

4.2-Cyp19a1 gene expression and regulation in the brain of amphibians

In amphibians also, there is now clear evidence that the brain is a steroidogenic organ, and that neurosteroids might play important developmental or physiological roles in the brain [START_REF] Rego | Steroid biosynthesis within the frog brain: a model of neuroendocrine regulation[END_REF][START_REF] Mensah--Nyagan | Regulation of neurosteroid biosynthesis in the frog diencephalon by GABA and endozepines[END_REF][START_REF] Vaudry | Neurosteroid biosynthesis in the brain of amphibians[END_REF]. Studies based on in vitro biochemical analyses performed with brain extracts identified estrogen synthesizing enzyme activity, and therefore possible neuroestrogen synthesis in various adult brain areas of some amphibian species. Briefly, aromatase activity has been detected in the preoptic area, hypothalamus and amygdala of Rana catesbeiana (Necturius maculosus) [START_REF] Callard | Phylogenetic distribution of aromatase and other androgen--converting enzymes in the central nervous system[END_REF]. A high level of aromatase activity was also detected in the hypothalamus of female Rana catesbeiana [START_REF] Guerriero | Estrogen receptors and aromatase activity in the hypothalamus of the female frog, Rana esculenta. Fluctuations throughout the reproductive cycle[END_REF].

The cytochrome P450 aromatase, is encoded by a single cyp19a1 gene in amphibians. In Xenopus laevis and Pleurodeles waltl, cyp19a1 gene expression leads to two gonadal and one brain transcripts differing in their 5-untranslated region (UTR), but containing an identical open reading frame [START_REF] Iwabuchi | Analysis of the p450 aromatase gene expression in the Xenopus brain and gonad[END_REF][START_REF] Kuntz | Cerebral and gonadal aromatase expressions are differently affected during sex differentiation of Pleurodeles waltl[END_REF]. During development, brain expression of cyp19a1 was investigated by RT-PCR experiments in Xenopus [START_REF] Iwabuchi | Analysis of the p450 aromatase gene expression in the Xenopus brain and gonad[END_REF][START_REF] Langlois | Low levels of the herbicide atrazine alter sex ratios and reduce metamorphic success in Rana pipiens tadpoles raised in outdoor mesocosms[END_REF][START_REF] Langlois | Expression profiles of reproduction--and thyroid hormone--related transcripts in the brains of chemically--induced intersex frogs, Sexual development : genetics, molecular biology, evolution, endocrinology, embryology, and pathology of sex determination and differentiation[END_REF][START_REF] Urbatzka | Aromatase, steroid--5--alpha--reductase type 1 and type 2 mRNA expression in gonads and in brain of Xenopus laevis during ontogeny[END_REF] and Pleurodeles waltl [START_REF] Kuntz | Expression of aromatase and steroidogenic factor 1 in the lung of the urodele amphibian Pleurodeles waltl[END_REF]. In both species, the cyp19a1 gene is expressed in the brain from early developmental stages to metamorphosis and no sex differences in aromatase activity was detected at any larval stages [START_REF] Kuntz | Expression of aromatase and steroidogenic factor 1 in the lung of the urodele amphibian Pleurodeles waltl[END_REF].

Recent results in Xenopus laevis provided the first detailed information on the distribution of cyp19a1 mRNAs in amphibian during pre-and in post-metamorphic development. It was found that cyp19a1 expression in the brain is initiated from early larval stage (stage NF42) and remains elevated until post-metamorphic stages [START_REF] Coumailleau | Cyp19a1 (Aromatase) Expression in the Xenopus Brain at Different Developmental Stages[END_REF].

The highest expression was detected during developmental stages in the preoptic area and in the caudal hypothalamus; two regions known for their implication in calling and sexual behavior. Minor sites of cyp19a1 expression were also detected in the septum, bed nucleus of the stria terminalis, amygdala and ventral thalamus.

Importantly, using various cellular markers, it was fond that cyp19a1 transcripts are strictly expressed in neuronal cells, and not in radial glial or progenitor cells located in the ventricular layers of developing Xenopus laevis brain. Recent data also provide evidence that adult males and females exhibit similar expression levels and distribution patterns (P. Coumailleau et al., unpublished). Several attempts were made to localize the aromatase protein, notably in Rana esculenta (Pelophylax esculentus) using commercial antibodies [START_REF] Burrone | Induced synthesis of P450 aromatase and 17beta--estradiol by D--aspartate in frog brain[END_REF]. These data, based on uncharacterized heterologous antibodies, do not fully match aboved in situ hybridization data based on the use of specific X.laevis riboprobe. It thus appears that similar to mammals, immunohistochemistry of aromatase is difficult to achieve in amphibians.

The molecular mechanism underlying the brain-specific cyp19a1 gene expression in Xenopus laevis is depending on a recently identified brain specific promoter, promoter I.f, exhibiting potential cis-elements for several transcriptional factors, such as Oct-1, c-Myc, the GATA gene family, but no estrogen-responsive element [START_REF] Nakagawa | Brain--specific promoter/exon I.f of the cyp19a1 (aromatase) gene in Xenopus laevis[END_REF]. Virtually nothing is known on the potential effects of steroids on brain sexual differentiation. It is admitted that gonadal aromatase plays a key role in the sex differentiation of Xenopus gonads [START_REF] Miyata | In vitro effects of estradiol and aromatase inhibitor treatment on sex differentiation in Xenopus laevis gonads[END_REF][START_REF] Okada | Differential display analysis of gene expression in female--to--male sex--reversing gonads of the frog Rana rugosa[END_REF][START_REF] Villalpando | Determination of the sensitive stages for gonadal sex--reversal in Xenopus laevis tadpoles[END_REF]. In this later species, aromatase activity level fluctuated throughout the reproductive cycle in females with the highest levels during the breeding and recovery periods [START_REF] Guerriero | Estrogen receptors and aromatase activity in the hypothalamus of the female frog, Rana esculenta. Fluctuations throughout the reproductive cycle[END_REF]. Sex steroids are involved in gonadal differentiation in frogs [START_REF] Hayes | Sex determination and primary sex differentiation in amphibians: genetic and developmental mechanisms[END_REF] and amphibians, including Xenopus laevis, are relatively sensitive to steroid hormones when exposed during the critical period of sexual determination and differentiation of the gonads [START_REF] Miyata | In vitro effects of estradiol and aromatase inhibitor treatment on sex differentiation in Xenopus laevis gonads[END_REF][START_REF] Villalpando | Determination of the sensitive stages for gonadal sex--reversal in Xenopus laevis tadpoles[END_REF]. Interestingly, in Xenopus laevis, cyp19a1 is expressed at 5-fold higher level in the brain than in the gonads at the time of sex differentiation [START_REF] Iwabuchi | Analysis of the p450 aromatase gene expression in the Xenopus brain and gonad[END_REF][START_REF] Urbatzka | Aromatase, steroid--5--alpha--reductase type 1 and type 2 mRNA expression in gonads and in brain of Xenopus laevis during ontogeny[END_REF].

4.3-Endocrine disrupting compounds (EDC) in amphibians

Aromatase is considered as one of the potential targets of the many environmental chemicals (EDC) that can interfere with the endocrine system affecting the reproductive biology of aquatic vertebrates [START_REF] Mackenzie | Gonadal differentiation in frogs exposed to estrogenic and antiestrogenic compounds[END_REF][START_REF] Kloas | Amphibians as a model for the study of endocrine disruptors[END_REF][START_REF] Hogan | Estrogenic exposure affects metamorphosis and alters sex ratios in the northern leopard frog (Rana pipiens): identifying critically vulnerable periods of development[END_REF]. In Xenopus, EDCs can interfere with the hypothalamic-pituitary-gonad (HPG) axis and affect various aspects of reproduction via (anti)estrogenic and (anti)androgenic modes of action [START_REF] Szymanski | Aromatase knockout mice show normal steroid--induced activation of gonadotrophin--releasing hormone neurones and luteinising hormone surges with a reduced population of kisspeptin neurones in the rostral hypothalamus[END_REF][START_REF] Kloas | Endocrine disruption in aquatic vertebrates[END_REF][START_REF] Cevasco | Endocrine disrupting chemicals (EDC) with (anti)estrogenic and (anti)androgenic modes of action affecting reproductive biology of Xenopus laevis: II. Effects on gonad histomorphology, Comparative biochemistry and physiology[END_REF].

In addition, a significant correlation exist between exposure to potent xenoestrogens and delay of metamorphosis in various amphibian species [START_REF] Hogan | Estrogenic exposure affects metamorphosis and alters sex ratios in the northern leopard frog (Rana pipiens): identifying critically vulnerable periods of development[END_REF][START_REF] Tompsett | Effects of exposure to 17alpha--ethynylestradiol during larval development on growth, sexual differentiation, and abundances of transcripts in the liver of the wood frog (Lithobates sylvaticus)[END_REF]. The mechanism by which estrogenic substances inhibit larval development might be due to a crosstalk between estrogen receptor and thyroid hormone receptor [START_REF] Hogan | Estrogenic exposure affects metamorphosis and alters sex ratios in the northern leopard frog (Rana pipiens): identifying critically vulnerable periods of development[END_REF][START_REF] Duarte--Guterman | Mechanisms of crosstalk between endocrine systems: Regulation of sex steroid hormone synthesis and action by thyroid hormones[END_REF].

-Evolutive consideration and conclusions

The data obtained recently in Xenopus [START_REF] Coumailleau | Cyp19a1 (Aromatase) Expression in the Xenopus Brain at Different Developmental Stages[END_REF] clearly highlight the apparently unique features of the teleost brain regarding aromatase expression and regulation in radial glial cells. There is a single report in the mammalian literature suggesting that aromatase and ER are expressed in RGCs of the developing cortex of mouse where estrogens would stimulate cell proliferation [167]. However, despite our efforts, we could not confirm theses observations. Thus, teleost fish remain the only vertebrates in which aromatase is so strongly and exclusively expressed in RGCs.

Unfortunately, very little is known in basal groups of vertebrates, which makes it difficult to know what exactly was the ancestral situation in basal vertebrates and how this situation might have evolved over time. Clearly, the role of aromatase and estrogen receptors in the brain of lampreys and sharks would bring valuable information from an evolutive point of view. To our knowledge, there is no report regarding aromatase in lamprey, but it is know that the dogfish exhibits significant aromatase and 5α-reductase activity in the brain [START_REF] Callard | Phylogenetic distribution of aromatase and other androgen--converting enzymes in the central nervous system[END_REF]. In the stingray, a chondrostean fish, a single gene has been cloned with expression in both the brain and the gonad.

In the brain, transcripts were found in all major brain divisions, however the precise sites of expression are unknown [START_REF] Ijiri | Characterization of gonadal and extra--gonadal forms of the cDNA encoding the Atlantic stingray (Dasyatis sabina) cytochrome P450 aromatase (CYP19)[END_REF].

Recently, information was released on the Japanese eel, a basal teleost species, belonging to the Elopomorph order in which there is a single cyp19a1 gene, which is expressed in both the gonads and the brain [START_REF] Ijiri | Characterization of a cDNA encoding P--450 aromatase (CYP19) from Japanese eel ovary and its expression in ovarian follicles during induced ovarian development[END_REF]. Specific antibodies could be produced against the aromatase protein produced by this single gene and it was found, very interestingly, that both cyp19a1 transcripts and protein were unambiguously present in RGCs of both males and females [START_REF] Jeng | Expression of aromatase in radial glial cells in the brain of the Japanese eel provides insight into the evolution of the cyp191a gene in Actinopterygians[END_REF]. It was also shown that aromatase expression in the brain is up-regulated by sex steroids notably estrogens [START_REF] Jeng | Differential expression of neural and gonadal aromatase enzymatic activities in relation to gonadal development in Japanese eel, Anguilla japonica, Journal of experimental zoology[END_REF][START_REF] Jeng | Differential regulation of the expression of cytochrome P450 aromatase, estrogen and androgen receptor subtypes in the brain--pituitary--ovarian axis of the Japanese eel (Anguilla japonica) reveals steroid dependent and independent mechanisms[END_REF]. Thus, this single gene most likely has a brain promoter driving expression and regulation of aromatase in a way similar to what is found in other teleosts with two cyp19a1 genes. As it is known that eels have 8 hox complexes [START_REF] Henkel | Primitive duplicate Hox clusters in the European eel's genome[END_REF] and thus underwent the teleost specific whole genome duplication, it is suggested that from an ancestral gene having brain and gonad functions similar to those found in other teleosts, the 3R whole genome duplication gave birth to two copies that evolved differently in Elopomorphs (Eels) and other teleosts. Soon after the duplication, eels probably lost one copy of the cyp19a1 gene and the remaining copy retained brain and gonad functions. In other teleost fishes, a subfunctionalization process probably occurred that led to partition of functions between the two copies, cyp19a1a (gonad) and cyp19a1b (brain) [START_REF] Jeng | Expression of aromatase in radial glial cells in the brain of the Japanese eel provides insight into the evolution of the cyp191a gene in Actinopterygians[END_REF]. This could mean that the ancestor of the cyp19a1 gene in basal actinopterygian fishes already had such characteristics. However, unfortunately, in the actinopterygian lineage, information is limited to teleosts and data are not available in chondrostei nor in holostei.

The only really clear function that has emerged since researchers started to delve into the potential roles of aromatase expression in the brain is its implication on the masculinization and defeminization of the developing rodent brain, causing irreversible sexualization [START_REF] Maclusky | Estrogen formation in the developing rat brain: sex differences in aromatase activity during early post--natal life[END_REF]. Clearly, this cannot apply to teleost fishes, in which one can find a very large sample of sexual strategies, ranging from strict gonochorism to simultaneous hermaphrodism. Some fish species can rapidly change sex depending on social cues or determine their sex according to the availability of partners [START_REF] Godwin | Neuroendocrinology of sexual plasticity in teleost fishes[END_REF].

Steroid or neurosteroid-dependent expression of aromatase in progenitors and effects of estrogens on neurogenesis certainly open the door to unique possibilities in terms of brain plasticity. However, until conditional knock-out of cyp19a1a and cyp19a1b are performed, it will be very difficult to fully understand what makes the fish so special compared to other vertebrates.

Another interesting point of discussion concerns the fact that several studies mentioned aromatase expression in astrocytes of birds and mammals following chemical or mechanical lesions [START_REF] Garcia--Segura | Aromatase expression by astrocytes after brain injury: implications for local estrogen formation in brain repair[END_REF][START_REF] Peterson | Aromatase expression and cell proliferation following injury of the adult zebra finch hippocampus[END_REF][START_REF] Peterson | Radial glia express aromatase in the injured zebra finch brain[END_REF]. In addition, there are reports indicating that reactive astrocytes can be neurogenic [START_REF] Choi | Distribution of the Immunoreactivity for Glycoprotein M6B in the Neurogenic Niche and Reactive Glia in the Injury Penumbra Following Traumatic Brain Injury in Mice[END_REF][START_REF] Goldshmit | Fgf2 improves functional recovery--decreasing gliosis and increasing radial glia and neural progenitor cells after spinal cord injury[END_REF][START_REF] Guo | The effects of astrocytes on differentiation of neural stem cells are influenced by knock--down of the glutamate transporter, GLT--1[END_REF][START_REF] Saha | Cortical lesion stimulates adult subventricular zone neural progenitor cell proliferation and migration to the site of injury[END_REF] and that astrocytes retain the property of generating neurons under some physiological situations [START_REF] Costa | What determines neurogenic competence in glia?[END_REF]. Teleost fishes, at least in the forebrain, do not possess bona fide astrocytes and it is likely that RGCs in fish share some common features with reactive astrocytes. Because reactive astrocytes are critical for the process of brain repair, we cannot exclude that the outstanding capacity of the teleost brain to regenerate after lesions is linked to these common characteristics.

Legends to figures:

Figure 1 (A-L): Expression of esr2a on a series of transverse sections of the telencephalon and preoptic area in an adult male (A-F) and female zebrafish (G-L) as shown by radioactive in situ hybridization. Note that the highest expression is detected in the preoptic area (BC) and the ventral hypothalamus (J). However, lower expression is observable in many other regions, in particular along the brain

  ventricles and their recessi (arrows). Bar= 100µm ATN: anterior tuberal nucleus; CA: anterior commissure; Cpop: Postoptic commissure; CM: Corpius mammilaris; CO: optic chiasma; CP: Posterior commissure; Dc: central zone of dorsal telencephalic area; Dl: lateral zone of dorsal telencephalic area; Dm, medial zone of dorsal telencephalic area; Dp: posterior zone of dorsal telencephalic area; Hav: ventral habenular nucleus; Hc: central zone of periventricular hypothalamus; Hv: ventral zone of periventricular hypothalamus; MT: Midbrain tegmentum; OT: Optic tract; PGa: anterior preglomerular nucleus; PGl, lateral preglomerular nucleus; PGm: medial preglomerular nucleus; PPa: parvocellular preoptic nucleus, anterior part; PPp: parvocellular preoptic nucleus, posterior part; PTN: posterior tuberal nucleus; RL: lateral recess; RP: Posterior recess; SC: Suprachiasmatic nucleus; TeO: Optic tectum; TLa: torus lateralis; TPp: periventricular nucleus of posterior tuberculum; Vd: dorsal nucleus of dorsal telencephalic area; Vv: ventral nucleus of dorsal telencephalic area; VM: ventromedial thalamic nucleus; Vl: lateral nucleus of dorsal telencephalic area; Zl: zona limitans.

Figure 2 :

 2 Figure 2: Aromatase B-positive cells in the brain of zebrafish exhibit active proliferation. Example of aromatase B positive cells (A) at the level of the nucleus recessi posterioris (NRP) surrounding the posterior recess (rp) of the third ventricle (III). (B) shows that 24 hours after BrdU exposure, BrdU-positive nuclei are visible at the level of the NRP and correspond to the nuclei of aromatase B positive cells as shown in (C) (arrowheads). Bar = 50µm.

Figure 3 :

 3 Figure 3: Schematic representation of aromatase positive-cells RGCs (green cells) in the brain of zebrafish. RGCs can proliferate and generate neuroblasts (red cells) that migrate along the radial processes and give birth to neurons (orange cells. RGCs can produce estrogens that can either act on neighboring neurons or on RGC themselves

to modulate their neurogenic activity. RGCs are also target for peripheral steroids and xeno-hormones.