The insulin signaling pathway is required for adreno-gonadal development

Thesis

Reference

The insulin signaling pathway is required for adreno-gonadal development

PITETTI, Jean-Luc

Abstract

Adrenals and gonads both emerge from a common embryonic structure referred to as the adreno-gonadal primordium (AGP). Insulin-like growth factors (IGFs) provide essential signals for the control of embryonic development. We showed that the in vivo deletion of both Insr and Igf1r is sufficient to induce a complete XY male-to-female sex reversal phenotype due to a failure of upregulating Sry and the subsequent testicular genetic program. In XX and XY mutant gonads, we observed a delay in ovarian differentiation and germ cell entry into meiosis as well as a complete absence of adrenal gland. Later, Sertoli cells (SCs), the only somatic constituents of the seminiferous epithelium are mainly committed to sustain spermatogenesis.

Importantly, the final testis size, the number of germ cell in the adult testis and the sperm output are directly linked to the total number of Sertoli cells. We also demonstrated that mice lacking both Insr and Igf1r specifically in Sertoli cells display a reduction of testicular weight, Sertoli cell number, tubule's length and sperm output by over 70%, probably due to a massive apoptotic wave during [...]

PITETTI, Jean-Luc. The insulin signaling pathway is required for adreno-gonadal development. Thèse de doctorat : Univ. Genève, 2010, no. Sc. 4270

URN : urn:nbn:ch:unige-145421

DOI : 10.13097/archive-ouverte/unige:14542

Available at:

http://archive-ouverte.unige.ch/unige:14542

UNIVERSITÉ DE GENÈVE

Département de Zoologie et FACULTÉ DES SCIENCES de Biologie Animale Professeur François Karch

Département de Médecine Génétique FACULTÉ DE MÉDECINE

et Développement Professeur Jean-Dominique Vassalli Docteur Serge Nef

The Insulin Signaling Pathway Is Required For Adreno-Gonadal Development

THÈSE

présentée à la faculté des sciences de l’Université de Genève pour obtenir le grade de Docteur ès sciences, mention biologie

par Jean-Luc Pitetti

de Genève (GE) Thèse n° 4270

Genève

Atelier d’impression ReproMail, Uni Mail

Contents

I Summary

II Résumé en français

III

Introduction

III.1 Embryonic development of the gonads 8

III.1.1 The adrenogenital primordium development 8

III.1.2 The primordial germ cells 10

III.1.3 The gonadal development 10

III.1.3.1 Sexual differentiation 10

III.1.3.2 Male gonadal development 10

III.1.3.2.1 Sertoli cell differentiation 11

III.1.3.2.2 Sertoli cell proliferation 13

III.1.3.2.3 Testis cord formation 13

III.1.3.2.4 Mesonephric cell migration 14

III.1.3.2.5 Vasculature development 16

III.1.3.2.6 Fetal leydig cell differentiation 18

III.1.3.2.7 Energy storage and consumption 18

III.1.3.3 Female gonadal development 19

III.1.3.4 Molecular factors that regulate the AGP development 20 III.1.3.4.1 The steroidogenic factor 1 (SF-1) 20

III.1.3.4.1.1 Sf1 structure 20

III.1.3.4.1.2 Regulation of Sf1 expression and activity 21 III.1.3.4.1.3 Sf1 insufficiency in mice and humans 21

III.1.3.4.2 Wt1 23

III.1.3.4.3 Odd1 23

III.1.3.4.4 Pbx1 23

III.1.3.4.5 Cited2 24

III.1.3.4.6 Cbx2/M33 24

III.1.3.4.7 Lhx9 24

III.1.4 Molecular pathways that mediate male and female

sex determination 25

III.1.4.1 Two antagonistic cascades 25

III.1.4.2 Molecular pathway regulating testicular development 25

III.1.4.2.1 Sry expression 25

III.1.4.2.2 Sox9 upregulation 27

III.1.4.2.3 SOX9-FGF9 feed forward loop 27

III.1.4.3 The molecular pathway regulating ovarian development 29 III.1.4.3.1 The R-spondin1/Wnt4/ -Cat pathway 29

III.1.4.3.2 The FoxL2 pathway 32

III.2 Postnatal development of the gonads 33

III.2.1 The phenotypic sex 33 III.2.2 Spermatogenesis 33 III.2.2.1 Hormonal regulation of spermatogenesis 33 III.2.3 Factors that regulate adult testis size 36

III.2.3.1 Perinatal SC proliferation 36 III.2.3.1.1 Regulation of SC number and activity by FSH 36

III.2.3.1.2 Regulation of perinatal SC proliferation by thyroid hormones 37

III.2.3.2 The Insulin signaling pathway 39 III.2.3.2.1 Receptor expression 39

III.2.3.2.2 Targeted deletion of the insulin signaling factors 40 III.2.3.2.3 Insulin signaling and sex determination 40

III.2.3.2.4 Insulin signaling and reproduction 40 III.2.3.2.5 Downstream of the insulin signaling pathway 41 III.2.3.2.5.1 The PI3K-AKT pathway 41 III.2.3.2.5.2 The MAPK pathway 42

IV Objectives of the project

V Results

VI Discussion

VII References

VIII Abbreviations

IX Additional results

X Remerciements

I Summary

Adrenals and gonads both emerge from a common embryonic structure referred to as the adreno-gonadal primordium (AGP). As development proceeds, the AGP separates into two distinct regions, the adrenal primordium and the bipotential gonads (Morohashi, 1997)(Morohashi, 1997). The adrenocortical primordium differentiates into the adrenal cortex in both sexes while the bipotential gonads subsequently differentiate into testis or ovary depending on the genetic sex. In XY gonads, the supporting precursor cells differentiate into Sertoli cells (SCs) that act as organizational centers to promote testicular differentiation at the time of sex determination. Later, SCs, the only somatic constituents of the seminiferous epithelium are mainly committed to sustain spermatogenesis. Importantly, individual Sertoli cell can only support a finite number of germ cells. Therefore, the final testis size, the number of germ cell in the adult testis and the sperm output are directly linked to the total number of Sertoli cells. A threshold number of Sertoli cell precursors is a prerequisite for male sex determination and the final number of adult Sertoli cells is the limiting factor for daily sperm production.

In recent years, increasing evidence has emerged that the insulin family of growth factors plays an essential role during gonadal development, as well as gonadal and adrenal function. The insulin signaling pathway is known to be involved in several cellular processes including metabolism, growth, proliferation, differentiation and survival. While specific ablations of the insulin receptor (Insr) and the IGF1 receptor (Igf1r) system, alone or in combination, are associated with embryonic growth retardation and lethality at birth, targeted deletion of the three receptors (Insr, Igf1r and the orphan receptor Irr) results in a complete XY sex reversal in mice.

This thesis project aimed to 1) identify the molecular mechanisms involved in the AGP development and sex determination that are regulated by the Insulin signaling system and 2) to study the paracrine/autocrine effects of the insulin signaling pathway on the establishment of the final pool of Sertoli cells. In the first study, we showed that the deletion of both Insr and Igf1r is sufficient to induce a complete XY male-to-female sex reversal phenotype and that Irr is not required for male sex determination. The absence of testis development in XY double mutant embryos is due to a failure of upregulating Sry and the subsequent testicular genetic program. In XX and XY mutant gonads, we observed a delay in ovarian differentiation and germ cell entry into meiosis. Expression analysis of SF1- positive gonadal cells during sex determination reveals that a significant fraction of the male and female genetic programs are prematurely altered which in itself could explain the incapacity of mutant gonads to develop either into ovaries or testes at the time of sex determination. In addition, both XX and XY mutants display a complete absence of adrenal glands due to the fact that adrenal primordium never formed. Our study suggests that the insulin/IGF signaling pathway is essential for AGP development, correct adrenal specification and gonadal differentiation.

In the second study, we investigated whether the insulin/IGF signaling pathway could play a role during Sertoli cell differentiation and maturation by the time of postnatal development. However, little is known about the molecular mechanisms underlying Sertoli cell differentiation and physiology during late embryogenesis and their subsequent perinatal proliferation, pubertal maturation and adult metabolic activity. Here, we demonstrated that mice lacking both Insr and Igf1r specifically in Sertoli cells display a reduction of testicular weight, Sertoli cell number, tubule’s length and sperm output by over 70%. We found that Sertoli cells undergo a massive apoptotic wave during the period of perinatal proliferation (P5) and that Sertoli cell maturation is delayed thereby affecting the synchrony of the first spermatogenic wave. Despite a severe reduction in testis size and desynchronization of the first wave of spermatogenesis, mutant mice remained fertile. These results showed that the insulin/IGF signaling pathway is the major system controling the number of Sertoli cells and by inference testis size and daily sperm production. Interestingly, expression analysis suggest that germ cells are the main source of IGF1 and IGF2 and may regulate themselves the final pool of Sertoli cells.

Overall, this work provides new evidence concerning the essential role played by the insulin-like growth factors in regulating adrenal development, gonadal differentiation, Sertoli cell number and daily sperm production. Overall, this work improve not only our understanding of the mechanisms mediating adrenal development and sex determination in mammals, but also provide important information on the

metabolic, proliferative and differentiating functions of insulin/IGF receptor family signaling pathways in the developing gonads. Another aspect of this proposal concerns the relevance to humans and the genetic basis of disorders related to sex determination and male reproduction. The underlying cause of such pathologies are too often unidentified, indicating that our understanding of the factors and signaling pathways mediating sex determination and spermatogenesis is far from complete. This work, based on mouse functional genomics, could positively impact the identification and characterization of new factors and pathways involved in adrenogenital development and reproduction. It may also result in the improvement of diagnostic and genetic counselling practices for individuals suffering from sexual, endocrine and reproductive disorders.

II Résumé en français

Les glandes surrénales et les gonades ont pour origine une structure embryonnaire commune, le primordium adréno-gonadique (AGP). Au cours du développement, cette structure se divise en deux ébauches, l'ébauche surrénalienne et la gonade bipotentielle, cette dernière ayant la capacité de se différencier en testicules ou en ovaires en fonction du sexe génétique de l'embryon. Les cellules qui composent les deux ébauches se différencient soit en cellules adréno-corticales du cortex surrénalien soit en cellules des lignées supportrice ou stéroidogénique dans les gonades. Finalement, les cellules supportrices se différencient en cellules de la granulosa dans l’ovaire ou en cellules de Sertoli dans le testicule. Ces dernières cellules jouent le rôle essentiel lors de la détermination sexuelle en fonctionnant comme centre organisationnel et, plus tard, lors de la puberté, en soutenant la maturation et la différenciation des cellules germinales lors de la spermatogénèse. Ainsi, les cellules de Sertoli sont donc essentielles non seulement pour la différenciation testiculaire mais leur nombre final détermine également la production quotidienne de spermatozoïdes au cours de la vie adulte.

La voie de signalisation de l'insuline est impliquée dans plusieurs processus cellulaires tels que le métabolisme, la croissance, la prolifération, la différenciation et la survie. Plusieurs études ont démontré le rôle de la signalisation de l'insuline et des IGFs au cours du développement embryonnaire et de la détermination sexuelle. Les délétions simples ou combinées des deux récepteurs à l’insuline (Insr) et au facteur de croissance de type insuline 1 (Igf1r) sont associées dans les deux cas à un retard de croissance embryonnaire et à une létalité néonatale. En outre, la délétion génique ciblée des trois récepteurs de cette voie de signalisation Insr, Igf1r et du récepteur orphelin (Irr) se traduit par un renversement sexuel complet du mâle vers la femelle chez la souris avec la présence d’ovaires chez les embryons XY. D’autre part, les actions paracrines/autocrines de l’insuline et des IGFs sur les cellules de Sertoli restent à ce jour très mal compris.

Les buts de ce projet de thèse sont i) d’identifier l’implication de la voie de signalisation à l’insuline et des IGFs dans les mécanismes moléculaires du développement adréno-génital et de la différenciation gonadique, et ii) d’étudier les effets paracrines/autocrines de la voie de signalisation de l’insuline sur le nombre final de cellules de Sertoli adultes et la fonction testiculaire.

Tout d’abord, nous avons spécifiquement inactivé les récepteurs à l'insuline (Insr) et au facteur de croissance de type insuline 1 (Igf1r) pendant la détermination sexuelle. Dans cette étude, nous avons montré que la suppression des deux récepteurs Insr et Igf1r est suffisante pour induire un reversement complet du mâle vers la femelle et, que Irr n'a pas de fonction dans la détermination sexuelle. Surtout, nous avons observé que les transcrits de Sry sont fortement réduits dans les crêtes génitales des souris mutantes XY. Par conséquent, le programme génétique mâle n’est pas initié. De manière similaire, le programme ovarien est également altéré de façon prématurée dans les crêtes génitales des souris mutantes XX ce qui provoque un retard de l’expression du programme génétique femelle. Néanmoins, à E16.5 le programme génétique femelle est activé à la fois dans les gonades mutantes XX et XY. De plus, les souris mutantes des deux sexes souffrent d’une aplasie des glandes surrénales. Notre étude suggère donc que les récepteurs INSR et IGF1R sont essentiels pour le développement de l’AGP probablement en régulant l'expression de nombreux facteurs importants pour l’organogenèse correcte des glandes surrénales et pour la détermination sexuelle.

Nous avons également investigué si la voie de signalisation de l'insuline et des IGFs pourrait jouer un rôle lors de la différenciation et la maturation des cellules de Sertoli au cours de la période de développement post-natal. De nombreuses études ont porté sur les causes génétiques et sur les facteurs endocrines/paracrines qui régulent le nombre et la fonction des cellules de Sertoli adultes. Toutefois, les mécanismes moléculaires qui sous-tendent la prolifération, la maturation et la différentiation des cellules de Sertoli restent à ce jour mal compris. Dans ce travail, nous avons démontré que les souris dépourvues de deux récepteurs Insr et Igf1r spécifiquement dans les cellules de Sertoli présentent une réduction du poids des testicules, du nombre de cellules de Sertoli, de la longueur des tubules et de la production de sperme de plus de 70%. Nous avons constaté que les cellules de Sertoli subissent une vague massive d’apoptose au cours de la période de prolifération périnatale (P5) et que la maturation des cellules de

Sertoli est retardée ce qui affecte la synchronisation de la première vague de spermatogenèse. En dépit de la réduction importante de la taille des testicules et de la désynchronisation de la première vague de spermatogenèse, les souris mutantes sont fertiles. Ces résultats suggèrent que se sont les récepteurs INSR et IGF1R qui contrôlent principalement le nombre final de cellules de Sertoli adultes et donc la production quotidienne de spermatozoïdes.

Ce travail de thèse met en évidence le rôle essentiel de la voie de signalisation de l'insuline et des IGFs au cours de la détermination du sexe et du développement des gonades. Plus particulièrement, nous avons identifié de nouvelles voies de signalisation régulées par les récepteurs INSR et IGF1R qui pourraient être impliquées dans la détermination du sexe aussi bien chez le mâle que chez la femelle (par exemple la voie de signalisation de l’acide rétinoïque). De plus, nos résultats suggèrent que les cellules germinales régulent elles-mêmes le nombre de cellules de Sertoli via la sécrétion d’IGF1 et IGF2 nécessaire à la survie et la maturation des cellules de Sertoli.

Ce travail soulève de nouvelles questions sur les mécanismes moléculaires, régulés par les facteurs de croissance de la famille de l’insuline, qui sont impliqués dans le développement du primordium adréno- gonadique et la différenciation des cellules de Sertoli. Il offre également de nouvelles perspectives dans la compréhension des mécanismes des développements sexuels et reproductifs ainsi que de leurs altérations potentielles (désordre du développement sexuel et infertilité).

III Introduction

The goal of this research project was to investigate the role of the Insulin signaling system in both male and female sex determination and reproduction. This introduction follows the developmental chronology of the gonads and is divided in two parts, although in reality they form a continuum. The first part describes the key embryonic processes of gonadal and adrenal development, and the second covers the factors that modulate both the size and the function of the adult gonads. As a central player in both these processes, Sertoli cells orchestrate various aspects of sex determination and go on to sustain spermatogenesis during adulthood. Several genetic factors have been identified in the normal development of both the testes and ovaries. In addition to genetic factors, several endocrine and paracrine signals and downstream pathways support sexual differentiation. Similarly, numerous investigations have focused on the genetic determinants as well as the endocrine and/or paracrine factors that regulate Sertoli cell number and function. Little is known, however, about the molecular mechanisms underlying Sertoli cell differentiation and physiology during embryogenesis and their subsequent perinatal proliferation, pubertal maturation and metabolic activity during adulthood. The insulin signaling pathway mediates both prenatal and postnatal growth. In addition, it is involved in several cellular processes such as metabolism, proliferation, cell survival and differentiation, potentially by coordinating numerous extracellular signals of the PI3K and the MAPK pathways. Evidence from various studies suggests that the insulin signaling pathway is involved in not only key developmental processes, but also in the normal function of fully matured cells.

III.1 Embryonic development of the gonads

III.1.1 The adrenogenital primordium development

In vertebrates, both the gonads and the adrenal cortex, the two main producers of steroids, originate from a common anlage known as the adrenogenital primordium (AGP). In mice, at embryonic day 9.0 (E9.0), the AGP is composed of precursor cells that derive from the coelomic epithelium of the embryonic mesoderm (Bland et al., 2003; Ikeda et al., 1994) . Importantly, these precursor cells all are immunoreactive for the Steroidogenic factor 1 (Sf1/Ad4BP), a key factor of gonad and adrenal development (Morohashi, 1997). In mice, both gonadal and adrenal primordia separate from each other progressively, between E9.5 and E10.5 (Keegan and Hammer, 2002; Morohashi, 1997). By E10.5, the gonadal primordium is composed of two types of cell: the somatic cells and the primordial germ cells (PGCs). At the same time, precursor cells within the adrenal primordium start to differentiate into the three layers of the adrenal cortex: the zonae glomerulosa, fasciculata and reticularis (Val et al., 2007).

Between E12.5 and E13.5, cells originating from the neural crest migrate toward the central part of the developing adrenal cortex and give rise to the medulla (Bland et al., 2003). This entire process finally breaks up at E13.0, by which time both gonads and adrenals have completely individualized, and become two distinct structures (Figure 1) (Hatano et al., 1996).

Figure1. Early adrenal and genital development in mouse embryos. Adrenal cortex and gonads derived from the same embryonic structure: the adrenogenital primordium (AGP, green cells). As development proceeds, adreno-cortical cells progressively separate from the AGP (blue light cells). Remaining cells of the AGP give rise to the bipotential gonad (pink cells).

Concommitantly with the separation of the adrenal primordium, primordial germ cells invade the bipotential gonad. The bipotential gonad develops as a testis upon Sry expression in XY embryos or as an ovary in XX embryos. After the separation from the AGP is completed, the adrenal primordium is encapsulated and colonized by neural crest cells that will give rise to the medulla. Some key factors involved in AGP, adrenal and gonadal development are shown. [Adapted from (Val and Swain, 2010)].

III.1.2 The primordial germ cells

The primordial germ cells (PGCs) originate from the epiblast (embryonic ectoderm), located at the base of the allantois in the region of the developing hindgut, as a group of cells expressing alkaline phosphatase (McLaren, 2000). At first, as hindgut morphogenesis proceeds, proliferating PGCs are drawn along its length. At E9.5, however, they leave the hindgut and begin actively migrating towards the bipotential gonads. The PGCs invade the developing gonads between E10.0 and E11.0, where they continue to proliferate for 2 to 3 days. By E12.5, they cease dividing and are thereafter referred to as gonocytes. It is interesting to note that gonocytes express meiotic factors as early as E13.5, which suggests an intrinsic ability to enter meiosis irrespective of the genetic sex of the gonad (Di Carlo et al., 2000). In spite of this, meiosis is suspended in male gonads and the gonocytes are arrested in G0/G1 of the mitotic cycle as pro-spermatogonia until after birth (McLaren, 1984). At this point, retinoic acid (RA), a diffusible factor produced by the nearby mesonephros, is responsible for inducing germ cell entry into meiosis (Bowles et al., 2006; Koubova et al., 2006). In the testis, SCs produce an enzyme of the p450 family known as CYP26B1, which breaks down the RA into inactive metabolites. This prevents the expression of the Stimulated by Retinoic Acid gene 8 (Stra8) and thus blocks the entry into meiosis. At E13.5, CYP26B1 levels start to decrease in the testis, and the germ cells prevent their own entry into meiosis by producing the factor NANOS2 (Suzuki and Saga, 2008). In contrast, none of the somatic cells of the developing ovary produces CYP26B1, and germ cells are subjected to a retinoic acid-rich environment (Bowles et al., 2006; Bowles and Koopman, 2007; Koubova et al., 2006). As a consequence, the Stra8 gene is upregulated in the ovarian germ cells, allowing them to progress into meiosis until the diplotene stage of meiosis I, at which point they remain until birth.

III.1.3 The gonadal development III.1.3.1 Sexual differentiation

In vertebrates, sex determination is defined as the induction, within the supporting cells of the bipotential gonads, of either a male or female genetic program. Several different mechanisms exist to induce sex determination: genetic mechanisms (e.g. Sry, Sox9 in mammals), environmental mechanisms (e.g.

temperature in crocodiles and turtles) and social mechanisms (population density in fish) (Barske and Capel, 2008). In mammals, sex determination is controlled by a genetic mechanism, and can be separated in three sequential phases. The first phase is genetic sex determination, which is established at fertilization by the donation of one or other of the sex chromosomes from the male gamete. An embryo which has inherited an X chromosome from the father develops as a female, while inheritance of the Y chromosome leads to development along the male pathway. In the second phase, the genetic information is translated into the gonadal sex, and promotes the differentiation of the uncommitted gonads into either a testis or an ovary. Finally, there is the phenotypic sex determination phase, which begins in fetal or early postnatal life, and then continues throughout puberty. This final phase depends on the secretion from the gonads of hormones which direct the differentiation of the accessory ducts, the external genitalia and secondary sexual characteristics.

III.1.3.2 Male gonadal development

As mentioned above, the bipotential gonads originate within the intermediate mesoderm and arise as paired structures of the coelomic epithelium in the region that covers the ventro-medial surface of the mesonephros (Capel, 2000; Nef and Vassalli, 2009a). In mice, the bipotential gonads are identifiable at E10.0, and are composed of two cell lineages: the somatic cell precursors and the PGCs. The precursor cells have the capacity to differentiate as testicular or ovarian cell types, and can give rise to either Sertoli (SCs) and Leydig cells (LCs), or granulosa and theca cells respectively (Albrecht and Eicher, 2001;

Wilhelm et al., 2007b). Depending on the genetic sex of the supporting cell lineage (so named because it sustains germ cell development and maturation in both sexes), PGCs have the ability to differentiate into either spermatogonia or oogonia (McLaren, 1995). The expression of key factors in the supporting cell precursors initiates sex determination, which continues with the activation of sex specific pathways that

promote one fate over another. Male sex determination depends on the expression of a Y-linked gene, Sry (sex determining region on the Y chromosome), which triggers the differentiation of a subpopulation of somatic precursor cells into SCs (Koopman et al., 1991). Sry is critically important in sex determination, being both necessary and sufficient to trigger the male pathway. Studies have shown that XY individuals with a mutation in Sry develop as phenotypically female with functional ovaries, whereas Sry expression in XX mice promotes the male pathway and testes development (Koopman et al., 1991; Lovell-Badge and Robertson, 1990).

III.1.3.2.1 Sertoli cell differentiation

SCs are the first cells to differentiate within the gonad, and this process is the defining event of gonad transition from an uncommitted state into testis development. Analysis of chimerical XXXY embryos revealed that the SC population displays a strong bias for the presence of the Y chromosome (90% of SCs are XY) (Palmer and Burgoyne, 1991; Patek et al., 1991). Sry expression was shown to be restricted to the Sertoli cell lineage and this finding confirms the idea that the unique role of Sry is to trigger the differentiation of the supporting cell precursors into SCs (Clarkson and Harley, 2002; Palmer and Burgoyne, 1991). Interestingly, the remaining 10% of SCs are XX, indicating that non-XY cells are recruited into a Sertoli fate in a paracrine manner (Brennan and Capel, 2004). In vitro experiments show that prostaglandin D2 (PGD2), a secreted factor produced by the SCs, is necessary and sufficient to induce expression of Sox9 in cells negative for Sry expression, and therefore to develop as SCs (Patek et al., 1991; Wilhelm et al., 2007b). This non autonomous recruitment of cells into a “Sertoli” fate has been hypothesized to provide a supplementary mechanism to guarantee that a sufficient number of SCs are present to ensure testicular differentiation (Figure 2). Chimera experiments have also revealed that a minimum of 20% of SC precursor population must be XY to allow development along the male pathway (Patek et al., 1991; Wilhelm et al., 2007b). Once differentiated, SCs act as master organizers and direct the differentiation of other cell types within the male gonad. Following SC differentiation, several testis- specific events take place which result in a thorough reorganization of the gonads. Intensive proliferation of interstitial cells, mesonephric cell migration, vasculature development, compartmentalization and Leydig cell differentiation all contribute to the morphological changes of the XY gonad (Brennan and Capel, 2004).

Figure 2. Model for cell autonomous and non cell autonomous differentiation of SC through PGD2 signalling. Sry induces Sox9 up-regulation in a cell autonomously manner 1). Sox9 then represses Sry expression 2) and maintains its own expression through an autoregulatory loop 3). In addition, both Sry and Sox9 up-regulate Pgds 4) which leads to PGD₂ synthesis and secretion. PGD2 then activate its receptor which leads to the upregulation of Sox9 in an autocrine/paracrine manner 5). This mechanism allows supporting cells that not reach the critical threshold of Sry to upregulate Sox9 and still differentiate into Sertoli cells. [Adapted from (Wilhelm et al., 2005)].

III.1.2.3.2 Sertoli cell proliferation

A specific feature of male development is the rapid increase in testis size as a consequence of an increase in somatic cell proliferation (Hunt and Mittwoch, 1987). This male-specific proliferation occurs in two consecutive steps (Schmahl et al., 2000). The first wave of proliferation occurs immediately before the peak of Sry expression, with the proliferation rate in XY gonads increasing twofold. On the other hand, no such increase is observed in XX gonads. Almost immediately after the first wave of proliferation, a second one occurs with proliferation continuing to increase. This second wave is even more pronounced in XY gonads than the first, when compared to the XX gonads baseline level. Importantly, the first wave of proliferation occurs within SF-1 positive cells at or near the coelomic epithelium, whereas the second wave is concentrated in the ceolomic surface cells negative for SF-1 expression (Karl and Capel, 1998;

Schmahl et al., 2000). Interestingly, both Sertoli and Leydig cells are derived from the Sf1-positive cell population, and when specific proliferation inhibitors are injected at the time of the peak of Sry expression, somatic cell proliferation is abrogated, Sox9 expression is impaired and testis cord formation never occurs (Brennan and Capel, 2004; Schmahl and Capel, 2003). The principal function of the male- specific proliferation of somatic cells is probably to increase the number of SC precursors necessary to reach the threshold of Sry-expressing cells required to initiate male differentiation. Although it is possible that rapid and asymmetrical divisions within the male genital ridges could be required for the SC lineage differentiation (Schmahl and Capel, 2003). However, to date, the factors that regulate the male specific proliferation are unknown.

III.1.3.2.3 Testis cord formation

The intensive proliferation of the coelomic epithelium precedes the first morphological change of the male gonad, namely the formation of rudimentary testis cords (Figure 3) (Schmahl et al., 2000), structures that give rise to seminiferous tubules in adult testis (Kaufman, 1995). Testis cord formation is initiated around E11.5 when SCs aggregate and enclose PGCs. This aggregation is characterized by a transition of SCs from a mesenchymal state to a structured epithelium, and is triggered by changes in both cell-cell interactions, and interactions between cells and the extracellular matrix (ECM) (Frojdman et al., 1992;

Frojdman and Pelliniemi, 1994). Another fundamental event making up part of the formation of testis cords is the polarization of SCs (Frojdman et al., 1989). This event coincides with the establishment of a close association between SCs and the peritubular myoid cells (PTM). It results in the production and accumulation of ECM proteins (e.g. collagens type III, IV and V, laminin, fibronectin) and basal membrane (BM) components at the basal surface of SCs, which actively interact with cytoskeletal components (actin filaments, vimentin, cytokeratin) accumulating at the basal pole of SCs (Kanai et al., 1991; Kanai et al., 1992; Pelliniemi and Frojdman, 2001). This complex association of ECM proteins and adhesion molecules gives rise to the basal lamina, which together with PTM cells defines the frontier between the developing interstitial compartment and the testis cords (Tung et al., 1984). This boundary performs the role of a non-specific barrier so as to prevent vasculature penetration, immune cell passage and macromolecule diffusion into the testis cords (Cool et al., 2008; Skinner et al., 1985). The synchronization of development and differentiation of SCs and PTM cells is of crucial importance to the formation of testis cords since their agenesis or dysgenesis can be linked with either SC- or PTM cell- associated problems. On the other hand, PGCs appear to have no role in, testis cord formation, which proceeds normally in the absence of PGCs (Buehr et al., 1993b).

To date, very few genes have been associated with the correct function of PTM cells. Dax1 (the dosage- sensitive sex reversal, adrenal hypoplasia congenital, critical region on the X chromosome, gene1) is involved in PTM cell differentiation, and therefore correct basal lamina deposition (Meeks et al., 2003).

Dax1^-/- mice display either a complete absence of or a delay in testis cord formation, a disruption of the basal lamina and an aberrant location of fetal LCs. In this case, testis cords formation fails due to the reduced number and altered differentiation process of PTM cells by E13.5.

An additional key factor, desert hedgehog (Dhh), is produced and secreted by SCs and plays a role in PTM function (Clark et al., 2000; Pierucci-Alves et al., 2001). Dhh, which belongs to the hedgehog family of genes, begins being expressed in male gonads at E10.5 (Bitgood and McMahon, 1995). DHH binds Patched (Ptc), its receptor, which is expressed only in male embryonic gonads at E11.5. In absence

of DHH, PTC normally interacts with SMOOTHENED (Smo) and represses its intracellular signalization.

Upon DHH binding however, Smo is released allowing the upregulation of GLI transcription factors (Capdevila and lzpisua Belmonte, 1999; Walterhouse et al., 1999). Interestingly, the disruption of Dhh leads to reduced testis size and absence of spermatogenesis in adult XY mutants. The PTM cells remain immature and fail to form a continuous layer of cells. As a consequence, the basal lamina is either breached or totally absent around testis cords, leading to the mislocalization of germ cells within the interstitial epithelium and their entry into meiosis (see section I.2).

III.1.3.2.4 Mesonephric cell migration

Testis cord formation within the XY gonad also requires the migration of somatic cells from the adjacent mesonephros. Significantly, the only cells migrating from the mesonephros into the genital ridges are cells with an endothelial origin (Combes et al., 2009; Cool et al., 2008). XY genital ridges cultured for several days in absence of the adjacent mesonephros fail to form testis cords. Similarly, when a semi- permeable membrane is placed between the XY genital ridge and the mesonephros, no defined testis cords develop (Buehr et al., 1993a; Martineau et al., 1997; Merchant-Larios et al., 1993). Gonad culture experiments have subsequently demonstrated that mesonephric cell migration is a male-specific event, and is dependent on Sry expression (Albrecht et al., 2000; Capel et al., 1999). Nevertheless, cell migration still occurs after Sry is switched off, indicating that the signal directing cell migration is not Sry itself but results from an indirect consequence of Sry expression. Moreover, gonad sandwich experiments, which involve coculturing genital ridges with mesonephros of distinct origin, clearly demonstrate that this signal is efficient at long ranges (100Pm) and acts as chemoattractant (figure 3) (Martineau et al., 1997).

Testis cord formation, while independent of gonadotropin signaling, requires paracrine factors secreted within the testis, which act as chemotactic agents to induce mesonephric cell migration (Colenbrander et al., 1979; Cupp et al., 2000). The factors involved in the migration of mesonephric cells are known to include neurotropin-3 (NT3) and platelet-derived growth factors (PDGFs). Neurotrophins and their cognate receptors are involved in mesenchymal-epithelial cell interactions during tissue remodeling and are expressed in the testis during sex determination (Cupp et al., 2000; Levine et al., 2000; Tessarollo et al., 1993). More precisely, SCs secrete NT3, and mesonephric migrating cells express its high-affinity receptor trkC. TrkC^-/- mice display a reduction in the number of testis cords during development, probably as a consequence of disturbed cell migration. Consistent with this interpretation, cell migration fails in male gonads cultured with a specific inhibitor of the trkC receptor (AG879) that impairs NT3 signaling (Cupp et al., 2002; Cupp et al., 2003). Other paracrine factors such as platelet-derived growth factors (PDGFs) are known to play a role in mesenchymal-epithelial interactions and endothelial cell migration (Betsholtz et al., 2001). PDGF signaling modulates several cellular processes including proliferation, migration, differentiation and chemotaxis, in multiple cell types. PDGFs bind to two distinct receptors – PDGFR and – and the resultant signaling depends on the different combinations of the receptor isoforms. For instance, - or -homodimers mainly mediate mitogenic effects, chemotaxis, actin remodeling, Ca²⁺ mobilization and cell survival; whereas heterodimers are only involved in the strong activation of proliferation and chemotaxis (Heldin and Westermark, 1999). Interestingly, in vitro experiments using an inhibitor of PDGFs (tyrphostin) showed that testis cords did form, but they were reduced in number with an enlarged appearance (Uzumcu et al., 2002). Unlike neurotropin blockers, inhibitors of PDGF signaling did not block testis cord formation, but altered their normal development and gross morphology. These results were confirmed using Pdgfr^-/- mutants, in which testis cord formation is delayed at E12.5, and by E13.5 only few dilated cord-like structures are seen. Strikingly, no fetal LCs differentiate in between the swollen cords, due to a delay in Dhh expression. Furthermore, a

A B

Figure 3. Schema of the gonad culture system. A. Urogenital ridges are dissected out from XX or XY embryos and mesonephroi and genital ridges are separated from each other and reassembled in agar blocks prior to be cultured for 24 to 48 hours. B. Cell migration from adjacent mesonephros only occurs if the gonad is XY but not if it is XX. [Adapted from (Martineau et al., 1997)].

large coelomic vessel forms but fails to subdivide into branches in between forming testis cords (Brennan et al., 2002; Brennan et al., 2003). Taken together, these results show that the disruption of PDGF signaling impairs the migration of endothelial cell into the gonads, resulting in testis cord disorganization.

Mesonephric cell migration is therefore an essential mechanism involved in the process of testis morphogenesis (Brennan et al., 2003; Smith et al., 2005).

III.1.3.2.5 Vasculature development

The future testis, as one of the main producers of androgens, must develop a substantial vasculature in order to export sufficient testosterone to the rest of the embryo to promote its subsequent masculinization.

At E11.5, when Sry reaches its peak of expression, both XX and XY vasculature developments are identical. Following Sry expression, however, endothelial cells migrate within the testis and then aggregate near the coelomic surface to form the major testicular artery: the coelomic vessel (Brennan et al., 2002; Combes et al., 2009; Coveney et al., 2008). More precisely, after entering the gonad, pioneer migrating cells randomly extend filipodia, some of which connect to the coelomic surface. The cellular extensions that do make connections will be maintained, and serve as a guide to the cell body. On the other hand, filipodia that do not succeed in making contact with the coelomic surface are retracted. Once initial paths have been established by the pioneer cells, other cells follow, resulting in the partitioning of the gonad to around 10 sub-compartments devoid of vasculature. Within these compartments, SCs aggregate and enclose germ cells so as to form the nascent testis cords. These findings have led to the conceptualization of a model in which SCs together with migrating endothelial cells orchestrate testis morphogenesis. The migrating endothelial cells have an instructive role in modifying the components of the ECM, thereby facilitating the path of subsequent migrating cells (Figure 4).

Figure 4. Model of testis cord development controled by Sertoli and endothelial cells. A. In early genital ridges Sertoli and germ cells are randomly distributed. B. Nascent testis cords appeared by the time Sertoli cells surround cluster of germ cells. C.

These latters are subsequently definitely partitioned into testis cords when endothelial cells migrate to form the coelomic vessel.

D. Finally, Testis cords associated with peritubular myoid cells so as to form multiple maturing testis cords. g, gonad; m, mesonepros. [Adapted from (Combes et al., 2009)].

III.1.3.2.6 Fetal Leydig cells

Another testis-specific event is the differentiation of the steroidogenic cell lineage known as the Leydig cells (LCs). LCs are essential because they produce androgens, mainly by converting cholesterol into testosterone. In mammals, at least two distinct populations of LCs arise sequentially during the life of an individual. Initially, during development, the fetal LCs are required for the masculinization of the rest of the embryo. During puberty, these will be replaced by adult LCs which are required to sustain spermatogenesis and male reproductive function (Habert et al., 2001; O'Shaughnessy et al., 2006).

Once testis cord formation has been attained, fetal LC differentiation occurs within the interstitial compartment, where they begin to synthesize testosterone (Byskov, 1986; Habert et al., 2001). To date, fetal LC origin remains obscure, although cells with either a mesonephrotic or a neural crest origin have been hypothesized to be fetal LCs precursors (Buehr et al., 1993a; Jeays-Ward et al., 2003; Mayerhofer et al., 1996). In contrast, Barsoum and Yao suggested that a pool of cells within the interstitial compartment, which remain SF1-positive, give rise to fetal LCs (Barsoum and Yao, 2010).

The differentiation of fetal LCs requires two Sertoli-secreted factors: Dhh and Pdgfa. After its secretion by SCs, DHH binds its specific receptor, Patched (Ptc), on the LC surface. This leads to the upregulation of both Sf1 and P450 side chain cleavage (P450SCC), which thus specifies Leydig cell fate (Yao et al., 2002)., When Dhh is disrupted in mice, fetal LCs consistently fail to differentiate or mature into adult LCs (Figure 3). Another critical factor involved in fetal LC differentiation is Pdgfr- (Brennan et al., 2003). In Pdgfr-^-/- XY gonads, the precursor cells of the Leydig lineage do not undergo fetal LC differentiation. As a consequence the testis size is reduced and spermatogenesis is prematurely arrested, most likely due to lower levels of testosterone. These differentiating factors are antagonized by the Notch signaling pathway which, in contrast, promotes the maintenance of precursor cells in a progenitor state and inhibits their differentiation into fetal LCs (Tang et al., 2008). Indeed, the inhibition of the Notch signaling pathway within the precursor cell population induces an increase in the number of fully differentiated fetal LCs (Tang et al., 2008).

III.1.3.2.7 Energy storage and consumption

For all these morphological changes to occur, developing XY gonads require a sizeable amount of storable energy. Glycogen is a readily available source of energy, and is frequently stored by cells prior to the onset of morphological, physiological and/or developmental changes (Ferrer et al., 2003). It is therefore noteworthy that one of the earliest events downstream of Sry expression, and concomitant with interstitial cell proliferation, is the accumulation of glycogen within SC precursors. This accumulation is a prerequisite for testis cord formation (Matoba et al., 2005). Interestingly, XY-explants of male gonads cultured in glucose deprived medium (GD) fail to accumulate glycogen within pre-SCs. In accordance with this, E11.5 XY genital ridges cultured in GD medium show a complete absence of testis cords, which correlates with a reduction of Sox9 expression. Moreover, both laminin and collagen IX alpha 3 (Col9a3), two genes whose products participate in basal lamina deposition, have reduced expression levels. Notably, this glycogen storage is mediated mainly by the PI3K/Akt pathway in pre-SCs. Taken together this data shows that a massive energy source, in the form of glycogen storage within the pre-SCs, is a prerequisite for the expression of Sox9, and subsequent SC differentiation and testis cord formation.

Besides the signaling pathways that have been identified as dimorphic, it appears that energy metabolism is also dimorphic between males and females during sex determination. However, the signaling pathway that mediates glycogen synthesis and storage is still unknown.

III.1.3.3 Female gonadal development

In the absence of a Y chromosome, the bipotential gonad develops as an ovary. In contrast to testis development, the presence of germ cells is absolutely required for the normal development of an ovary (Couse et al., 1999; Guigon and Magre, 2006; Hashimoto et al., 1990; McLaren, 1991). Indeed, XX embryos deficient for germ cells display either ovarian dysgenesis or granulosa cell trans-differentiation into Sertoli-like cells. The latter is associated with the development of testis-like cords (TLCs) (McLaren, 1991). In adult female mice, it has been hypothesized that germ cells are required to maintain the ovarian phenotype. However both Dazl (Deleted in azoospermia-like) and Fig (Factor in the germline alpha) mutant female mice display germ cell loss but no loss of granulosa cell identity (McNeilly et al., 2000;

Soyal et al., 2000). Moreover, recent findings have demonstrated that the loss of germ cells in 8-week old sexually mature female mice does not disrupt the identity of granulosa cells and the ovarian phenotype (Uhlenhaut et al., 2009).

Similarly to the somatic cells of the undifferentiated gonads, the male and female germ cells remain indistinguishable prior to sex determination (Adams and McLaren, 2002; Eicher and Washburn, 1986;

Palmer and Burgoyne, 1991). This cellular plasticity is well illustrated in aggregating experiments, where XY germ cells enter meiois in XXXY female chimeras and mature as oocytes whereas XX germ cells enter mitotic arrest in XXXY male chimeras and develop as prospermatogonia. This shows that embryonic germ cells differentiate according to the sex of the gonad rather than their own chromosomal constitution (McLaren, 1995) .

For a long time, it was believed that the development of an ovary as opposed to a testis was the result of a default pathway followed in the absence of Sry expression. However, Nef et al demonstrated that ovarian development is set in motion long before the first morphological changes are seen (Nef et al., 2005). In fact, a large-scale transcriptional analysis revealed that a robust genetic program is activated in XX gonads as early as E11.5, and becomes strengthened throughout the process of ovarian differentiation.

The first morphological changes are seen between E12.0 and E13.5, during which period loose cord like structures, referred to as ovigerous cords, appear in the developing ovary (Loffler and Koopman, 2002;

Odor and Blandau, 1969). These structures are composed of mesenchymal cells, the pre-granulosa cells that surround clusters of PGCs called oogonia (Pepling, 2006). The oogonia remain connected by intercellular bridges and are organized in a structure known as the cyst. This cyst organization allows synchronous mitotic divisions of oogonia, and the transport of organelles and mRNA between daughter germ cells.

Between E13.5 and E15.5, oogonia stop proliferating, enter meiosis and are thereafter referred to as oocytes (McLaren, 2000). Then, around E17.5, oocytes progress through the stage of prophase I and arrest at the diplotene stage of the first meiotic division (MI). This quiescent state is preserved at least until postnatal day five (P5) in mice (Borum, 1961). In mammals, only a small fraction of the initial pool of oocytes survives to form individual primordial follicles. The massive loss of oocytes, which occurs all along ovary development, contributes to the elimination of germ cells bearing either chromosomal abnormalities or defective mitochondria. Another reason for this loss is that dying germ cells function as nurse cells, providing oocytes with mRNAs, proteins and organelles (Baker, 1972; Pepling, 2006). At birth, germ cell cysts progressively undergo cyst breakdown. During this step, pre-granulosa cells enclosing the germ cell cyst extend cytoplasmic processes between the germ cells and gradually isolate oocytes from each other. As a result, each individualized oocyte becomes surrounded by a single layer of flattened pre-granulosa. The deposition of a basal lamina around the forming follicle results in the separation of the oocyte from the stromal cells and defines the functional unit of the ovary: the primordial follicle (Loffler and Koopman, 2002; Merchant-Larios and Chimal-Monroy, 1989).

III.1.3.4. Molecular factors that regulate the AGP development

The following section describes the main transcription factors involved in the AGP development. Several studies have revealed the central role of Sf-1 in both gonadal and adrenal development. For this reason, the first part of this section is entirely devoted to Sf-1 and its interactions with other fundamental factors such as Wt-1, Odd-1, Pbx-1, M33, Cited2 and Lhx9.

Since both the gonads and the adrenals are derived from the AGP, mutations within the genes involved in its developmental processes often result in agenesis or dysgenesis of both gonadal and adrenal tissues (Else and Hammer, 2005). The differentiation and maintenance of the AGP is directly linked to Sf-1 expression. Indeed in mice, Sf-1 haploinsufficiency impairs adrenal development (Bland et al., 2000).

Moreover, Sf-1^-/- mice display a complete regression by apoptosis of both gonads and adrenals between E12.0-E12.5 (Luo et al., 1994; Sadovsky et al., 1995). The Wilms’ tumor gene (Wt-1), the Odd-skipped related 1 gene (Odd1), the Pre-B-cell-leukemia gene (Pbx1), the mouse polycomb M33 factor gene (M33/Cbx2), the CPB/p300-interacting transactivator with ED-rich tailed 2 gene (Cited2) and the LIM homeobox gene 9 (Lhx9) were shown to be essential for urogenital ridge formation, Mutations in any of these genes result in adrenal aplasia and/or gonadal defects (Birk et al., 2000; Buaas et al., 2009; Katoh- Fukui et al., 2005; Moore et al., 1999; Schnabel et al., 2003; Val et al., 2007; Wagner et al., 2003; Wang et al., 2005).

III.1.3.4.1. The steroidogenic factor 1 (SF-1)

SF-1 (NR5A1, AD4BP1, FTZ1-F1) is a master regulator of both gonadal and adrenal development and thereafter regulates the transcriptional programs that lead to testicular and ovarian differentiation.

Originally, Sf-1 was identified as a key modulator of the expression of genes involved in steroidogenesis (Honda et al., 1990; Rice et al., 1990). Consistent with this function, Sf-1 expression is observed in steroidogenic tissues such as the testis, the ovary and the adrenal cortex (Morohashi, 1999; Morohashi et al., 1994). In addition, Sf-1 is expressed in the pituitary gland and in few neurons of the hypothalamus (Schimmer and White, 2010). During development, Sf-1 can be detected as early as E8.5 in the AGP (Hatano et al., 1994). Sf-1 expression is then maintained at high levels long after adrenal differentiation, both during adrenal maturation and throughout adult life. Initially, the SF-1 expression profile is similar between males and females. After the onset of sex determination, however, Sf-1 expression is upregulated in testes at around E12.5, whereas it is downregulated in ovaries (Ikeda et al., 1994). This sex specific pattern of expression is preserved until birth, at which point the levels of Sf-1 increase in neonatal ovaries to a magnitude equivalent to that found in testes (Morohashi and Omura, 1996; Val et al., 2003).

III.1.3.4.1.1. SF-1 structure

SF-1 is an orphan nuclear receptor that shares common features with all other members of the family: a zinc finger DNA binding domain (DBD), an A-box, a hinge region, and a ligand binding domain (LBD) (Lin and Achermann, 2008). Within the DBD of nuclear receptors, the presence of a proximal-box (P- box) confers their ability to recognize specific DNA target sequences (Evans, 1988) (Figure 5). The A- box of SF-1 confers DNA binding stability, and is probably the reason SF-1 binds to DNA as monomer, unlike other nuclear receptors (Ueda et al., 1992; Wilson et al., 1992). The hinge region plays crucial roles in post-translational regulation of SF-1 function (e.g. by phosphorylation, sumoylation – see I.3.4.1.2). It has been demonstrated that the phosphorylation of a unique serine (S²⁰³) within the activation function 1 (AF-1) domain of SF-1 results in the recruitment of cofactors that may regulate expression of target genes (Hammer et al., 1999). In contrast, repression of SF-1 activity is achieved through its sumoylation (Lee et al., 2005). Finally, the LBD is involved in co-factor recruitment, as it can form an activation function structure (AF-2) (Figure 5) (Lin and Achermann, 2008).

III.1.3.4.1.2 Regulation of SF-1 expression and activity

Sf-1 expression is complex and highly regulated both at the transcriptional and post-transcriptional levels and its promoter is bound by numerous factors such as WT-1, LHX9, M33 or SOX9 (Katoh-Fukui et al., 2005; Shen and Ingraham, 2002; Wilhelm and Englert, 2002). The Sf-1 promoter contains four binding sites for WT-1 and a single binding site for LHX9. Of the different isoforms of WT-1, only the WT-1(- KTS) isoform can bind and activate the Sf-1 promoter. This activation is enhanced when WT-1 and LHX9 both bind to the Sf-1 promoter (Wilhelm and Englert, 2002). In Y1 adrenocortical cells, the transcription factor encoded by the mouse polycomb gene M33 binds two distinct regions within the SF-1 locus – one region upstream of the first exon and another region just downstream of the last exon – suggesting that M33 may regulate Sf-1 expression (Katoh-Fukui et al., 2005). The Sf-1 proximal promoter also contains a Sox protein binding site (Sox-BS), which is strongly bound and activated by SOX9, thus providing a positive feed back to maintain the expression of its own partner Sox9 (Shen and Ingraham, 2002).

In addition to the manipulation of Sf-1 expression by transcription factors, other forms of regulation exist.

During development, the spatiotemporal expression of many key genes is under the control of epigenetic mechanisms (Mohn and Schubeler, 2009). DNA methylation of CpG islands within promoters is an efficient mechanism for transcriptional repression. The proximal promoter of Sf-1 is subjected to DNA methylation, and Sf-1 gene transcription is inversely proportional to its promoter methylation. It has been reported that the Sf-1 proximal promoter is unmethylated within both male and female developing gonads as well as in adrenocortical cells, but completely methylated in fetal medulla cells (Hoivik et al., 2008).

Furthermore, the DNA methylation pattern is established early in embryonic development and is faithfully conserved as growth proceeds.

SF-1 is also subject to post-translational modifications that change its activity. SF-1 carries one consensus site for protein kinase A (PKA) and the phosphorylation of this serine (S²⁰³) within the AF-1 domain of Sf-1 results in the recruitment of cofactors that may help regulate the expression of target genes (Hammer et al., 1999). In vitro studies reveal that both the extracellular signal-regulated kinase (Erk2) and the cyclin dependant kinase 7 (CDK7) are also able to phosphorylate the S²⁰³ residue (Hammer et al., 2005;

Lewis et al., 2008). Upon S²⁰³ phosphorylation, SF-1 enhances its interactions with coactivators or corepressors, such as the glucorticoid receptor-interacting protein 1 (GRIP1) and silencing mediator of retinoid and thyroid hormone receptor (SMRT), thereby increasing or reducing target gene expression (Hammer, Krylova et al. 1999). Finally, the sumoylation of SF-1 within the DBD domain inhibits S²⁰³ phosphorylation, and impairs SF-1 interaction with cofactors. However, acetylation of lysine residues increases its activity (Lee et al., 2005). Altogether, these modifications mediate the integration of several extra- and intra-cellular signals, and thereby allow the appropriate fine-tuning of the cellular response.

III.1.3.4.1.3 SF-1 insufficiency in mice and humans

In mice, targeted deletion of the Sf-1 gene results in the complete agenesis of adrenal glands and gonads.

Mutant pups die a few days after birth due to glucocorticoid and mineralocorticoid deficiency (Luo, Ikeda et al. 1994). The formation of the AGP and initial stages of adrenal and gonadal development occur normally. However, by E12.0, and prior to the onset of hormone production, the gonads degenerate by apoptosis, leading to a XY sex reversal of both internal and external genitalia. Moreover, a normal gene dosage of Sf-1 is required, as mice bearing just one functional allele of Sf-1 display adrenal dysgenesis, which leads to adrenal insufficiency (Bland et al., 2004; Bland et al., 2000). In humans, several cases of XY sex reversal and adrenal aplasia were reported with a homozygous mutation in Sf-1 (Achermann et al., 1999; Ozisik et al., 2002). Sf-1 haploinsufficiency is associated with adrenal insufficiency, XY sex reversal, and ovarian failure in human patients (Tajima et al., 2009).

Figure 5. A. Diagram of the genomic structure of SF-1. B. The key structural domains of SF-1 are schematically represented. C.

Comparison of the crystal structures of mouse and human SF-1 ligand-binding domain. [Adapted from (Lin and Achermann, 2008)].

III.1.3.4.2 WT-1

Wt-1 can give rise to 24 distinct protein isoforms, which are produced by alternative splicing, RNA editing or alternative translation start sites (Wagner et al., 2003). Among them, two major variants encoded by Wt-1, the +KTS and -KTS isoforms, are of particular interest. These two isoforms result from the use of alternative splice donor sites within exon 9. When donor site one is used, the KTS sequence is omitted (WT-1 (-KTS)), whereas use of donor site 2 leads to the inclusion of the KTS sequence between zinc fingers 3 and 4 (WT-1 (+KTS)) (Wagner et al., 2003). Interestingly, WT-1 (-KTS) is known to promote the transcriptional activity of genes involved in differentiation or proliferation. On the other hand the WT-1 (+KTS) isoform binds RNA with higher affinity than DNA, and is thought to stabilize target transcripts (Bor et al., 2006; Lee and Haber, 2001).

WT-1 is a transcription factor bearing four Zn finger domains, which either activates or represses target genes depending on the cellular context (Roberts, 2005). It is expressed in several tissues during embryonic development, such as the urogenital system, the adrenal gland and the proliferating coelomic epithelium (Armstrong et al., 1993; Moore et al., 1999; Moore et al., 1998). WT-1 is involved in the regulation of numerous genes that participate in urogenital development, including Amhr2, Sry, Sox9, Sf-1 and Wnt4 (Gao et al., 2006; Hossain and Saunders, 2001; Klattig et al., 2007; Shimamura et al., 1997;

Wilhelm and Englert, 2002).

Analysis of a Wt-1 knockout model demonstrated that Wt-1 is necessary for the development of several organs, including gonads and adrenals (Moore et al., 1999; Patek et al., 1999). In addition, mutations within the Wt-1 gene are linked to both Frasier and Denis-Drash syndrome, which are characterized by degenerative renal disease, pseudohermaphroditism, complete male to female sex reversal, and an elevated risk for the development of Wilms’ tumors, (Koziell and Grundy, 1999). Mice lacking the WT-1 (-KTS) isoform display both a drastic reduction in kidney size, and streak gonads in which cells fail to survive during embryonic development (Bradford et al., 2009; Wagner et al., 2003). It has been shown that the WT-1 (-KTS) variant can bind to and transactivate the Sf-1 promoter together with LHX9 (Wilhelm and Englert, 2002). Wt-1 (+KTS)^-/- mice display a less severe phenotype. Although kidney development is also impaired, bipotential gonads do form. However, XX and XY individuals develop along the female pathway owing to low levels of Sry expression (25% of WT animals) (Bradford et al., 2009; Hammes et al., 2001). Other transcription factors interact synergistically with WT-1, or otherwise affect Wt-1 expression, which ultimately results in the modification of Sf-1 expression.

III.1.3.4.3 Odd1

The Odd1 gene encodes a putative transcription factor bearing four Zn fingers that plays an essential role in embryonic development, differentiation and morphogenesis (Wang et al., 2005). Odd1 transcripts are detected throughout the intermediate mesoderm at E8.5, but are later found in a broad range of other tissues (So and Danielian, 1999). Mutant embryos generally die early on during embryonic development, at around E11.5-E12.5, although some survive to late embryonic stages. All Odd1^-/- embryos that survive to late gestation lack kidneys, adrenals and gonads, due to increased apoptosis of nephrogenic tissue and retarded development of urogenital ridges. Odd1 and Wt-1 share common expression patterns and are thought to interact synergistically in the molecular pathways that regulate urogenital development. Hence, Wt-1 expression is downregulated in Odd1^-/- mice, as are Pax2 and Lhx1, genes essential for normal intermediate mesoderm differentiation(Wang et al., 2005). Whether Odd1 acts upstream of Wt-1, or both act together to induce the differentiation of the intermediate mesoderm still remains an open question.

III.1.3.4.4 Pbx1

Pbx1 codes for a TALE (three amino acid loop extension) class homeodomain protein, which dimerizes with other TALE proteins and then forms a trimeric complex together with Hox proteins (Ferretti et al., 2000; Schnabel et al., 2003). These trimers subsequently activate downstream targets that contain a Pbx- responsive element within the promoter. During embryonic development, Pbx1 is first expressed at E10.0, within the nephrogenic cord and the coelomic epithelium, as well as the AGP (Schnabel et al., 2003).

Thereafter, Pbx1 expression is observed inter alia in the bipotential gonads, the Wolffian ducts, the

Müllerian ducts and the interstitium of both testis and ovary. Several studies have demonstrated that Pbx1 is essential for the development of numerous organs including the kidneys and urogenital tract (Schnabel et al., 2000; Schnabel et al., 2003). Pbx1^-/- mice lack adrenals and exhibit reduced metanephric kidneys as well as rudimentary male and female gonads. Interestingly, the cells within the AGP proliferate at slower rate than in control animals (65% of control). Subsequently, during genital and adrenal primordia differentiation, SF-1 positive cells are detected in the defective gonads, although adrenocortical cells are completely absent. In addition, cell proliferation is reduced within the genital ridges of both XX and XY mutants, and testis cord formation fails. As a consequence, at E14.5, both XX and XY gonads are histologically indistinguishable from one another. The lack of adrenal development correlates with the reduced expression of Sf-1 at E10.0 and confirms the greater sensitivity of the adrenal cortex to Sf-1 dosage effects (Bland et al., 2004).

III.1.3.4.5 Cited2

The Cited2 gene encodes a growth factor that cannot itself bind DNA, but which physically interacts with the transcription factor AP-2 (TFAP2) and functions as a co-activator (Braganca et al., 2003). Cited2 is expressed in the AGP and in the bipotential gonads at E10.5. Recently it was shown that CITED2 interacts directly with WT-1 on the promoter of SF-1 and together they stimulate its expression in the AGP, ensuring adrenal differentiation and gonadal development (Val et al., 2007). At E12.0 almost Cited2 no expression is detected within the developing gonads, although it is present in the adrenal cortex and, by E13.5, becomes restricted to this location (Val et al., 2007).

Previous studies have revealed the essential role of Cited2 during right-left patterning, heart, neural tube, and adrenal development and both testis and ovary differentiation (Bamforth et al., 2001; Buaas et al., 2009; Combes et al., 2009). Cited2^-/- mice display exencephaly, cardiac malformations, neural crest defects, gonads dysgenesis and a complete absence of adrenal glands. Notably, the expression levels of Sf-1 in mutant mice are reduced by 64%. This impairs adrenal development but permits gonad development, although male and female sexual differentiation is altered. Indeed, Sry, Sox9 and Fgf9 expression levels are reduced and delayed in XY Cited2^-/- gonads. Interestingly, in the ovaries of mutant females it appears that the male pathway is not fully repressed (Combes et al., 2009). At E10.5, Wnt4 is down regulated whereas Fgf9 is upregulated, and one day later, Foxl2 and RspoI in their turn appear to be down regulated. However, at E12.5, the female genetic program recovers normal levels of Wnt4, RspoI and Foxl2.

Nevertheless, Cited2 appears to be a key factor in early AGP development as well as adrenal specification and sex determination. At this time, however, the factors that regulate Cited2 expression are still unknown.

III.1.3.4.6 M33

M33 belongs to the polycomb gene family, the prototype of which was first described as a repressor of Hox gene transcription in Drosophila (Pirrotta, 1997). M33 is expressed in somatic mesodermal tissues during development, where it regulates the expression of specific Hox genes (Bel et al., 1998). Disruption of M33 in mice results in the reduced size of the adrenals and spleen, which correlates with the reduction of Sf-1 expression levels. Furthermore, M33 forms a complex with other polycomb genes (complex 2) and binds two regions of Sf-1, one upstream of the first exon and the second immediately downstream of the last exon. Whether or not the interaction between M33 and Sf-1 accounts for the reduction in the latter’s expression level remains to be demonstrated (Katoh-Fukui et al., 2005).

III.1.3.4.7 Lhx9

Lhx9 is a transcription factor characterized by two LIM domains, which are involved in protein-protein interactions, and a DNA binding homeodomain (Wilhelm et al., 2007b). Lhx9 is expressed within urogenital ridges from E9.5 onwards, in a region that will later give rise to the presumptive gonads (Birk et al., 2000). In Lhx9^-/- mice, germ cells migrate to and reach the genital ridges correctly, but gonads fail to develop due to a somatic cell proliferation defect. As a consequence, androgens and AMH are not