Pierre Val

Amanda Swain

Gene dosage effects and transcriptional regulation of early mammalian adrenal cortex development

Overview of adrenal development

. The present review will focus on the early events of adrenal development with emphasis on formation and maintenance of the adreno-gonadal primordium, foetal adrenal cell fate determination and the emerging role of signalling molecules in the control of proliferation, differentiation and shaping of the cortex.

In mammals, the major steroid producing organs are the gonads and the adrenal cortex. Enzymes involved in the first steps of steroidogenesis are common to both gonads and adrenals (Cyp11a1 and 3HSD) but the final steps are catalyzed by organ specific enzymes: Cyp21, Cyp11b1 and Cyp11b2 in the adrenal; Cyp19, 17-HSD and Cyp17 in the gonads, although the latter is expressed in primates' adrenals. Even though the specificity of expression is relatively strict in adult tissues, numerous reports have shown that some gonadspecific enzymes are expressed in the adrenal during development. Conversely, adrenal enzymes were observed in the developing gonad [START_REF] Hatano | Identical origin of adrenal cortex and gonad revealed by expression profiles of Ad4BP/SF-1[END_REF][START_REF] Heikkila | Wnt-4 deficiency alters mouse adrenal cortex function, reducing aldosterone production[END_REF][START_REF] O'shaughnessy | Adrenocorticotropic hormone directly stimulates testosterone production by the fetal and neonatal mouse testis[END_REF][START_REF] Wang | Expression of 11- hydroxylase in rat Leydig cells[END_REF][START_REF] Hu | Expression of Cyp21a1 and Cyp11b1 in the fetal mouse testis[END_REF][START_REF] Val | Identification of a novel population of adrenallike cells in the mammalian testis[END_REF]. This suggests that, in agreement with histological studies, the adrenal cortex and gonads have a common embryological origin. Consistent with this, mice deficient for steroidogenic factor 1 (Sf-1), an essential regulator of steroidogenesis [START_REF] Val | SF-1 a key player in the development and differentiation of steroidogenic tissues[END_REF][START_REF] Parker | Steroidogenic factor 1: an essential mediator of endocrine development[END_REF], lack both adrenals and gonads [START_REF] Luo | A cell-specific nuclear receptor is essential for adrenal and gonadal development and sexual differentiation[END_REF][START_REF] Sadovsky | Mice deficient in the orphan receptor steroidogenic factor 1 lack adrenal glands and gonads but express P450 side-chain-cleavage enzyme in the placenta and have normal embryonic serum levels of corticosteroids[END_REF]. Detailed analysis of Sf-1 expression in rat embryos identified a group of positive cells that form the presumed adrenogonadal primordium or AGP, the shared precursor of both gonads and adrenals. These studies showed symmetrical lines of Sf-1-positive cells extending from the dorsal aorta to the dorsal coelomic epithelium of the E11.5 rat urogenital ridges. At E12.5, the adrenal primordium separated from the gonadal primordium on the dorsal aortal side, in the rostral area of the AGP [START_REF] Hatano | Identical origin of adrenal cortex and gonad revealed by expression profiles of Ad4BP/SF-1[END_REF]. In mouse, Sf-1 was detected as early as E9.0. The process of AGP development was similar and the adrenal anlage separated from the bipotential precursor at E10.5 (10-11 tail somites) [START_REF] Ikeda | Developmental expression of mouse steroidogenic factor-1, an essential regulator of the steroid hydroxylases[END_REF][START_REF] Val | Adrenal development is initiated by Cited2 and Wt1 through modulation of Sf-1 dosage[END_REF] (Fig1).

Several genes have been implicated in the development of the AGP. Mice lacking these factors have defects in both gonad and adrenal development.

Steroidogenic Factor 1:

Steroidogenic Factor 1 / Adrenal 4 Binding Protein (Sf-1/Ad4BP) was initially identified as the transcription factor binding to the Ad4 sequence found in the promoters of genes encoding several steroidogenic enzymes. It belongs to the nuclear hormone receptor super-family and has been shown to act as a master regulator of steroidogenic cell differentiation and function as well as of male sexual differentiation [START_REF] Val | SF-1 a key player in the development and differentiation of steroidogenic tissues[END_REF]. Consistent with the central role of Sf-1 in steroidogenic tissue differentiation, stable transfection of ES cells or adipose tissue-derived and bone marrow-derived mesenchymal cells with Sf-1 induces their differentiation to a steroidogenic fate [START_REF] Crawford | Nuclear receptor steroidogenic factor 1 directs embryonic stem cells toward the steroidogenic lineage[END_REF][START_REF] Tanaka | Steroidogenic factor 1/adrenal 4 binding protein transforms human bone marrow mesenchymal cells into steroidogenic cells[END_REF][START_REF] Gondo | Adipose tissue-derived and bone marrow-derived mesenchymal cells develop into different lineage of steroidogenic cells by forced expression of steroidogenic factor 1[END_REF]. Mice lacking Sf-1 show early adrenal and gonadal development, however, the two anlagen regress by apoptosis between E11.5 and E12.5 [START_REF] Luo | A cell-specific nuclear receptor is essential for adrenal and gonadal development and sexual differentiation[END_REF][START_REF] Sadovsky | Mice deficient in the orphan receptor steroidogenic factor 1 lack adrenal glands and gonads but express P450 side-chain-cleavage enzyme in the placenta and have normal embryonic serum levels of corticosteroids[END_REF]. This indicates that Sf-1 is essential for early development and maintenance of adrenal and gonadal primordia. Although there is increasing evidence that Sf-1 regulates proliferation in the adrenal primordium and adult adrenal, the mechanisms involved in apoptosis of the primordia in Sf-1-/-mice are not known [START_REF] Beuschlein | Steroidogenic factor-1 is essential for compensatory adrenal growth following unilateral adrenalectomy[END_REF][START_REF] Bland | Differential requirement for steroidogenic factor-1 gene dosage in adrenal development versus endocrine function[END_REF][START_REF] Doghman | Increased steroidogenic factor-1 dosage triggers adrenocortical cell proliferation and cancer[END_REF]. SF-1 has recently been shown to repress expression of NOV/CCN3, a factor with pro-apoptotic activity in H295R human adrenal carcinoma cell lines. Interestingly, NOV is expressed in foetal adrenal cells [START_REF] Doghman | Nephroblastoma overexpressed/cysteine-rich protein 61/connective tissue growth factor/nephroblastoma overexpressed gene-3 (NOV/CCN3), a selective adrenocortical cell proapoptotic factor, is down-regulated in childhood adrenocortical tumors[END_REF], suggesting that it may be involved in the apoptotic phenotype in Sf-1 deficient mice.

Wilm's Tumour 1

The Wilm's Tumour suppressor gene (WT1) was initially identified as a gene that was mutated in a subset of childhood kidney tumours. Patients with mutations in WT1 also showed various urogenital abnormalities that in some extreme cases led to male to female sex reversal. The WT1 gene encodes at least 24 different isoforms of a zinc finger containing protein through use of alternative promoters, alternative splicing and RNA editing. WT1 can bind DNA through its four zinc fingers DNA binding domain, but can also associate with A c c e p t e d M a n u s c r i p t spliceosomes in the presence of a three amino-acid motif (KTS) between the third and fourth zinc fingers [START_REF] Hastie | Life, sex, and WT1 isoforms--three amino acids can make all the difference[END_REF][START_REF] Reddy | The WT1 Wilms' Tumor suppressor gene : How much do we really know ?[END_REF]. In mouse urogenital ridges, Wt1 is expressed as early as E9.5. Mice deficient for Wt1 lacked kidneys and although a thickening of the early urogenital ridge was observed, this did not give rise to gonadal or adrenal development [START_REF] Kreidberg | WT-1 is required for early kidney development[END_REF]. Interestingly, mice lacking either the +KTS or the -KTS isoform of Wt1 develop an adrenal, suggesting that the two isoforms can compensate for each other in adrenal development. However, mice mutant for the -KTS isoform show an increase in apoptosis at E11.5 in the anterior region of the gonad, which is reminiscent of Sf-1 knockout mice [START_REF] Hammes | Two splice variants of the Wilms' tumor 1 gene have distinct functions during sex determination and nephron formation[END_REF]. This and the early urogenital phenotype of Wt1 deficient mice suggested that Wt1 acted upstream of Sf-1 in the genetic cascade leading to AGP development. Consistent with this, Wilhelm and Englert showed that Sf-1 expression was completely abrogated as early as E9.5 in Wt1-/-urogenital ridges, although expression of Lhx9, a gonadal marker, was unaltered. Using transgenic mice, they showed that Wt1 was directly activating transcription of Sf-1 through four Wt1 responsive elements found in a 674 bp promoter fragment that recapitulated Sf-1 expression in the urogenital ridges [START_REF] Wilhelm | The Wilms tumor suppressor WT1 regulates early gonad development by activation of Sf1[END_REF]. We have recently demonstrated that Wt1, in association with Cited2, plays an essential role in modulating Sf-1 dosage and triggering adrenal cortex differentiation ( [START_REF] Val | Adrenal development is initiated by Cited2 and Wt1 through modulation of Sf-1 dosage[END_REF], see chapter II).

Odd-Skipped related 1:

Odd-Skipped related 1 is a presumptive transcription factor with three zinc fingers that has homology with the odd-skipped family of developmental genes in drosophila [START_REF] So | Cloning and expression analysis of a mouse gene related to Drosophila odd-skipped[END_REF]. It is expressed in intermediate mesoderm and precedes Wt1 expression in the urogenital ridges. Odd1 deficient mice have urogenital phenotypes similar to Wt1 mutant mice with absence of kidney development, blunted genital ridge thickening and no overt adrenal and gonadal differentiation. Interestingly, Wt1 expression is down-regulated in the E9.5 urogenital ridges of Odd1-/-embryos [START_REF] Wang | Odd-skipped related (Odd1) is an essential regulator of heart and urogenital development[END_REF]. This suggests that Odd-1 acts upstream of Wt1 and controls its expression in intermediate mesoderm derivatives. Consistent with this, Odd1 deficient mice have a more severe kidney phenotype (absence of metanephric mesenchyme condensation) than Wt1 mutant mice.

II-Adrenal fate determination:

At around E10.5 in the mouse, the bipotent AGP divides into the adrenal cortex anlage and the sexually undetermined gonad. The adrenal primordium progressively separates from the gonad in the rostral region. Analysis of genetically modified mice has identified two factors, Lxh9 and Emx2, as being important in specifying gonad differentiation. Mice mutant for either gene show adrenal development. However, gonad development is markedly impaired [START_REF] Birk | The LIM homeobox gene Lhx9 is essential for mouse gonad formation[END_REF][START_REF] Miyamoto | Defects of urogenital development in mice lacking Emx2[END_REF]. This indicates that these factors are specifically required to trigger gonad as opposed to adrenal development, although the underlying molecular mechanisms have not been identified. Until recently, factors involved in the determination of adrenal fate from the AGP were unknown.

Cited2, Wt1 and Sf-1 dosage:

Cited2 is a transcription cofactor devoid of DNA binding activity. It can act as a coactivator for transcription factors such as Lhx2, Tfap2 and the Ppar nuclear receptors by recruiting Cbp/p300 to the promoter. It has also been shown to act as a corepressor for Hif1 by competing for binding to Cbp/p300. Mice lacking Cited2 were shown to lack adrenal glands at E17.5 although their gonads were apparently normal. These features were part of a more complex phenotype including neural crest migration defects, neural tube closure defects, abnormal body patterning and defective heart development [START_REF] Tien | Identification of the CBP/p300 interacting protein CITED2 as a PPARCoregulator[END_REF][START_REF] Bhattacharya | Functional role of p35srj, a novel p300/CBP binding protein, during transactivation by HIF-1[END_REF][START_REF] Bamforth | Cardiac malformations, adrenal agenesis, neural crest defects and exencephaly in mice lacking Cited2, a new Tfap2 co-activator[END_REF][START_REF] Bamforth | Cited2 controls left-right patterning and heart development through a Nodal-Pitx2c pathway[END_REF][START_REF] Bragança | Physical and functional interactions among AP-2 transcription factors, p300/CREB-binding protein, and CITED-2[END_REF][START_REF] Weninger | Cited2 is required both for heart morphogenesis and establishment of the left-right axis in mouse development[END_REF][START_REF] Yin | The essential role of Cited2, a negative regulator for HIF1-alpha, in heart development and neurulation[END_REF]. We recently conducted a A c c e p t e d M a n u s c r i p t 4 study of urogenital development in Cited2 deficient mice. We showed that Cited2 was expressed at high levels in the AGP and adrenal anlage and at lower levels in the overtly differentiated gonad. Consistent with this expression pattern, adrenal cortical development was not initiated in Cited2 homozygous mutants. We demonstrated that this essential role of Cited2 was dependent on its physical and functional interaction with Wt1 in the AGP, prior to adrenal formation. This interaction resulted in increased Sf-1 promoter activity and Sf-1 expression [20] (Fig2). Other studies have shown that modulations of Sf-1 dosage can affect gonadal and adrenal development to different degrees. In E13.5 Sf-1 heterozygous embryos, adrenal size was decreased by twelve fold compared to wild-type [START_REF] Bland | Differential requirement for steroidogenic factor-1 gene dosage in adrenal development versus endocrine function[END_REF]. Increased proliferation later in development and augmented steroidogenic capacity per cell allowed partial recovery but adrenal responsiveness to stress was still markedly impaired in adults [START_REF] Bland | Haploinsufficiency of steroidogenic factor-1 in mice disrupts adrenal development leading to an impaired stress response[END_REF][START_REF] Babu | Interaction between Dax-1 and steroidogenic factor-1 in vivo: increased adrenal responsiveness to ACTH in the absence of Dax-1[END_REF]. In contrast, although early male gonad differentiation was affected in Sf-1+/-embryos, the adult gonad was fully functional [START_REF] Park | Nuclear Receptors Sf1 and Dax1 function cooperatively to mediate somatic cell differentiation during testis development[END_REF]. This suggested that adrenal development in mouse was more sensitive to decreased Sf-1 dosage than gonadal development. Consistent with this, forced transgenic expression of Sf-1 in Sf-1 mutant mice rescued gonad development but not adrenal development, even though the transgene was expressed in both tissues in control animals [START_REF] Fatchiyah | Differential gene dosage effects of Ad4BP/SF-1 on target tissue development[END_REF].

In our experiments, Sf-1 expression levels in the E10.5 AGP correlated with the severity of the adrenal phenotype. With 36% of wild-type dosage, as seen in Cited2-/-embryos, adrenal development was not initiated, whereas with 45% of wild-type levels, as seen in Cited2+/-Wt1+/-embryos, adrenal development was initiated but adrenal growth was markedly impaired. Therefore we proposed that Sf-1 dosage had to reach a critical threshold, at a particular stage, to trigger adrenal development from the AGP and that Cited2, together with Wt1, were required to increase Sf-1 expression above that threshold (Fig2) [START_REF] Val | Adrenal development is initiated by Cited2 and Wt1 through modulation of Sf-1 dosage[END_REF]. It is intriguing that Wt1, which is not found in the overtly differentiated adrenal [START_REF] Val | Adrenal development is initiated by Cited2 and Wt1 through modulation of Sf-1 dosage[END_REF][START_REF] Nachtigal | Wilms' tumor 1 and Dax-1 modulate the orphan nuclear receptor SF-1 in sex-specific gene expression[END_REF], plays an essential role for its determination from the AGP. These data are confirmatory evidence of the existence of the AGP and the common origin of the gonad and adrenal cortex. Also consistent is the early gonad phenotype observed in Cited2-/-embryos, characterised by decreased expression of Cyp11a1 and Sox9 in the male gonad at E11.5. Preliminary studies reveal that this defect can lead to male to female sex reversal in different genetic backgrounds (unpublished work).

Sf-1 dosage, mouse versus human:

Consistent with the phenotype in Sf-1 homozygous mutant mice, patients with SF-1 mutations that were initially identified, were shown to have both adrenal insufficiency and gonadal dysgenesis [START_REF] Achermann | A mutation in the gene encoding steroidogenic factor-1 causes XY sex reversal and adrenal failure in humans[END_REF][START_REF] Achermann | Gonadal determination and adrenal development are regulated by the orphan nuclear receptor steroidogenic factor-1, in a dose-dependent manner[END_REF]. Recent reports have described patients with heterozygous nonsense mutations in SF-1 that result in premature translation termination and undetectable protein production from the mutant allele [START_REF] Hasegawa | Testicular dysgensis without adrenal insufficiency in a 46, XY patient with a heterozygous inactivae mutation of steroidogenic factor-1[END_REF][START_REF] Mallet | Gonadal Dysgenesis without adrenal insufficiency in a 46, XY patient heterozygous for the nonsense C16X mutation: a case of SF1 haploinsufficiency[END_REF]. These patients are thought to be true models of SF-1 haploinsufficiency in humans. Surprisingly, they show gonadal dysgenesis but normal adrenal function. Several heterozygous missense mutations have also been shown to cause gonadal dysgenesis despite normal adrenal function [START_REF] Coutant | Heterozygous mutation of steroidogenic factor-1 in 46,XY subjects may mimic partial androgen insensitivity syndrome[END_REF][START_REF] Kohler | Five novel mutations in steroidogenic factor 1 (SF1, NR5A1) in 46,XY patients with severe underandrogenization but without adrenal insufficiency[END_REF][START_REF] Reuter | A novel mutation in the accessory DNA-binding domain of human steroidogenic factor 1 causes XY gonadal dysgenesis without adrenal insufficiency[END_REF][START_REF] Lin | Heterozygous missense mutations in steroidogenic factor 1 (SF1/Ad4BP, NR5A1) are associated with 46,XY disorders of sex development with normal adrenal function[END_REF]. Although the latter observation may reflect impairment of tissue-specific cofactor recruitment, this suggests that in humans, as opposed to mouse, gonad development is more sensitive to reductions in SF-1 dosage than adrenal development.. Although this may seem incompatible with our model of adrenal differentiation, several considerations must be taken into account. One major difference between these studies is that human patients are assessed after birth when compensatory growth and differentiation events have masked some of the embryonic phenotype. Therefore dissimilar phenotypes might reflect a difference in compensation or overall adrenal functional robustness rather than a difference in the initial events of determination and differentiation. Another important point is the effect of the genetic background on the sensitivity of these subtle gene dosage systems. For instance, we have shown that the levels of Sf-1 in AGP of A c c e p t e d M a n u s c r i p t mice with a mixed genetic background was 1.4 fold higher than in mice with a pure C57/Bl6 background [START_REF] Val | Adrenal development is initiated by Cited2 and Wt1 through modulation of Sf-1 dosage[END_REF]. Consistent with this, we observed signs of adrenal development in Cited2 mutant embryos on a mixed genetic background (unpublished observations). It is thus conceivable that genetic modifiers are impinging upon the effects of SF-1 dosage in the human population, giving rise to different phenotypes in patients with similar mutations. Further analyses of adrenal development in Sf-1 or Cited2 mutant animals on different genetic backgrounds might uncover such modifiers.

It is now clear that Sf-1 dosage is crucial for steroidogenic cell fate determination, differentiation and proliferation control (Fig3). One of the main challenges is now to define which Sf-1 target genes are involved in these processes and how Sf-1 dosage is transcriptionally interpreted in order to trigger expression of tissue-specific pathways.

III-Regulation of Sf-1 dosage

Sf-1 locus: transcriptional enhancer elements

Because of the dosage-sensitive nature of Sf-1 activity, its spatio-temporal expression has to be tightly controlled. Analyses in transgenic mice have allowed the identification of promoter fragments and enhancers that control expression of Sf-1 in different tissues.

Adreno-gonadal primordium enhancer element

Studies in transgenic mice with the green fluorescent protein reporter gene, have shown that fifty kilobases of Sf-1 upstream regulatory regions and intragenic sequences (first exon and first intron) were sufficient to recapitulate endogenous Sf-1 expression pattern excepting in the anterior pituitary [START_REF] Stallings | Development of a transgenic green fluorescent protein lineage marker for steroidogenic factor 1[END_REF]. Within this genomic locus, 674 bp of a sequence upstream of the transcriptional start site were sufficient to drive expression of a LacZ reporter gene to the AGP of transgenic mice [START_REF] Wilhelm | The Wilms tumor suppressor WT1 regulates early gonad development by activation of Sf1[END_REF]. Mutation analysis showed that this region harboured four Wt1 responsive elements that were essential for promoter activity in the AGP as well as responsiveness to Cited2 [START_REF] Val | Adrenal development is initiated by Cited2 and Wt1 through modulation of Sf-1 dosage[END_REF][START_REF] Wilhelm | The Wilms tumor suppressor WT1 regulates early gonad development by activation of Sf1[END_REF]. However, this region was not sufficient to drive expression in the adrenal after its overt differentiation from the AGP.

Fetal Adrenal Enhancer (FAdE)

Zubair and colleagues have recently identified an Sf-1 regulatory element that is highly conserved between mouse and human. This 600 bp enhancer fragment is contained in the fourth intron of Sf-1 and was shown to drive specific reporter gene expression to the adrenal primordium of transgenic mice, as early as E10.5 [START_REF] Zubair | Two-step regulation of Ad4BP/SF-1 gene transcription during fetal adrenal development: initiation by a Hox-Pbx1-Prep1 complex and maintenance via autoregulation by Ad4BP/SF-1[END_REF]. Therefore, this DNA region was called Fetal Adrenal Enhancer or FAdE. Among the conserved transcription factor binding sites in FAdE were two Sf-1 responsive elements (two others were mouse-specific), one Pbx/Prep and one Pbx/Hox binding site. All Sf-1 responsive elements were functional in binding assays, suggesting that Sf-1 could auto-regulate its own expression. However, expression of the reporter was shown to persist in the E11.5 adrenal anlage when all four sites were mutated or when the wild-type transgene was assessed in Sf-1-/-mice. This indicated that Sf-1 was dispensable for the initiation of expression of its own fetal adrenal enhancer. It was nonetheless required at later stages of development as shown by the absence of expression of the mutated transgene in the E17.5 adrenal [START_REF] Zubair | Two-step regulation of Ad4BP/SF-1 gene transcription during fetal adrenal development: initiation by a Hox-Pbx1-Prep1 complex and maintenance via autoregulation by Ad4BP/SF-1[END_REF]. Interestingly, mice lacking Pbx1, a member of the TALE family of homeodomain transcription factors, lacked adrenals at E14.5, had rudimentary gonads (but no sex reversal) and showed renal abnormalities. Pbx1-/-embryos showed decreased expression of Sf-1 in the urogenital ridge at E10.0 although Wt1 expression was similar to wild-type levels. This suggested that Pbx1 was acting either at the same level or downstream of Wt1 in the genetic cascade controlling Sf-1 expression in the AGP.

A c c e p t e d M a n u s c r i p t 6 However, the marked decrease in proliferation observed in the E10.5 urogenital ridge of Pbx mutant embryos could also account for reduced expression of Sf-1 [START_REF] Schnabel | Pbx1 is essential for adrenal development and urogenital differentiation[END_REF]. In order to address this question, Zubair and colleagues analysed the function of the Pbx/Prep and Pbx/Hox binding sites within the FAdE sequence. Using electrophoretic mobility shift assays, these sites were shown to allow cooperative binding of Hox, Pbx1 and Prep factors. When one or the other site was mutated in the context of the FAdE sequence in transgenic mice, expression of the reporter gene was decreased in the adrenal anlage. When both sites were mutated, expression of the reporter was abrogated in the E11.5 adrenal primordium. Altogether, these observations indicated that Pbx1 was required to initiate Sf-1 enhancer activity in the foetal adrenal and that, at later stages, Sf-1 maintained FAdE activity by binding to its cognate responsive elements [START_REF] Zubair | Two-step regulation of Ad4BP/SF-1 gene transcription during fetal adrenal development: initiation by a Hox-Pbx1-Prep1 complex and maintenance via autoregulation by Ad4BP/SF-1[END_REF]. Interestingly, Sf-1 expression was not altered in Pbx1+/-adult adrenals. However, Sf-1 transfection in Y1 adrenocortical cells increased Pbx1 expression. Conversely, Pbx1 expression was decreased in Sf-1 haploinsufficient animals. This indicated that Sf-1 could stimulate Pbx1 expression in the adult adrenal [START_REF] Lichtenauer | Pre-B-cell transcription factor 1 and steroidogenic factor 1 synergistically regulate adrenocortical growth and steroidogenesis[END_REF]. Whether a similar feedforward loop exists in the foetal adrenal is still unknown. The identification of sites that bind both Pbx1 and Hox factors within FAdE indicate that members of the Hox gene family are important in controlling Sf-1 levels in the adrenal. Hox proteins have been shown to control the patterning of different structures during embryonic development by regulating the expression of subsets of target genes [START_REF] Zakany | The role of Hox genes during vertebrate limb development[END_REF][START_REF] Zacchetti | Hox gene function in vertebrate gut morphogenesis: the case of the caecum[END_REF]. It is thus likely that Hox factors define the antero-posterior area where the adrenal anlage arises from the AGP. Cell sorting experiments with mice expressing GFP under the control of the FAdE sequence have identified Hoxb5, Hoxb9 and Hoxc5 as potential candidates in this process [START_REF] Zubair | Two-step regulation of Ad4BP/SF-1 gene transcription during fetal adrenal development: initiation by a Hox-Pbx1-Prep1 complex and maintenance via autoregulation by Ad4BP/SF-1[END_REF].

Altogether these results suggest a three-step model for the control of Sf-1 expression in adrenal precursors and the foetal adrenal: 1) Wt1 induces Sf-1 proximal promoter activity in cells of the AGP. Cited2 increases Sf-1 expression through interaction with Wt1, triggering adrenal differentiation in a subset of cells 2) FAdE is activated in the defined adrenal cortex precursor cells through Pbx1 action, increasing Sf-1 production 3) Sf-1 maintains activity of its own enhancer by binding to its cognate sites. One question that remains open to investigation is the potential role of Cited2 on FAdE activity. Also, the enhancer responsible for Sf-1 expression in the definitive adrenal cortex remains to be identified.

Sf-1 dosage modulators

M33

M33 is a mammalian member of the Polycomb Group proteins that have been shown to repress Hox gene transcription at the level of chromatin structure. M33 deficient mice have partial male-to-female sex reversal and smaller adrenals [START_REF] Katoh-Fukui | Male-to-female sex reversal in M33 mutant mice[END_REF][START_REF] Katoh-Fukui | Mouse Polycomb M33 is required for splenic vascular and adrenal gland formation through regulating ad4BP/SF1 expression[END_REF]. Consistent with these phenotypes, gonad development is markedly impaired at E11.5 and expression of Sf-1 is reduced to 50% of wild-type levels in the E16.5 adrenals. This suggests that M33 controls Sf-1 expression in the AGP. Indeed, ChIP assays showed that M33 binds to Sf-1 upstream regulatory regions in mouse Y1 adrenocortical cells. These regions did not overlap with the 674bp AGP promoter or with the FAdE [START_REF] Katoh-Fukui | Mouse Polycomb M33 is required for splenic vascular and adrenal gland formation through regulating ad4BP/SF1 expression[END_REF]. The role of M33 in the control of Sf-1 expression in the AGP or the adrenal primordium thus remains unclear, although it could be implicated in the formation of intergenic boundaries impinging upon expression of the whole locus.

Pod1

Pod1 (or Tcf21) is a bHLH transcription factor that has been shown to play important roles in the development of numerous tissues [START_REF] Quaggin | The basic-helix-loop-helix protein pod1 is critically important for kidney and lung organogenesis[END_REF]. It is expressed in the urogenital ridges as early as A c c e p t e d M a n u s c r i p t 7 E9.5 and delineates the boundary between the gonad and mesonephros in both sexes at E11.5. At later stages Pod1 expression becomes sexually dimorphic and it is found in the testis interstitium but not within the ovary [START_REF] Tamura | Pod-1/Capsulin shows a sex-and stage-dependent expression pattern in the mouse gonad development and represses expression of Ad4BP/SF-1[END_REF]. Pod1 was shown to repress Sf-1 expression in tissue culture experiments [START_REF] Tamura | Pod-1/Capsulin shows a sex-and stage-dependent expression pattern in the mouse gonad development and represses expression of Ad4BP/SF-1[END_REF][START_REF] Cui | Disrupted gonadogenesis and male-to-female sex reversal in Pod1 knockout mice[END_REF][START_REF] Nomura | An E box element is required for the expression of the ad4bp gene, a mammalian homologue of ftz-f1 gene, which is essential for adrenal and gonadal development[END_REF]. Consistent with this, Pod1 deficient embryos showed an expanded Sf-1 expression domain at the boundary between the gonad and mesonephros. The number of Sf-1 positive cells in the gonad of both sexes was also increased and gonad development and testis differentiation were affected in these mice. Although Pod1 was not directly involved in adrenal development, its disruption prevented the physical separation between the gonad and adrenal primordia. Interestingly, there was no increase in adrenal markers expression in the gonad which suggested that differentiation and sorting of the precursors were not affected [START_REF] Cui | Disrupted gonadogenesis and male-to-female sex reversal in Pod1 knockout mice[END_REF]. Thus Pod1 acts to restrict Sf-1 expression such that the adrenal and gonad primordia are separated during development. This suggests that the mechanism of appearance of the adrenal primordium from the AGP is one of expansion of the AGP that is then restricted to form the two anlagen. However, a model where AGP cells migrate to the region where the adrenal forms cannot be excluded with the available information.

Dax1

The Dax1 gene encodes a putative transcription factor that belongs to the nuclear hormone receptor family although it is devoid of the classical zinc finger DNA binding domain (reviewed in [START_REF] Lalli | DAX-1, an unusual orphan receptor at the crossroads of steroidogenic function and sexual differentiation[END_REF]). DAX1 was initially identified as the gene present in the region of the X chromosome found duplicated in patients with male to female sex reversal. Mutations or deletions of regions of the X chromosome containing DAX1 in humans cause hypogonadotrophic hypogonadism and adrenal hypoplasia congenita, characterised by the persistence of the foetal adrenal zone [START_REF] Achermann | Phenotypic spectrum of mutations in DAX-1 and SF-1[END_REF]. Sf-1 and Dax1 are coexpressed in steroidogenic tissues and other areas early in development [START_REF] Ikeda | Steroidogenic factor 1 and Dax-1 colocalize in multiple cell lineages: potential links in endocrine development[END_REF][START_REF] Swain | Mouse Dax1 expression is consistent with a role in sex determination as well as in adrenal and hypothalamus function[END_REF]. In vitro studies have shown that Dax1 acts as a repressor of steroidogenesis and of Sf-1 transcriptional activity by recruiting corepressors to Sf-1 target promoters (reviewed in [START_REF] Lalli | DAX-1, an unusual orphan receptor at the crossroads of steroidogenic function and sexual differentiation[END_REF][START_REF] Iyer | Molecular mechanisms of DAX1 action[END_REF]). However, recent observations show that Dax-1 can also function as a coactivator for Sf-1 by recruiting the RNA coactivator SRA and TIF2 [START_REF] Xu | DAX-1 and Steroid Receptor RNA Activator (SRA) Function as Transcriptional Coactivators for Steroidogenic Factor-1 in Steroidogenesis[END_REF]. Thus depending on the cellular context, Dax-1 has the potential to repress or stimulate Sf-1 transcriptional activity. Deletion of the C-terminal domain of Dax1 in mouse gives rise to a mild adrenal phenotype characterised by the persistence of a presumptive foetal adrenal cortex in adults, reminiscent of the human pathology, but no adrenal hypoplasia [START_REF] Yu | Role of Ahch in gonadal development and gametogenesis[END_REF]. Studies with this mutant allele show partial rescue of histological and functional defects in Sf-1 haploinsufficient mice, in agreement with the repressive activity of Dax1 on Sf-1-mediated gene transcription [START_REF] Babu | Interaction between Dax-1 and steroidogenic factor-1 in vivo: increased adrenal responsiveness to ACTH in the absence of Dax-1[END_REF]. These observations suggest that Dax1 does not play a major role in early adrenal development. However, these mice do not represent a complete loss of Dax1 function as truncated Dax1 protein may be produced from the mutant locus. It is likely that such a protein would retain some of Dax1 biological activity, which would affect the interpretation of the mutant phenotypes. Recent lineage tracing experiments have suggested that Dax1 acts to restrict adrenal differentiation from the AGP. When the Sf-1 foetal adrenal enhancer sequence (FAdE) was used to drive expression of CRE, recombination was not only observed within the adrenal but in a few cells in the anterior region of the gonad primordium at E11.5 [START_REF] Zubair | Developmental links between the fetal and adult adrenal cortex revealed by lineage tracing[END_REF]. These cells were not observed in transgenic mice where the same enhancer was used to drive LacZ expression [START_REF] Zubair | Two-step regulation of Ad4BP/SF-1 gene transcription during fetal adrenal development: initiation by a Hox-Pbx1-Prep1 complex and maintenance via autoregulation by Ad4BP/SF-1[END_REF]. This indicated that cells within the gonad had been induced to become adrenal precursors but that their differentiation was subsequently prevented. Interestingly, careful analysis of Dax1 expression in the region showed that it was found in the gonad but not the adrenal compartment at E10.5. In vitro studies also showed that Dax1 could repress FAdE activity [START_REF] Zubair | Developmental links between the fetal and adult adrenal cortex revealed by lineage tracing[END_REF].

A c c e p t e d M a n u s c r i p t 8 Therefore it is tempting to speculate that Dax1 prevents adrenal differentiation in the cells of the AGP that are targeted to gonad fate. We previously described cells with adrenal markers expression -the so-called adrenal-like cells-within the testicular interstitium during embryonic development and after birth [START_REF] Val | Identification of a novel population of adrenallike cells in the mammalian testis[END_REF]. It would be interesting to investigate If Dax1 prevents adrenal differentiation in the AGP, by analysing the population of adrenal-like cells in the Dax1 mutant gonads of both sexes, which should be expanded. Thorough analysis of the early stages of adreno-gonadal development in Dax1 knockout mice may clarify this point.

IV-Signalling molecules and adrenal development

Various signalling pathways have been shown to be crucial for cell interactions that modulate patterning, differentiation and proliferation in numerous tissues of the embryo. Recent genetic studies in mouse have implicated members of the Wnt, Hedgehog and Fgf signalling pathways in the development of the adrenal cortex.

Wnt/catenin

The Wnt or 'wingless-like MMTV integration site' family of morphogens signal through an evolutionarily conserved pathway that has been implicated in many processes in embryonic development, regeneration, stem cell maintenance and cancer. The canonical Wnt pathway modulates the posttranslational regulation of -catenin, by controlling the activity of a multiprotein complex that targets -catenin for ubiquitin dependent degradation via the proteasome. -catenin is a multifunctional protein with one of its main functions being to act as a transcriptional cofactor for members of the Lef/Tcf family of transcription factors. In this capacity, -catenin acts as a final downstream target of the action of the Wnt family of secreted signalling molecules (for an in depth review of this pathway read [START_REF] Fuerer | Wnt signalling in development and disease. Max Delbruck Center for Molecular Medicine meeting on Wnt signaling in Development and Disease[END_REF]) A role for -catenin in adrenal development has recently been demonstrated by the study of mice with a conditional allele of this gene and a transgene containing Cre recombinase driven by Sf1 regulatory sequences [START_REF] Kim | Targeted disruption of beta-catenin in Sf1-expressing cells impairs development and maintenance of the adrenal cortex[END_REF]. In this study two transgenic Cre lines were used: one carried one copy of the transgene and had low recombination efficiency in the adrenal cortex (Sf1/Cre Low ); the other line carried five copies of the same transgene and displayed high recombination efficiency (Sf1/Cre High ). The Sf-1/Cre High transgene was shown to induce complete inactivation of -catenin as early as E12.5. This resulted in a drastic decrease in proliferation and expression of Sf-1. Expression of the steroidogenic enzymes Cyp11a1 and 3HSD was also markedly reduced. By E18.5, most of the adrenal gland (cortex and medulla) had disappeared in -catenin deficient embryos. These observations showed that -catenin was required to stimulate and/or maintain proliferation of adrenal cortical cells during embryonic development. In contrast, partial inactivation of -catenin with the Sf-1/Cre Low transgene had no major effect on adrenal histology and differentiation until 30 weeks post partum. At this stage, a subset of mutant mice presented thinning and disorganisation of the adrenal cortex as well as increased apoptosis. These also showed decreased expression of Sf-1 and steroidogenic markers. At 45 weeks post partum, the thinning phenotype was evident in all -catenin deficient mice. This suggested that renewal of adrenal cortical cells over time was markedly impaired in -catenin deficient mice. -catenin has been shown to play important roles in progenitor cell expansion and lineage decisions in multiple tissues [START_REF] Grigoryan | Deciphering the function of canonical Wnt signals in development and disease: conditional loss-and gain-of-function mutations of betacatenin in mice[END_REF]. In the adult adrenal,-catenin was found to be expressed in the subcapsular area [START_REF] Kim | Targeted disruption of beta-catenin in Sf1-expressing cells impairs development and maintenance of the adrenal cortex[END_REF] which has been postulated to contain adrenal progenitor cells [START_REF] Kim | Adrenocortical cells with stem/progenitor cell properties: recent advances[END_REF]. It is thus tempting to speculate that catenin plays a role in renewal and/or commitment of progenitors, in order to maintain adrenal cortical function throughout life.

A c c e p t e d M a n u s c r i p t 9

The impairment of proliferation in the embryonic adrenal cortex of -catenin deficient mice is consistent with clinical observations suggesting that constitutive ectopic activation of catenin in adult adrenals could be involved in the pathogenesis of benign and malignant adrenal cortical tumours [START_REF] Tissier | Mutations of beta-catenin in adrenocortical tumors: activation of the Wnt signaling pathway is a frequent event in both benign and malignant adrenocortical tumors[END_REF][START_REF] Gaujoux | Wnt/beta-catenin and 3',5'-cyclic adenosine 5'monophosphate/protein kinase A signaling pathways alterations and somatic betacatenin gene mutations in the progression of adrenocortical tumors[END_REF]. Analysis of Sf-1 haplo-insufficient mice suggested that Sf-1 was involved in the control of proliferation in the adrenal cortex [START_REF] Bland | Differential requirement for steroidogenic factor-1 gene dosage in adrenal development versus endocrine function[END_REF][START_REF] Bland | Haploinsufficiency of steroidogenic factor-1 in mice disrupts adrenal development leading to an impaired stress response[END_REF][START_REF] Bland | Gene dosage effects of steroidogenic factor 1 (SF-1) in adrenal development and the stress[END_REF]. Sf-1 was overexpressed in certain types of adrenal tumours, suggesting that increased Sf-1 dosage could participate to the tumorigenic process [START_REF] Figueiredo | Amplification of the Steroidogenic Factor 1 (SF-1) gene in childhood adrenocortical tumors[END_REF][START_REF] Digiammarino | A novel mechanism of tumorigenesis involving pHdependent destabilization of a mutant p53 tetramer[END_REF][START_REF] Ribeiro | An inherited p53 mutation that contributes in a tissue-specific manner to pediatric adrenal cortical carcinoma[END_REF][START_REF] Lefrancois-Martinez | Decreased expression of cyclic adenosine monophosphate-regulated aldose reductase (AKR1B1) is associated with malignancy in human sporadic adrenocortical tumors[END_REF]. Consistent with this, when Sf-1 was overexpressed in transgenic mice, a two fold increase in Sf-1 dosage was sufficient to induce adrenal tumour development soon after birth. Tumours in these mice first developed by expansion of capsular/sub-capsular cells and presented a mixed adrenal and gonadal phenotype [START_REF] Doghman | Increased steroidogenic factor-1 dosage triggers adrenocortical cell proliferation and cancer[END_REF]. Interestingly, Sf-1 and -catenin have been shown to co-ordinately control expression of multiple genes [START_REF] Gummow | Convergence of Wnt signaling and steroidogenic factor-1 (SF-1) on transcription of the rat inhibin alpha gene[END_REF][START_REF] Jordan | Wnt4 overexpression disrupts normal testicular vasculature and inhibits testosterone synthesis by repressing steroidogenic factor 1/-catenin synergy[END_REF][START_REF] Mizusaki | Dax-1 Gene Transcription is Regulated by Wnt4 in the Female Developing Gonad[END_REF]. Furthermore, -catenin and Lrh-1, a nuclear receptor closely related to Sf-1 that binds to the same DNA response element [START_REF] Fayard | LRH-1: an orphan nuclear receptor involved in development, metabolism and steroidogenesis[END_REF], cooperate to stimulate expression of Cyclin D1 and Cyclin E1 in intestinal crypts [START_REF] Schoonjans | Liver receptor homolog 1 contributes to intestinal tumor formation through effects on cell cycle and inflammation[END_REF][START_REF] Botrugno | Synergy between LRH-1 and beta-catenin induces G1 cyclinmediated cell proliferation[END_REF]. Therefore, it is likely that -catenin and Sf-1 cooperate to control cell proliferation and progenitor cell commitment in the adrenal cortex. The only Wnt molecule reported to be expressed in the adrenal is Wnt4, which is thought not to signal through the canonical pathway [START_REF] Heikkila | Wnt-4 deficiency alters mouse adrenal cortex function, reducing aldosterone production[END_REF]. However, studies in the gonad showed that Wnt4 can act through -catenin as Wnt4 mutant embryos show a similar phenotype to catenin mutants in the developing ovary [START_REF] Chassot | Activation of beta-catenin signaling by Rspo1 controls differentiation of the mammalian ovary[END_REF][START_REF] Liu | Sex-Specific Roles of {beta}-catenin in Mouse Gonadal Development[END_REF][START_REF] Jeays-Ward | Endothelial and steroidogenic cell migration are regulated by WNT4 in the developing mammalian gonad[END_REF]. Wnt4 was shown to be expressed in the early adrenal and restricted to the outermost area of the cortex as early as E14.5. Analysis of the adrenal phenotype of Wnt4 mutant embryos showed a milder phenotype than the catenin mutants, in that adrenal development did occur but zona glomerulosa differentiation was defective with reduced Cyp11b2 expression and low aldosterone production [START_REF] Heikkila | Wnt-4 deficiency alters mouse adrenal cortex function, reducing aldosterone production[END_REF]. This suggested that other -catenin activating signalling molecules were present in the adrenal. A phenotype shared by Wnt4 and -catenin mutant embryos was the presence of cells expressing the adrenal specific marker, Cyp21 within the anterior region of the ovary. Cell migration assays showed that these cells migrated from the adrenal area to the mutant gonad in Wnt4 mutant embryos [START_REF] Heikkila | Wnt-4 deficiency alters mouse adrenal cortex function, reducing aldosterone production[END_REF][START_REF] Jeays-Ward | Endothelial and steroidogenic cell migration are regulated by WNT4 in the developing mammalian gonad[END_REF]. This indicated that this pathway was required to prevent migration of adrenal cells to the gonad. Reciprocally, Wnt4 mutant male adrenals showed ectopic expression of Cyp17. This 'gonadal' steroidogenic enzyme is normally expressed in the female but not the male developing adrenal and is never found in the adrenal of rodents after birth [START_REF] Heikkila | Wnt-4 deficiency alters mouse adrenal cortex function, reducing aldosterone production[END_REF]. Whether this observation reflects abnormal migration of gonadal cells to the adrenal or de-repression of the gonad differentiation programme in the adrenal is unknown.

Sonic Hedgehog (Shh)

The hedgehog signalling pathway is involved in essential processes such as patterning of tissues during development, cell proliferation and differentiation. Activity of the pathway relies on three ligands: Sonic Hedgehog (Shh), Indian Hedgehog (Ihh) and Desert Hedgehog (Dhh). These bind the Patched1 and Patched2 (Ptch1, Ptch2) membrane receptors on Hedgehog responding cells. Binding of Hedgehogs to their receptors triggers a complex signalling cascade that culminates with modulation of Gli transcription factor activity (For an in depth review of this pathway, read [START_REF] King | Hedgehog signalling in endocrine development and disease[END_REF]). Hedgehog signalling has been implicated in steroidogenic cell activation within the gonad. Yao and colleagues showed that in the developing testis, Dhh was expressed by Sertoli cells at E11.5 whereas Ptch1 was present in the interstitium at E12.5, suggesting that Hedgehog signalling could be involved in the development of Leydig cells. Consistent with this, genetic ablation of Dhh or treatment with the hedgehog signalling inhibitor cyclopamine, resulted in an impairment of Leydig cells differentiation, characterised by decreased expression of Sf-1 and Cyp11a1 [START_REF] Yao | Desert Hedgehog/Patched 1 signaling specifies fetal Leydig cell fate in testis organogenesis[END_REF]. Although an effect of hedgehog signalling on Leydig cells precursors A c c e p t e d M a n u s c r i p t recruitment could not be ruled out, this suggested that hedgehog signalling was required for up-regulation of Sf-1 expression (either direct or indirect) and subsequent Leydig cell differentiation. The first hint that Hedgehog signalling may be involved in adrenal development came from the observation of adrenal hypoplasia in infants with Pallister-Hall syndrome (PHS, OMIM #146510), an autosomal dominant disorder caused by truncations of GLI3 [START_REF] Hall | Congenital hypothalamic hamartoblastoma, hypopituitarism, imperforate anus and postaxial polydactyly--a new syndrome? Part I: clinical, causal, and pathogenetic considerations[END_REF][START_REF] Kang | GLI3 frameshift mutations cause autosomal dominant Pallister-Hall syndrome[END_REF]. Consistent with this, transgenic mouse models with a similar truncation of Gli3 also showed adrenal agenesis at late gestation [START_REF] Bose | Pallister-Hall syndrome phenotype in mice mutant for Gli3[END_REF]. Preliminary experiments in mouse embryos showed that Shh was the only member of the family to be expressed at the periphery of the cortex as early as E14.5 and in the subcapsular area after birth. Ptch1 was found in an adjacent external layer of cortical cells, suggesting that active hedgehog signalling occurred in adrenal areas that contain progenitor cells ([95, 100] and our unpublished observations). Consistent with this, preliminary observations by King and colleagues show that adrenal gland development is impaired as early as E12.5 in Shh deficient mice [START_REF] King | Hedgehog signalling in endocrine development and disease[END_REF]. Whether this affects proliferation, differentiation, zonal patterning or renewal of the adrenal cortex has yet to be determined. The overlap of Shh and -catenin expression in the subcapsular area suggests that the two pathways may interact to control progenitor cells renewal and commitment as observed in other development and cancer models [101-104]. Detailed genetic analysis of Hedgehog and -catenin signalling interactions should provide insight into the role of these pathways in adrenal gland development and homeostasis.

Fibroblast Growth Factors (FGFs) and FGF-receptor type II (FGFR2)

FGFs constitute a vast family of signalling proteins (at least 28 members) involved in diverse processes such as cell differentiation, proliferation, angiogenesis, and wound healing. FGFs mediate their activities by binding to a family of four receptor tyrosine kinases (FGFR1-4). This triggers intra-cellular signalling through the MAP kinase (MAPK), the phosphatidyl inositol 3 kinase (PI3K) and protein kinase C (PKC) pathways (for review see [105]). FGF9, through FGF-receptor type 2 is essential for male sex determination by controlling proliferation of pre-sertoli cells in the testis [106-108]. Interestingly, mice in which Fgfr2 was conditionally deleted using a Sf-1-driven Cre line had barely detectable adrenal development at E15. 5 [108]. The factor responsible for this effect on adrenal growth is unknown. However basic FGF (bFGF/FGF2) which was shown to stimulate proliferation in a variety of adrenal cell types is a likely candidate [109-112]. In adult adrenals, bFgf expression is stimulated by ACTH and it was proposed to mediate the mitogenic effects of ACTH [109,110,113]. Although ACTH is involved in adrenal growth during development [START_REF] Mesiano | Developmental and functional biology of the primate fetal adrenal cortex[END_REF], a potential impairment of ACTH mitogenicity owing to Fgfr2 deficiency is unlikely to account for adrenal agenesis in Fgfr2-/-embryos at E15.5. Indeed, although adrenals in mice deficient for POMC, the peptide precursor of ACTH, are hypoplastic at birth, their size and histology can be restored upon chronic ACTH treatment [114][115][116]. This indicates that embryonic adrenal development is only mildly impaired in the absence of ACTH. Therefore FGFs must have a role during early adrenal development beyond growth response to ACTH. Interestingly, in adult rat adrenals, bFgf production was detected in the zona glomerulosa and bFgf binding sites were found in the zona glomerulosa and in cells of the capsule [110,111]. This subcapsular localisation is reminiscent of both -catenin and Shh and suggests a role for Fgf signalling in the control of adrenal progenitor cells homeostasis.

The role of signalling pathways in adrenal development was overlooked for a long time. Recent analysis of the mutant mouse models presented in this chapter has emphasised the need for in-depth research in this area. Although the published data mainly points to a role of -catenin, hedgehog and Fgf signalling in the stimulation of adrenal proliferation it is likely A c c e p t e d M a n u s c r i p t 11 that these pathways also participate in the differentiation and zonal shaping of the adrenal cortex. Other signalling pathways may also be involved, for example the TGF/activin/BMP signalling pathway has been shown to prevent adrenal tumorigenesis and gonadal differentiation of adrenal progenitor cells [117,118]. Although this data relies on observations in adult mice, it is possible that this pathway could play a role during specification of the adrenal cortex at earlier stages.

V-Conclusion

Although the adrenal gland was first described by Eustachius in the 16 th century, our understanding of the molecular and cellular mechanisms required for its embryonic development is still limited. The identification of Sf-1 and the analysis of the phenotype of mice with a genetic ablation of this gene in 1994 undoubtedly enhanced our knowledge of how adrenal cortex development and physiology were regulated at the molecular level. More recent work has shown that Sf-1 dosage is of crucial importance to specify adrenal cell fate from the bipotent AGP and has identified some of the mechanisms required to ensure that the proper levels are available to ensure adrenal differentiation. Other than Sf-1, very little is known about what factors are required to determine the differentiation of the adrenal cortex. Sf-1 target genes involved in this process and zone specific factors that interact with Sf-1 providing specificity have still not been identified. An in depth study of the different signalling pathways that have been shown to be required for adrenal cortex development will be essential not only in establishing the role of these factors but in identifying cellular interactions that are important in this process. The key events of adrenal development are shown. Adrenal cortex and gonads are derived from the adreno-gonadal primordium (AGP, green cells), a common progenitor cell population. At E10-E10.5 foetal adrenal cortical cells progressively separate from the AGP in the rostral area (light blue cells). Remaining cells of the AGP form the bipotential gonad anlage (pink cells). This differentiates as a testis upon expression of Sry in the male embryo or as an ovary in the female embryo. After separation from the AGP, the adrenal anlage is encapsulated and colonised by neural crest cells that will form the medulla. Lineage tracing experiments have shown that foetal adrenal cells derived from the AGP give rise to definitive adrenal cells (dark blue cells) that will form the adult cortex. The first signs of functional adrenocortical zonation appear at around E16 but it is not until the third week post partum that all four cortical zones (glomerulosa, fasciculata, reticularis, X-zone) are clearly defined. The X-zone is thought to represent a remnant of the foetal adrenal cortex. It regresses after the first pregnancy in females or at puberty in males. Molecular and genetic analysis of Cited2 mutant mice has shown that this cofactor interacts with Wt1 in order to modulate Sf-1 dosage in the AGP, before overt adrenal differentiation. In wild-type mouse AGP, Cited2 and Wt1 stimulate Sf-1 expression above the threshold required to trigger adrenal development. In Cited2 deficient mice, Sf-1 expression is decreased to 36% of wild-type levels. This level is sufficient to trigger development of the gonad although its later differentiation is impaired. However, Sf-1 dosage does not reach the threshold required to trigger adrenal formation. Adrenal primordium development from the AGP does not occur. Pictures show RNA in situ hybridisation for Sf-1 in wild-type (left panel) or Cited2-/-embryos (right panel). Abbreviations: AGP, adreno-gonadal primordium; Ad, adrenal primordium; Go, gonad primordium.

A c c e p t e d M a n u s c r i p t

A c c e p t e d M a n u s c r i p t 2 I

 2 -The Adreno-Gonadal Primordium (AGP)

Figure 1 .

 1 Figure 1. Adrenal development in mouse embryos.The key events of adrenal development are shown. Adrenal cortex and gonads are derived from the adreno-gonadal primordium (AGP, green cells), a common progenitor cell population. At E10-E10.5 foetal adrenal cortical cells progressively separate from the AGP in the rostral area (light blue cells). Remaining cells of the AGP form the bipotential gonad anlage (pink cells). This differentiates as a testis upon expression of Sry in the male embryo or as an ovary in the female embryo. After separation from the AGP, the adrenal anlage is encapsulated and colonised by neural crest cells that will form the medulla. Lineage tracing experiments have shown that foetal adrenal cells derived from the AGP give rise to definitive adrenal cells (dark blue cells) that will form the adult cortex. The first signs of functional adrenocortical zonation appear at around E16 but it is not until the third week post partum that all four cortical zones (glomerulosa, fasciculata, reticularis, X-zone) are clearly defined. The X-zone is thought to represent a remnant of the foetal adrenal cortex. It regresses after the first pregnancy in females or at puberty in males. Factors involved in AGP development as well as adrenal and gonadal fate determination are shown. See text for a detailed description of their function. Abbreviations: E, Embryonic day; dpp, days post partum.

  Figure 1. Adrenal development in mouse embryos.The key events of adrenal development are shown. Adrenal cortex and gonads are derived from the adreno-gonadal primordium (AGP, green cells), a common progenitor cell population. At E10-E10.5 foetal adrenal cortical cells progressively separate from the AGP in the rostral area (light blue cells). Remaining cells of the AGP form the bipotential gonad anlage (pink cells). This differentiates as a testis upon expression of Sry in the male embryo or as an ovary in the female embryo. After separation from the AGP, the adrenal anlage is encapsulated and colonised by neural crest cells that will form the medulla. Lineage tracing experiments have shown that foetal adrenal cells derived from the AGP give rise to definitive adrenal cells (dark blue cells) that will form the adult cortex. The first signs of functional adrenocortical zonation appear at around E16 but it is not until the third week post partum that all four cortical zones (glomerulosa, fasciculata, reticularis, X-zone) are clearly defined. The X-zone is thought to represent a remnant of the foetal adrenal cortex. It regresses after the first pregnancy in females or at puberty in males. Factors involved in AGP development as well as adrenal and gonadal fate determination are shown. See text for a detailed description of their function. Abbreviations: E, Embryonic day; dpp, days post partum.

Figure 2 .

 2 Figure 2. Effect of Cited2 on Sf-1 dosage and adrenal fate determination in the AGP.Molecular and genetic analysis of Cited2 mutant mice has shown that this cofactor interacts with Wt1 in order to modulate Sf-1 dosage in the AGP, before overt adrenal differentiation. In wild-type mouse AGP, Cited2 and Wt1 stimulate Sf-1 expression above the threshold required to trigger adrenal development. In Cited2 deficient mice, Sf-1 expression is decreased to 36% of wild-type levels. This level is sufficient to trigger development of the gonad although its later differentiation is impaired. However, Sf-1 dosage does not reach the threshold required to trigger adrenal formation. Adrenal primordium development from the AGP does not occur. Pictures show RNA in situ hybridisation for Sf-1 in wild-type (left panel) or Cited2-/-embryos (right panel). Abbreviations: AGP, adreno-gonadal primordium; Ad, adrenal primordium; Go, gonad primordium. Pictures were reproduced with the kind permission of The Company of Biologists Ltd. They originate from Val et al., Development 134[START_REF] Wang | Expression of 11- hydroxylase in rat Leydig cells[END_REF]:2349-2358.

  Figure 2. Effect of Cited2 on Sf-1 dosage and adrenal fate determination in the AGP.Molecular and genetic analysis of Cited2 mutant mice has shown that this cofactor interacts with Wt1 in order to modulate Sf-1 dosage in the AGP, before overt adrenal differentiation. In wild-type mouse AGP, Cited2 and Wt1 stimulate Sf-1 expression above the threshold required to trigger adrenal development. In Cited2 deficient mice, Sf-1 expression is decreased to 36% of wild-type levels. This level is sufficient to trigger development of the gonad although its later differentiation is impaired. However, Sf-1 dosage does not reach the threshold required to trigger adrenal formation. Adrenal primordium development from the AGP does not occur. Pictures show RNA in situ hybridisation for Sf-1 in wild-type (left panel) or Cited2-/-embryos (right panel). Abbreviations: AGP, adreno-gonadal primordium; Ad, adrenal primordium; Go, gonad primordium. Pictures were reproduced with the kind permission of The Company of Biologists Ltd. They originate from Val et al., Development 134[START_REF] Wang | Expression of 11- hydroxylase in rat Leydig cells[END_REF]:2349-2358.

Figure 3 .

 3 Figure 3. Factors controlling Sf-1 dosage in early embryonic development. Factors that stimulate Sf-1 expression in the AGP (Wt1 and Cited2) through the 'AGP enhancer' or in the foetal adrenal (Pbx1/Hox) through the foetal adrenal enhancer (FAdE) are shown on the left (red arrows). Whether Cited2 cooperates with Pbx1 or other factors to stimulate FAdE activity is not known. How M33 stimulates Sf-1 expression is yet unclear. Factors that decrease Sf-1 dosage either at the transcription level (Pod1) or by inhibitory protein-protein interactions (Dax1) are shown on the right (blue arrows). Increased Sf-1 dosage is essential for adrenal fate determination. It results in steroidogenic differentiation, increased cell proliferation and inhibition of apoptosis. Sf-1 targets involved in cell fate determination, proliferation and apoptosis are mostly unidentified.

  s c r i p t A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t


Lamiae Grimaldi-Bensouda

Lucien Abenhaim

Laurent Michaud

Olivier Mouterde

Annie-Pierre Jonville-Bera

Bruno Giraudeau

Benoît David

Elisabeth Autret-Leca
email: autret-leca@med.univ-tours.fr

Annie Pierre

A P Jonville

Clinical features and risk factors for upper gastrointestinal bleeding in children: a case-crossover study

Keywords: Upper gastrointestinal bleeding, Nonsteroidal anti-inflammatory drug, Children, Casecrossover, Ibuprofen

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Introduction

Upper gastrointestinal bleeding (UGIB) is a rare but severe occurrence in children that often requires emergency treatment. It is generally agreed that the disease presents itself with haematemesis or maelena that it is favoured by a medical history of ulcer, coagulation disorders, portal hypertension and Mallory-Weiss syndrome and that aspirin and some other nonsteroidal anti-inflammatory drugs (NSAIDs) play a causative role [START_REF] Laine | Proton pump inhibitor co-therapy with nonsteroidal anti-inflammatory drugs-nice or necessary?[END_REF][START_REF] Van Leerdam | Epidemiology of acute upper gastrointestinal bleeding[END_REF][START_REF] Hollenz | NSAIDassociated dyspepsia and ulcers: a prospective cohort study in primary care[END_REF][START_REF] Cagnoni | Gastrointestinal bleeding in hemophilia as a complication of the use of over the counter non-steroidal anti-inflammatory drugs[END_REF]. Data pertaining to children are, however, limited [START_REF] Fox | Gastrointestinal bleeding in infancy and childhood[END_REF], and the evidence is based mainly on case reports, series and relatively small cohorts [START_REF] Mouterde | Les hémorragies digestives chez l'enfant, à propos de 485 endoscopies[END_REF][START_REF] Keenan | Clinically significant gastropathy associated with nonsteroidal antiinflammatory drug use in children with juvenile rheumatoid arthritis[END_REF][START_REF] Titchen | Adverse drug reactions to nonsteroidal anti-inflammatory drugs, COX-2 inhibitors and paracetamol in a paediatric hospital[END_REF]. The actual distribution of anatomical locations and lesion presentation and the part of the disease attributable to risk factors and drugs are poorly understood in children. The role of ibuprofen has been dismissed after a large randomised double-blind trial of febrile children treated by paracetamol or ibuprofen concluded there was no statistical difference in disease risk between the drugs [START_REF] Lesko | An assessment of the safety of pediatric ibuprofen. A practitioner-based randomized clinical trial[END_REF]. However, the four cases of UGIB observed during the study were in the ibuprofen arm [START_REF] Lesko | An assessment of the safety of pediatric ibuprofen. A practitioner-based randomized clinical trial[END_REF]. In clinical practice, the observation of patients exposed to this drug is relatively frequent, but the use of the drug is also extremely frequent. Thus, it is still questionable as to whether ibuprofen is or is not a risk factor for this disease, and if so, at what dosage. To our knowledge, no population-based study of drug risk factors and UGIB in children has been reported. We conducted this study to provide a formal description of the location and clinical presentation of UGIB in children and describe the relative contribution of several risk factors, including drugs. The study was powered with the primary objective of assessing the potential contribution of NSAIDs.

Methods

Cases

We conducted a national study in which paediatric gastroenterologists from all 32 university hospitals in France were identified and contacted. Twenty agreed to participate. Two large regional hospitals were subsequently added. Potential participants were informed of the study objectives, and when they agreed to participate, they were visited for study implementation. Patients to be recruited were children aged between 2 months and 16 years of both genders with UGIB defined as oesophageal and/or gastric and/or duodenal bleeding and presenting with haematemesis, maelena or haemorrhagic shock. Gastroenterologists were requested to report all cases identified in their centres during the study period and to describe clinical presentation using clinical research forms. Among the items of interest were previous medical history, symptoms and signs at presentation, diagnostic procedures applied, severity of the bleeding and treatment to manage UGIB. Physicians were required to describe endoscopy findings, if performed. A priori risk factors included portal hypertension, history of UGIB or gastric or duodenal ulcer, gastrooesophageal reflux and coagulation disorders. Consent was obtained by the physicians from the parent of participating children.

Interviews

Parents were given a self-assessment questionnaire by their child's physicians to fill out in preparation for the interview. Parents were questioned by two independent trained interviewers who contacted them at home by telephone and confirmed their consent. The interview was conducted in the 10 days following UGIB. Information on children's drug intake in the 4 weeks before hospitalisation for UGIB was requested, and parents were asked to have at hand the written prescriptions and drug packaging information. To minimize recall bias, parents were informed that drug use as far as 1 month prior to onset of bleeding was just as important as recent drug use. Parents were asked to report the drug(s) used by their children, without any list provided to them a priori. For each drug reported within the 4 weeks prior to hospitalisation, indication of use, dosage, duration of use and date of initiation were requested. Dosages were recorded as prescribed, usually in milligrams (mg)/kilogram (kg) per day. Those reported in mg/day were converted to mg/kg per day (three patients). Drugs were first coded as reported (usually brand or generic name) and then by class (antibiotics, antacids, NSAIDs, corticoids, others). We considered NSAIDs used at the time of the study (aspirin, ibuprofen, ketoprofen, mefenamic acid, fenamate, tiaprofenic acid) and the others. Paracetamol was coded individually. Drug exposure was defined by at least one drug intake during risk or control periods (described below).

Sample size

The study was powered to detect a hypothetical relative risk (RR) of NSAID-related UGIB of 3, with 95% confidence interval (CI) and 80% power, assuming a prevalence of NSAID use in children of 4%. The odds ratio (OR) of 3 was chosen because it is usually reported in adults treated with ibuprofen, the most frequently used NSAID in children [START_REF] Rodriguez | Relative risk of upper gastrointestinal complications among users of acetaminophen and nonsteroïdal anti-inflammatory drugs[END_REF]. The prevalence of NSAID use of 4% in the paediatric population was chosen assuming children are exposed 2 weeks per year to NSAIDs (personal data). A target sample size of 187 children with UGIB was calculated as required for such a goal. Thirty centres were initially thought to be necessary for the recruitment.

Design

A case-crossover comparison for the presence of transient risk factors was chosen for this study. In this design, cases are taken as their own controls [START_REF] Maclure | The case-crossover design: a method for studying transient effects on the risk of acute events[END_REF] and exposures compared for different individual time windows considered as at risk or not at risk for developing the disease as a consequence of exposure. The predefined periods for comparisons were the 7 days preceding the date of hospitalisation (period at risk, or week 1) and the 21st to the 28th days before that date (control period, or week 4).

Statistical analysis

Crude and adjusted case-crossover ORs were calculated for exposure to any NSAID during the risk or control periods. Crude ORs were estimated with methods for matched-pair studies, by N 01 /N 10 (Mantel-Haenszel estimate for matched-pair analyses) where N 01 is the number of cases exposed during the risk period and not the control period, and N 10 the number of cases not exposed during the risk period and exposed during the control period [START_REF] Maclure | The case-crossover design: a method for studying transient effects on the risk of acute events[END_REF]. Sets where exposures occurred in both periods were considered as concordant pairs. Potential confounders known to influence the disease (portal hypertension, history of UGIB or gastric or duodenal ulcer and coagulation disorders) were defined a priori. Children with and without those risk factors were compared regarding NSAID use. Multivariate conditional logistic regression was performed with all a priori confounders cited above. All NSAIDs and the three most used classes of drugs other than NSAIDs were included into the final model. Results are presented as means, percentages, and ORs with 95% two-sided CI with beta set at 20%. Statistical analysis and estimation of OR and their CI were performed using SAS software version 9.1 (SAS Institute, Inc., NC, USA). The fraction of cases attributable to an exposure was calculated by the following formula: (OR-1)/OR. Percentages are rounded to the closest integer in the text and to the closest decimal in the tables; 95% CI to OR are presented in parentheses in the text.

Results

Fourteen university hospitals and two regional general hospitals recruited cases. Among the 185 cases reported in 24 months, the parents of three children were not able to be contacted by telephone, and five secondarily declined the interview. The final data set included 177 patients aged 2 months to 16 years.

Clinical features

The proportion of children 2 months to 2 years old (33%) reported was relatively more important than those of other age groups. Haematemesis was present in almost all children, with the exception of a few younger ones, and maelena was more frequent in the 2 months to 2 years age group (24% vs. 9%). Most patients (80%) had undergone endoscopic examination, which showed difference in lesion location according to age: 37% of children in the youngest age group presented with gastric lesions and 52% with oesophageal lesions and 65% and 23%, respectively, in older children. Duodenal lesions were rare in all age groups. Clinical presentation was similar in girls and boys (data not shown). The treatment required was antiulcer and antacid drugs in 60% of children, transfusion in 11%, miscellaneous in 18% and none in 21% (Tables 1 and2).

Risk factors

Thirty-three children had at least one medical risk factor of bleeding, such as previous medical history of portal hypertension, personal history of ulcer or UGIB, coagulation disorders or rheumatoid purpura. Details of medical risk factors are presented in Table 1. Among them, 21.2% had also used an NSAID at some point during the 4 previous weeks, which is significantly different from the proportion of NSAID use in children without medical risk factor (p =0.001).

In the 4-week period prior to bleeding, 79.1% of children had taken at least one drug. Eighty-three children (46.9%) had taken at least one NSAID at some point during these 4 weeks, which was ibuprofen in 70% of cases, aspirin in 31% and another NSAID in 11% (Table 3). Other NSAIDs used were fenamate, ketoprofen, flurbiprofen and naproxen. This use had occurred mostly in the week at risk. The 3-to 7-year-old age group was the most exposed to NSAIDs. Fever was reported 69 times (83%) as the reason for NSAID uses, moderate pain 18 times (22%), rheumatic indication once (1%) and reasons unknown 11 (13%). NSAIDs dosages used were indeed mainly those corresponding to the recommended doses for antipyretic and analgesic treatments: 18.4±11 mg/kg per day for ibuprofen and 24±16 mg/kg per day for aspirin, 31.75±17 mg/kg per day for fenamates, 1.75±0.25 mg/kg per day for ketoprofen, 2 mg/kg per day for flurbiprofen and 11.5 mg/kg per day for naproxen. The mean duration of NSAID use in the 4 weeks prior to bleeding was short and in accordance with their indication (1.9±1.5 days); it varied from 1.6±1.4 days for aspirin to 1.8±1.4 for ibuprofen. Other drugs most frequently reported were paracetamol (37%) and antibiotics (29%).

Table 4 shows the crude and adjusted OR (with 95% CI) for NSAIDs use during the risk and control periods in the 177 children studied, controlling for the most frequently used drugs. The adjusted OR of exposure to NSAIDs was 8.2 (2.6-26.0) for NSAIDs altogether, and this was 10.0 92.0-51.0) for ibuprofen and 7.3 (0.9-59.4) for aspirin. The adjusted OR of exposure to NSAIDs related to oesophageal lesions was 1.0 (0.2-7.2) and related to gastric or duodenal lesions 7.9 (1.7-37.5).

The OR for NSAID use and UGIB was 14.1 (2.8-71.2) and 3.4 (0.6-21.3) in the 2 months to 7 years and 8-16 years age groups, respectively (p for trend<0.001).

Discussion

To our knowledge, this is the first published populationbased survey of UGIB in children that explores drug risk factors. Almost half (45%) of the university hospitals of the country participated, plus two very large regional hospitals. Recruitment per centre was very close to plans (5.5 vs. 6 cases per centre per year), and the study finally had the power to assess the risk associated with different risk factors and drugs by age, gender and clinical presentation. One case out of five occurred in patients with a medical risk factor or medical history, and 40% or more of the children had taken an NSAID in the week prior to hospitalisation, three quarters of which were ibuprofen. The frequency of children who took aspirin is not surprising, as in contrast to most European countries, aspirin is not contraindicated for use in children in France. In fact, only a caution of use is included in aspirin summary of product characteristics (SPC) in cases of viral infections (flu, chicken pox). In approximately two thirds of cases, no risk factor was found. Boys and girls had very similar presentations, and the risk seemed to decline with age. The results show, for the first time, that in children, as in adults, there is apparently no excess risk of oesophageal lesion associated with NSAIDS [START_REF] Rodriguez | Relative risk of upper gastrointestinal complications among users of acetaminophen and nonsteroïdal anti-inflammatory drugs[END_REF][START_REF] Holvoet | Relation of uppergastrointestinal bleeding to non-steroidal anti-inflammatory drugs and aspirin: a casecontrol study[END_REF][START_REF] Laporte | Upper gastrointestinal bleeding associated with the use of NSAIDs: newer versus older agents[END_REF][START_REF] Gutthann | Individual non-steroidal anti-inflammatory drugs and other risk factors for upper gastrointestinal bleeding and perforation[END_REF]. According to the findings, there would be an increased risk of gastric lesions with these drugs, with no apparent difference between ibuprofen and aspirin, taken at analgesic or antipyretic doses. The study confirms that UGIB is rare, so that the absolute risk associated with these drugs may remain small altogether. Before exploring these findings further, biases that may have participated to the observations are discussed. First, parents may better recall drug intake in their offspring in the week preceding hospitalisation than those that occurred several weeks earlier. The fact that, as expected, there was no increased risk associated with NSAIDs use observed in a The 2 months to 2 years and 3-to 7-years age groups were aggregated due to small numbers b OR were adjusted for the use of paracetamol, corticosteroids, antibiotics, antacids and antiulcer; an adjusted model was applied separately for each drug or group of drugs studied

NC not calculated due to small numbers children with oesophageal lesions (OR =1.0) militates against an important recall bias, as parents would not know this difference. Also, there was no significant change observed in the risk estimates when a more recent time window (week 3) was used as control (data not shown).

Second, physicians may be more likely to diagnose UGIB in children exposed to NSAIDs: endoscopic examination was slightly less frequently performed in children exposed to NSAIDs than in non-NSAIDs users (72% vs. 84%, respectively), but NSAIDs users were diagnosed with severe signs as frequently as nonusers (63% vs. 66%, respectively); the OR for NSAIDs use in children who had undergone confirmatory endoscopy were similar to those observed in the sample as a whole: 7.7 (2.1-28.0) vs. 8.2 (2.6-26.0), respectively. Third, the notoriety of the risk of UGIB side effects of NSAIDs may have inclined centres to more frequently report cases recently exposed to NSAIDs than those that were not. Important efforts were made to minimize this bias by insisting very frequently on the necessity to report all cases of UGIB observed. The average number of cases reported by centre and per year (n=5.5) over the study period was very close to the number planned before the study started (n=6) ,and there was no significant variation in the percentage of exposure recorded per centre.

All in all, we believe that the results are not likely to be explained fully by these biases. The case-crossover design has the advantage of controlling efficiently for nontransient individual confounders, such as genetic, biological or environmental factors, and has been shown to be efficient in pharmacoepidemiology [START_REF] Destefano | Vaccinations and risk of central nervous system demyelinating diseases in adults[END_REF][START_REF] Viboud | Comparison of the statistical efficiency of case-crossover and case-control designs: Application to severe cutaneous adverse reactions[END_REF][START_REF] Etienney | Nonsteroidal anti-inflammatory drugs as a risk factor for acute diarrhoea: a case crossover study[END_REF]. Within the NSAIDs category, the risk was similar for ibuprofen, aspirin or all NSAIDs (adjusted OR 10.0, 7.3 and 8.2, respectively). There was not sufficient use of other NSAIDs to conclude on their risks individually, but one cannot exclude the possibility that others may be risk factors also. However, as they represented a small fraction of drugs used, the risk for all NSAIDs reflect mainly that of ibuprofen and aspirin. These results are not inconsistent with the few data published on NSAIDs and UGIB in children mentioned in the "Introduction". Indeed, in a published series of 231 children who underwent endoscopy for UGIB, 15% took NSAIDs [START_REF] Mouterde | Les hémorragies digestives chez l'enfant, à propos de 485 endoscopies[END_REF]; from an Australian hospital adverse drug reactions database, four haematemesis with NSAIDs were collected over 5 years, three of which were associated with ibuprofen (used at the higher end of the usual range of dose) and one with celecoxib at too high a dose [START_REF] Titchen | Adverse drug reactions to nonsteroidal anti-inflammatory drugs, COX-2 inhibitors and paracetamol in a paediatric hospital[END_REF]. The only randomised trial is the Lesko study [START_REF] Lesko | An assessment of the safety of pediatric ibuprofen. A practitioner-based randomized clinical trial[END_REF], which reported more cases of UGIB with ibuprofen than with paracetamol (four vs. zero cases) but may have lacked power (although its sample size was large).

The RR estimated here with NSAIDs use seems higher than that usually observed in adults (eight vs. two to four). This may be due to chance or to the fact that children may have a higher sensitivity to NSAIDs side effects. Alternative explanations may be that primary exposure to the drugs may be associated with higher risk, which is the case in adults [START_REF] Levy | Major upper gastrointestinal tract bleeding. Relation to the use of aspirin and other nonnarcotic analgesics[END_REF], or to the so-called depletion of susceptible effect that we previously demonstrated to occur with NSAIDs and UGIB in adults [START_REF] Moride | Evidence of the depletion of susceptibles effect in non-experimental pharmacoepidemiologic research[END_REF] (people at risk tend to be withdrawn from the opportunity to exposure and to subsequently develop disease more and more with time). The number of cases reported in the younger age groups was indeed proportionally much higher than in older children. The fact that younger patients may be more readily hospitalised in the presence of bleeding is not supported by the similar severity and percentage of confirmed lesions between age groups. The study confirms that the number of UGIB seen in paediatric units of university or regional hospitals is few (one case every other month). Not all cases of this disease are referred to these hospitals, as some may be seen in general hospitals without paediatric units. Typically, nonuniversity or nonregional general hospitals accrue a lesser number of cases per year. Using the numbers observed and applying different hypothesis yields estimates between 1,000 and 2,000 cases per year in France, which is an incidence in the vicinity of 1-2 per 10,000 children per year in the 2 months to 16 years old age group (12,000,000 in the country). According to this study, the fraction of cases attributable to NSAIDs would be 88% of the 41% exposed in the week before occurrence, which is 36%.

To conclude, this study confirms that UGIB is rare, occurs more likely in children of younger age and shows that it is attributable to the use of NSAIDs, including ibuprofen and aspirin, in approximately one third of cases. Some of these cases may be avoided by using alternative drugs such as paracetamol for analgesic or antipyretic purposes, particularly in patients with medical risk factors for the disease. The findings from this study call for more caution in prescribing NSAIDs to children.

Table 1

 1 Patient gender and clinical features by age

	Age groups
	2months to <2years	> 3years to <7years	> 7years to < 16years	Total
	N=58 (32.8%)	N=60 (33.9%)	N=59 (33.3%)	N=177

Table 2

 2 Endoscopy findings (numbers are not exclusive)

		2months to <2years N=58	> 3years to <7years N=60	> 7years to < 16years N=59	Total N=177
		(32.8%)	(33.9%)	(33.3%)	(%)
	Endoscopy performed	46 (79.3)	46 (76.7)	49 (83.1)	141 (79.7)
	Endoscopy: normal	8 (17.4)	11 (23.9)	5 (10.2)	24 (17.0)
	Endoscopy: unknown	2 (4.3)	1 (2.2)	1 (2.0)	4 (2.8)
	Oesophageal lesions	24 (52.2)	10 (22.7)	18 (36.7)	52 (36.9)
	-Erythematous	2	5	10	17
	oesophagitis
	-Erosive oesophagitis	5	2	5	12
	-Ulcerated oesophagitis 5	4	4	13
	-Varices	3	2	2	7
	-Other	3	0	5	8
	Gastric lesions	17 (37.0)	30 (65.2)	32 (65.3)	79 (56.0)
	-Erythematous gastritis	3	10	11	24
	-Erosive gastritis	4	3	5	12
	-Ulcerations	0	4	6

Table 4

 4 Crude and adjusted odds ratios (OR) and confidence intervals (CI) for the use of NSAIDs and other classes of drugs in the 4 weeks before bleeding in the 177 children included in the study

	Exposure	Week 1 before	Percent	Week 4 before	Percent	Crude OR	Adjusted OR b
		bleeding (at-risk) N		bleeding (control) N		(95% CI)	(95% CI)
	All NSAIDs	73	41.2%	8	4.5%	17.3 (6.3-47.3)	8.2 (2.6-26.0)
	Oesophageal lesions	11	6.2%	5	2.8%	3.0 (0.6-11.0)	1.0 (0.2-7.2)
	Gastric or duodenal lesions	38	21.5%	5	2.8%	11.9 (3.7-39.0)	7.9 (1.7-37.5)
	Ibuprofen	50	28.2%	5	2.8%	23.5 (5.7-96.7)	10.0 (2.0-51.0)
	Aspirin	23	13.0%	2	1.1%	22.0 (3.0-163)	7.3 (0.9-59.4)
	Other NSAIDs	7	4.0%	2	1.1%	3.5 (0.7-16.8)	NC
	All NSAIDs in the 2 months	56	31.6%	4	2.3%	27.0 (6.6-110)	14.1 (2.8-71.2)
	to 7 years a age group
	All NSAIDs in the 8 to 16 years	17	9.6%	4	2.3%	7.5 (1.7-32.8)	3.4 (0.6-21.3)
	age group

Table 3

 3 Drugs used in the 4 weeks before bleeding, by age Fenamates 6, ketoprofen 2, flurbiprofen 1 and naproxen 1. One child took two types of nonsteroidal anti-inflammatory drugs (NSAIDs)

		Age groups
		2m to 2years N=58 (32.8%)	3to 7years N=60 (33.9%)	8to 16years N=59 (33.3%)	Total N=177 (%)
	Drugs
	-Any NSAIDs	17	40	26	83 (46.9)
	• Ibuprofen	12	31	15	58 (32.8)
	• Aspirin	5	11	10	26 (14.7)
	• Other NSAIDs a	1	4	4	9 (5.1)
	-Paracetamol	17	28	20	65 (36.7)
	-Antibiotics	15	24	12	51 (28.8)
	-Antacids and antiulcer	23	7	10	40 (22.6)
	-Other drugs	29	27	22	78 (44.1)

a

Eur J Clin Pharmacol (2010) 66:831-837

Acknowledgements The authors thank Richard Medeiros (Rouen University Hospital Medical Editor) for editing the manuscript.

Funds This study was supported by research grants from the French Agency of Medical Security of Health Products (AFSSAPS).

Conflict of interest statements None of the authors have financial or personal relationships, academic competition or intellectual commitments that might bias the work or interfere with objective judgment.

Access to data Lamiae Grimaldi-Bensouda, Elisabeth Autret-Leca and Benoit David had full access to all data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.