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23 Heine, P.A. et al. (2000) Increased adipose tissue in male and female estrogen receptor-α-knockout mice. Proc. Natl. Acad. Sci. U. S. A. 97, 12729–12734 24 Schmidt, A. et al. (1999) Femoral bone density and length in male and female estrogen receptor-α (ERα)-knockout mice. J. Bone Miner. Res. SU098, S456 25 Gentile, M.A. et al. (2001) Bone response to estrogen replacement in βERKO (estrogen receptor-β) null mice. J. Bone Miner. Res. SA331, S282 26 Lindberg, M. et al. (2001) Estrogen exerts both estrogen receptor α/β dependent and

independent effects. J. Bone Miner. Res. F501, S230 27 Sims, N.A. et al. (2001) Estrogen receptor α is the major receptor regulating bone response to estradiol in gonadectomized female mice and testosterone plays a role in intact male and female ERα-knockout mice. J. Bone Miner. Res. SU515, S431 28 Windahl, S.H. et al. (2001) In the absence of estrogen receptor α, estrogen promotes bone resorption via estrogen receptor β in female mice. J. Bone Miner. Res. M107, S551

29 Gentile, M.A. et al. (2001) Bone response to estrogen replacement in ovx double estrogen receptor-(α and β)-knockout mice. J. Bone Miner. Res. 1040, S146 30 Vandenput, L. et al. (2001) Testosterone prevents orchidectomy-induced bone loss in estrogen receptor-α-knockout mice. Biochem. Biophys. Res. Commun. 285, 70–76 31 Lindberg, M.K. et al. (2002) Two different pathways for the maintenance of trabecular bone in adult male mice. J. Bone Miner. Res. 17, 555–562

Recent insights into organogenesis of the adrenal cortex Catherine E. Keegan and Gary D. Hammer The primary endocrine organs responsible for steroid hormone biosynthesis – the adrenal cortex and gonads – are derived from the urogenital ridge. Several recent discoveries in human and mouse genetics have begun to unravel the complex genetic cascade that dictates adrenocortical cell lineage, proliferation and differentiation. The factors that regulate adrenocortical organogenesis and the maintenance of growth promote or block a cascade of transcription factors that differentially coordinate the proliferation and differentiation of the gland. Here, we outline the developmental milestones of the adrenal cortex with recent contributions to the field, focusing on factors that have been shown to play a role in vivo in humans and mice. Published online: 30 April 2002

Catherine E. Keegan Dept Pediatrics, Division of Genetics, University of Michigan Medical School, 5552 MSRB II, 1150 West Medical Center Drive, Ann Arbor, MI 48109-0678, USA. Gary D. Hammer* Dept Physiology and Internal Medicine, Division of Endocrinology and Metabolism, University of Michigan Medical School, 5560 MSRB II, 1150 West Medical Center Drive, Ann Arbor, MI 48109-0678, USA. *e-mail: ghammer@ umich.edu

The adrenal cortex is a crucial component of the hypothalamic–pituitary–adrenal axis, which coordinates the mammalian stress response. The early events of adrenal morphogenesis have only recently been explored in detail. Studies of the regulatory factors that dictate the proliferation and differentiation of adrenal steroidogenic cells have focused on: (1) human diseases of adrenocortical formation; and (2) cellular, molecular and genetic analyses in mice. Although there are undeniably species-specific differences in the structural and functional organization of the mammalian adrenal cortex, the basic principles of adrenocortical development that regulate its proper growth and differentiation share many common features. Here, we summarize the important events in adrenocortical development, from the initial condensation of coelomic epithelial cells into the adrenogonadal primordium to the growth and differentiation of the fully zonated adrenal cortex. Examples of mouse models and human patients with developmental abnormalities of the adrenal cortex, as manifested by congenital adrenal insufficiency, are used to illustrate the importance of known molecules in the adrenal developmental cascade. http://tem.trends.com

Adrenocortical function Adult zones

The adrenal cortex is functionally defined by the presence of three distinct cell layers categorized by the expression of specific steroidogenic enzymes and the ability to respond to specific peptide hormones [outer zona glomerulosa (ZG), central zona fasciculata (ZF) and inner zona reticularis (ZR)] (Table 1; Fig. 1). Developmental zones

There is also evidence for the presence of a stem cell layer between the ZG and ZF, defined by the absence of phenotypic markers of mature adrenocortical cells (i.e. the steroidogenic enzymes) [1]. The human and mouse adrenal possesses a transient developmental zone between the functional cortical zones and the adrenal medulla – the fetal or X zone, respectively [2,3]. Fetal and X-zone cells contain lipid droplets, peculiar mitochondrial complexes and smooth endoplasmic reticulum, characteristic of steroid-secreting cells [4]. The human fetal zone produces dehydroepiandrosterone sulfate (DHEAS) for placental conversion to estradiol, a hormone crucial for sustaining pregnancy [2]. The function of the mouse X zone, which develops postnatally, remains unclear. Adrenocortical disorders in humans Adrenal hyperplasia

Most congenital primary adrenal insufficiency in humans results from congenital adrenal hyperplasia, caused by defects in steroid hydroxylase enzymes, which catalyze various steps of the glucocorticoid synthesis pathway (Table 2). In general, individuals with these disorders have glucocorticoid deficiency, with compensatory adrenocorticotropic hormone (ACTH)-driven hyperplasia, indicating a primary defect in steroidogenesis. The most common form of

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a

Table 1. Functional zones of the adult adrenal cortex Zona glomerulosa

Zona fasciculata

Location Stimulus Primary membrane receptor Specific enzyme activity

Outer Angiotensin II (ACTH) Angiotensin II receptor CYP11B2

Central ACTH MC2R b CYP17 ; CYP11B1

Hormone product Function Deficiency

Mineralocorticoids (aldosterone) Regulation of intravascular volume Hyponatremia; hyperkalemia; hypotension

Zona reticularis

Middle ACTH MC2R b CYP17 (17OH and 17,20 lyase) Glucocorticoids (cortisol) Sex steroids (DHEAS) c c Glucose homeostasis; mobilization of energy stores Adrenarche ; well-being Hypoglycemia; lack of stress response Unknown

a

Abbreviations: ACTH, adrenocorticotropic hormone; CYP11B2, aldosterone synthase; CYP17, 17-hydroxylase (17OH); CYP11B1, 11β-hydroxylase; DHEAS, b dehydroepiandrosterone sulfate; MC2R, ACTH receptor. The production of both cortisol and androgens requires multiple functional activities and cofactors of the enzyme CYP17 (hydroxylase, lyase and cofactor oxidoreductase). The differential regulation of these enzymatic activities in the ZF and ZR is an area of active research [90]. Although the adult mouse adrenal gland does not produce cortisol and adrenal androgens, a single report documents expression of CYP17 in the developing mouse c adrenal gland [91]. Thus, corticosterone is the primary glucocorticoid in the mouse. Although the function of adrenal androgens remains controversial, growing evidence points to a role for the primary adrenal androgen, DHEAS, in adrenarche and brain function [92,93].

congenital adrenal hyperplasia is caused by 21-hydroxylase (encoded by CYP21) deficiency, with an incidence of 1 in 15 000 newborns [5]. Adrenal hypoplasia

Human patients have been described with adrenal insufficiency caused by a primary growth defect of the

Aldosterone p450aldo

p450c11B2 – 18βHSD Cholesterol StAR p450scc Pregnenolone

3βHSD

18OHCS p450c11B2 – 18OH p450c11B2

p450c21 Progesterone

DOCS

17OH p450c21 3βHSD 17OHPregnenolone 17OHProgesterone

Glomerulosa

CS

p450c17

17OH

17,20 lyase DHEA

17,20 lyase 3βHSD

Reticularis p450Aro

Androstenediol

3βHSD

Cortisol

Androstenedione 17βHSD

17βHSD

Fasciculata

p450c11B1 DOC

Estrone 17βHSD

Periphery

Testosterone p450Aro

Estradiol

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Fig. 1. Steroidogenesis in the human adult adrenal cortex. The biosynthetic pathways for mineralocorticoid, glucocorticoid and adrenal androgen production from the common precursor cholesterol are outlined for the three zones of the adrenal cortex (outer glomerulosa, middle fasciculata and inner reticularis). Each functional zone is phenotypically defined by a unique anatomical position in the layered gland, a distinct histological appearance and the production of a unique steroid hormone in response to specific peptide hormone stimulation. Zone- and cell-specific steroid production is accomplished by the restricted synthesis of (1) the specific peptide hormone receptors and (2) a specific set of steroidogenic enzymes unique to a given steroid hormone biosynthetic pathway. In addition to aldosterone and cortisol, the adrenal cortex synthesizes large amounts of the adrenal androgens – androstenedione and DHEA (uniquely sulfated to DHEAS in the adrenal) – which can both serve as substrates for the formation of testosterone and aromatization to estrogens in the periphery. Abbreviations: CS, corticosterone; DHEA, dehydroepiandrosterone; DOC deoxycortisol; DOCS deoxycorticosterone; 18OHCS, 18-hydroxylase corticosterone; p450scc, side-chain cleavage enzyme; p450c17, enzyme complex containing 17-hydroxylase (17OH) and 17,20 lyase activities; p450c21, 21-hydroxylase; p450Aro, aromatase; p450aldo, aldosterone synthase, the enzyme complex containing 11β-hydroxylase (p450c11B2), 18-hydroxylase (18OH) and 18β-hydroxysteroid dehydrogenase (18βHSD) activities; p450c11B1, 11β1-hydroxylase; 3βHSD, 3β-hydroxysteroid dehydrogenase; 17βHSD, 17β-hydroxysteroid dehydrogenase; StAR, steroidogenic acute regulatory protein.

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adrenal gland. The formation of the adrenal cortex in humans involves the migration and coalescence of two discrete developmental structures, the fetal zone, which regresses soon after birth, and the definitive zone, which later gives rise to the classic three steroidogenic zones of the cortex. Two major histological types of congenital adrenal hypoplasia have been described in relation to these two structures: the cytomegalic form and the miniature adult form [6]. The cytomegalic form typically presents in early infancy with adrenal insufficiency, skin hyperpigmentation and, ultimately, delayed puberty [6]. Males are most commonly affected, and several families exhibit an X-linked recessive pattern of inheritance. The cortical cells are enlarged and often vacuolated. The definitive cortex is small or absent, and the fetal zone persists [6]. This form of congenital adrenal hypoplasia is usually the result of mutations in the DAX1 gene (dosage-sensitive sex reversal–adrenal hypoplasia congenita critical region on the X chromosome) [7,8]. The miniature adult form of congenital adrenal hypoplasia is less common and is thought to be autosomal recessive because it has been reported in both male and female siblings [9,10]. Although it is often associated with defects of the central nervous system (CNS) and/or pituitary, such as anencephaly or panhypopituitarism (with subsequent corticotropin-releasing hormone and/or ACTH deficiency), the miniature adult form of congenital adrenal hypoplasia can lack CNS abnormalities [9,10]. Pathological studies show small adrenal size and the presence of all adrenocortical zones of the definitive cortex, although the fetal zone is absent. These patients present in early infancy and often die within the first few days of life. The genetic defect is unknown, and genetic heterogeneity is possible. Development of the urogenital ridge and associated structures

Adrenocortical cells derive from a condensation of coelomic epithelial cells (the urogenital ridge), which is also the origin of gonadal and kidney

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a

Table 2. Genetic disorders of adrenocortical growth and differentiation b

Mutant gene Rodent mutant or human disease Phenotype

Refs

Urogenital ridge Wt1 KO WT1 Human heterozygous deletion Frasier syndrome

[14] [94] [13]

Denys–Drash syndrome Wnt4 WNT4 acd

KO Duplication of chromosome 1p31–35 Spontaneous mutation

Foxd2

KO

Lethal: lacks kidneys, gonads and adrenals Wilms’ tumor, genital abnormalities and no known changes in adrenal phenotype XY sex reversal, renal failure, no Wilms’ tumor and no known changes in adrenal phenotype Urogenital abnormalities, renal failure, increased risk for Wilms’ tumor and no known changes in adrenal phenotype Abnormal kidney development, masculinization of XX females and adrenal defect Adrenal insufficiency and XY sex reversal Adrenocortical dysplasia, adrenal insufficiency, lack of X-zone development, hydronephrosis and hypogonadism Adrenal and kidney hypoplasia with reduced penetrance

[12] [18] [21] [84] [86]

Adrenogonadal primordia Sf1 KO SF1 Human heterozygous mutation Dax1 KO Transgenic overexpression DAX1 Human mutation

Lethal: lacks adrenals and gonads [35,36] Adrenal insufficiency, sex reversal in males and normal female development [37] Lack of X-zone regression and male infertility [43] XY sex reversal on certain genetic backgrounds [45] Cytomegalic congenital adrenal hypoplasia, persistence of fetal zone, adrenal [7,8] insufficiency, hyperpigmentation and hypogonadism Duplication of chromosome Xp21 XY sex reversal [44] Desrt KO Reduced thickness of ZR (X zone), reproductive abnormalities and transient immune [87] abnormalities Adrenal cortex Pomc/POMC Human mutation and mouse KO Atrophy of adrenals, obesity and hypopigmentation [66,67] MC2R Human mutation Adrenal insufficiency, hyperpigmentation and preservation of mineralocorticoid function [95,96] StAR Human mutation and mouse KO Congenital lipoid adrenal hyperplasia, adrenal insufficiency and XY sex reversal [5,97] CYP11A Human mutation Adrenal hyperplasia, adrenal insufficiency and XY sex reversal [98] HSD3B2 Human mutation Adrenal hyperplasia and adrenal insufficiency [5] CYP21 Human mutation Adrenal hyperplasia, adrenal insufficiency and masculinization of XX females [5] CYP17 Human mutation Adrenal hyperplasia, XY sex reversal or ambiguous genitalia and hypertension [5] CYP11B1 Human mutation Adrenal hyperplasia, glucocorticoid deficiency, hypertension and masculinization of XX [5] females AAAS Allgrove (AAA) syndrome Adrenal insufficiency, alacrima and achalasia [99,100] Unknown Miniature adult congenital adrenal Variably lethal, lack of fetal zone and small definitive zone [9] hypoplasia (human) Cited2 KO Complete adrenal aplasia, cardiac and brain defects [81] Ezg Spontaneous mutation Reduced thickness of zona glomerulosa [83] Ex Spontaneous mutation Premature X-zone regression [82] Casp2 KO Partial lack of X-zone regression [59] Smpd1 KO Complete lack of X-zone formation and presence of abnormal zone adjacent to medulla [101] a

Abbreviations: AAAS, AAA syndrome (alacrima-achalasia-adrenal insufficiency) gene; acd, adrenocortical dysplasia; Casp2, caspase 2; Cited2, CBP/p300-interacting transactivator with ED-rich tail 2; CYP11A, side-chain cleavage enzyme; CYP11B1, 11β-hydroxylase; CYP17, 17-hydroxylase; CYP21, 21-hydroxylase; Dax1/DAX1, dosage sensitive sex reversal–adrenal hypoplasia congenita critical region on the X chromosome 1; Desrt, developmentally and sexually retarded with transient immune abnormalities; Ezg, extent of zona glomerulosa; Ex, earlier X-zone degeneration; Foxd2, Forkhead box-D2; HSD3B2, 3β-hydroxysteroid dehydrogenase, type II; KO, knockout; MC2R, melanocortin 2 receptor (ACTH receptor); Pomc/POMC, proopiomelanocortin; Sf1/SF1, steroidogenic factor 1; Smpd1, sphingomyelin phosphodiesterase 1, acid lysosomal; StAR, steroidogenic acute regulatory protein; Wnt4/WNT4, wingless-related mouse mammary tumor virus integration site 4; b Wt1/WT1, Wilms’ tumor 1. Human genes are in capital letters; only the first letter of mouse gene is capitalized.

structures (Fig. 2) [2]. Several genes are crucial for urogenital ridge development, including the gene encoding Wilms’ tumor 1 (WT1), a complex gene that probably functions as a transcriptional regulator, and WNT4, a member of the WNT family of developmentally regulated signaling molecules. Wilms’ tumor 1

WT1 was first identified by positional cloning methods as a tumor suppressor gene, mutated in a significant number of cases of Wilms’ tumor [11]. WT1 is deleted in the WAGR microdeletion complex, which manifests phenotypically as Wilms’ tumor, aniridia, genital abnormalities and mental retardation [11]. The http://tem.trends.com

WT1 gene is mutated in the combined kidney and gonadal disorders – Denys–Drash syndrome and Frasier syndrome (Table 2) [12,13]. The importance of Wt1 in urogenital ridge development was further evaluated with gene targeting techniques in mice [14]. Homozygosity for a null allele of the mouse Wt1 gene results in midgestational death, with kidney and gonadal aplasia. Thus, WT-1 is crucial for the specification of both kidney and gonadal cell lineages. WT-1 also plays a role in adrenal development [15,16]. Use of a yeast artificial chromosome clone containing the Wt1 gene and a lacZ reporter gene in transgenic mice to rescue Wt1-knockout mice showed that, although mice continued to die of kidney failure, the

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Adult adrenal

Definitive zone Fetal adrenal Adrenogonadal primordia

Fetal zone

Neural crest cells Testis

SF1 Urogenital ridge DAX1 coelomic epithelium

AMH SF1 SOX9 SRY

LIM1

Primordial germ cells

WT1 WNT4

Bipotential gonad

PAX2 RET GDNF

DAX1 WNT4 Ovary

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Fig. 2. The urogenital ridge and adrenogonadal primordium. Urogenital ridge development results in formation of the kidneys, gonads and adrenal cortex. Specification of the metanephric kidney and adrenogonadal primordium occurs first. Following contact with primordial germ cells, the fetal adrenal (composed of fetal zone and definitive zone) and bipotential gonad then separate. The bipotential gonad differentiates into a testis or an ovary, depending on the genetic sex of the individual. The adrenal gland continues to develop as the neural crest cells that will ultimately become the adrenal medulla migrate to the center of the gland, and the process of cortical zonation is initiated. The important regulatory genes at each step in the pathway are highlighted in red. Abbreviations: AMH, anti-Müllerian hormone; DAX1, dosage-sensitive sex reversal–adrenal hypoplasia congenita critical region on the X chromosome 1; GDNF, glial-derived neurotropic factor; LIM1, LIM homeodomain transcription factor 1; PAX2, paired-box gene 2; RET, GDNF receptor; SF1, steroidogenic factor 1; SOX9, SRY-box gene 9; SRY, testis-determining factor encoded on the Y chromosome; WNT4, wingless-related mouse mammary tumor virus integration site 4; WT1, Wilms’ tumor 1.0

developing adrenal glands were smaller than in wildtype littermates and showed reduced expression of the gene encoding side-chain cleavage enzyme (CYP11A) in the adrenal primordium [15]. Furthermore, when the Wt1-knockout allele was bred on to a different genetic background, the homozygous mutant mice lacked adrenals in addition to kidneys and gonads [16]. These studies demonstrate that Wt1 is developmentally one of the earliest known genes that specifies kidney, gonadal and adrenal cell lineages. WNT4

The WNT (wingless-related mouse mammary tumor virus integration site) proteins are secreted glycoproteins that play an important role in the development of several embryonic structures [17]. They act via the frizzled receptor family to initiate a cascade of intracellular signals leading to β-catenin accumulation and subsequent transcriptional activation of downstream target genes. Wnt4 is expressed in developing tubules of the kidney, and Wnt4-knockout mice have aberrant kidney development and die shortly after birth from renal failure [18]. The kidneys in these mice showed lack of differentiation of the mesenchyme and absent glomeruli [18]. The fact that the Wt1 gene is expressed normally in Wnt4-mutant mice suggests that Wt1 expression precedes Wnt4 in this developmental cascade, or that these two molecules work in http://tem.trends.com

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parallel [19]. The Wnt4-knockout mice also have an uncharacterized adrenal defect, and XX females are masculinized, with an absence of the Müllerian duct and continued development of the Wolffian duct [20]. This masculinization is the result of ectopic activation of testosterone biosynthesis caused by expression of the gene encoding 17-hydroxylase (Cyp17) in the gonads of Wnt4-knockout females [20]. Interestingly, a patient has been described with a duplication of chromosome 1p31–35, who had male to female sex reversal and adrenal insufficiency [21]. This region was shown to contain the human WNT4 gene, suggesting that these anomalies resulted from altered gene dosage of WNT4. Another WNT gene, WNT11, is also expressed in the developing adrenal cortex, kidneys and gonads [22]. In the kidney, Wnt11 precedes Wnt4 expression and is localized at the tips of the ureteric buds [19]. However, mice lacking the Wnt11 gene have no kidney abnormalities [19]. The complete phenotype of these mice has yet to be published in detail, but will probably reveal additional clues to the importance of WNT molecules in urogenital ridge development. The adrenogonadal primordium

Several molecules are important for the specific development of the adrenogonadal primordium, giving rise to the adrenal cortex and gonads, but notably not to the kidney. In particular, two transcription factors, steroidogenic factor 1 (SF-1) and DAX-1, are essential for the development and differentiation of the adrenal cortex and gonads (Table 2). The presence of a true common embryonic precursor (adrenogonadal primordium) was demonstrated by immunohistochemical staining for SF-1, which revealed a single population of SF-1-positive cells located in the developing urogenital ridge from the coelomic epithelium to the dorsal aorta at embryonic day 11.5 (E11.5) of rat development [23]. These cells subsequently differentiate into two distinct populations of cells, which ultimately become the adrenal and gonadal primordia, respectively (Fig. 2). This occurs when the primordial germ cells migrate into contact with the adrenogonadal primordium [23]. The gonadal primordium goes on to differentiate into a testis or ovary, depending on the genetic sex of the organism, and the adrenal primordium differentiates into the adrenal cortex. Steroidogenic factor 1

SF-1 was initially identified as a transcription factor regulating expression of the various adrenal hydroxylase genes responsible for steroid production [24]. SF-1 is a member of the steroid receptor superfamily. It contains a classic zinc finger DNA-binding domain (DBD) in its N-terminal region, and a conserved ligand-binding domain (LBD) in the C-terminal region [25]. Because SF-1 has no known ligand, it is an orphan receptor. In addition to a conserved AF-2 (activation function 2) sequence in the distal C-terminus, SF-1 contains an extended hinge

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region (AF-1) between the DBD and the LBD that is uniquely phosphorylated by mitogen-activated protein kinase and facilitates cofactor recruitment [26]. Together, these two domains dictate recruitment of cofactors, including both coactivators (CREB-binding protein, WT1 and nuclear receptor coactivator 1) and corepressors (nuclear receptor corepressor, DEAD/H box polypeptide 20, COP9 constitutive photomorphogenic, subunit 2, nuclear receptor-interacting protein 1 and DAX-1) [25,27–31]. These interactions are presumed ultimately to regulate transcriptional activation. In addition to the adrenal cortex and gonads, Sf1 is also expressed in the gonadotropes of the anterior pituitary and in the ventromedial nucleus of the hypothalamus (VMH) [25]. Sf1 also exhibits sexually dimorphic expression in the gonads [32]. Although high levels of SF-1 persist in the embryonic testis throughout development, Sf1 transcripts are absent from the developing ovary between E13.5 and E16.5, and reappear only at E18.5. Thus, activation of the anti-Müllerian hormone (AMH) promoter by SF-1 in male embryos facilitates the male-specific developmental program. SF-1 in the anterior pituitary is important for specification of the gonadotrope lineage [33], and it regulates feeding and satiety in the VMH [34]. Sf1-null mice surprisingly have a complete absence of adrenal glands and gonads (in addition to loss of the VMH and impaired gene expression in gonadotropes), thus demonstrating the importance of SF-1 in both adrenal and gonadal development [35,36]. Heterozygous SF1 mutations have since been demonstrated in three human patients. The first had adrenal insufficiency, XY sex reversal and a G35E mutation in the first zinc finger of the DBD, which was shown to abrogate binding of SF-1 to target promoters [37]. The second was a phenotypically normal, although prepubertal, XX female with adrenal insufficiency, who had a mutation in the hinge region corresponding to the AF-1 domain of SF-1 [38]. A third SF-1 mutation causes XY sex reversal but has no effect on adrenal function [39], suggesting possible genotypic–phenotypic correlations. A fourth patient homozygous for a R92Q mutation has recently been described with adrenal insufficiency and XY sex reversal [40]. The parents and an unaffected sibling were heterozygous for the mutation, indicating that this mutation acts as a true recessive allele and demonstrating the importance of gene dosage in SF-1 function. Further molecular studies of these mutations will help to clarify the differential role of gene dosage of SF-1 in adrenal and gonadal development. DAX-1

DAX-1 is also a member of the orphan nuclear receptor family that has been postulated either to bind to hairpin loops of the steroidogenic acute regulatory protein (StAR) promoter and/or to function as an RNA-binding protein [41,42]. DAX1 was initially cloned as the gene responsible for human X-linked cytomegalic congenital adrenal hypoplasia, marked by http://tem.trends.com

an absent definitive zone and a persistent fetal adrenal zone [7,8]. Dax1-knockout mice do not exhibit adrenal insufficiency, but the X zone does not regress and males are infertile [43]. In humans, the discovery that the region of the X chromosome containing the DAX1 gene is duplicated in the dosage-sensitive sex reversal syndrome, causing XY sex reversal, led to the hypothesis that DAX-1 is important for normal female development [44]. Mice containing extra copies of the Dax1 gene (as a transgene under the control of its own promoter) showed XY sex reversal on genetic backgrounds with a weak SRY (the testis-determining factor encoded on the Y chromosome) allele [45]. However, this transgene was not expressed in the adrenal gland; thus, the phenotype of Dax1 overexpression in the adrenal cortex has yet to be determined. By contrast, female Dax1-knockout mice have normal gonadal development and are fertile [43], indicating that DAX-1, like SF-1, has sexually dimorphic roles in male and female gonadal development. Interestingly, a phenotypically normal XX female patient with a mutation in the DAX1 gene recently presented with isolated hypogonadotropic hypogonadism but, surprisingly, without a primary adrenal defect, indicating the possibility of an additional sexually dimorphic role of DAX-1 in adrenal function [46]. In addition, recent studies suggest that DAX1 is expressed at higher levels in females, a phenomenon thought to be mediated by androgens, which can downregulate DAX1 transcription [47]. Furthermore, DAX-1 has been shown to interact with both androgen and estrogen receptors [48,49]. This might explain some of the male–female differences in terms of X-zone regression and the phenotype of female Dax1-knockout mice. Because SF-1 and DAX-1 are colocalized in the urogenital ridge and adrenal primordia by E9.5 and subsequently in the developing adrenal cortex, gonads, anterior pituitary and VMH in the mouse [50], it is possible that these two molecules cooperatively activate the adrenal-specific developmental program. This is supported by the similar phenotype of adrenal hypoplasia in humans with mutations in either gene. Indeed, SF-1 can bind to the Dax1 promoter and compete with the chicken ovalbumin upstream promoter-transcription factor and ultimately activate transcription of the Dax1 gene [51]. However, there is accumulating evidence that DAX-1 inhibits SF-1-mediated transcription in the adrenal gland in cultured cell lines and in adult mice, probably by recruiting corepressors to the transcriptional complex [29,41,52–54]. Indeed, the absence of DAX-1 (Dax1-null allele) partially rescues the adrenal insufficiency in Sf1-haploinsufficient mice [54]. Interestingly, Dax1 expression is maintained in Sf1-knockout animals [50], and Sf1 expression is maintained in the adrenals of adult Dax1-knockout males [54]. As yet, it is not clear how the differential interplay between SF-1 and DAX-1 is regulated in adrenal organogenesis and in adult adrenocortical function in mice.

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Adrenal formation

(a)

(b)

Migration of coelomic epithelial cells

Urogenital ridge 5–7w

Formation of fetal zone

E9

Earliest expression of Sf-1 and Dax-1

7w

Formation of definitive zone

Adrenal primordia E11

8w

9w

Migration of mesenchymal capsular cells

Earliest expression of steroidogenic enzymes Migration of neural crest cells

E12–14

Migration of neural crest cells

Zonation P1–7

Regression of fetal zone

Formation of X zone

P1–6w Encapsulation of medulla

P14–21 P12–18m

Zonation

P35

Regression of X zone and encapsulation of medulla

10–20y TRENDS in Endocrinology & Metabolism

Fig. 3. The adrenocortical developmental cascade. (a) Human adrenocortical development showing progression from the urogenital ridge to final zonation. Two separate populations of cells migrate from the coelomic epithelium to form the fetal and definitive zones, which condense to form the adrenal primordia. The adrenal primordium is then encapsulated by mesenchymal cells, and neural crest cells migrate centrally to form the adrenal medulla. The fetal zone undergoes apoptosis after birth, followed by encapsulation of the medulla and final zonation of the adult cortex. The stage of development is shown under each structure. (b) Mouse adrenocortical development showing progression from the urogenital ridge to X-zone regression. Similar to humans, the adrenal primordium forms from a condensation of coelomic epithelial cells, followed by migration of mesenchymal capsular cells and migration of neural crest cells. Markers of early adrenal development include the nuclear receptors, Sf-1 and Dax-1, followed by steroidogenic enzymes. In contrast to humans, zonation of the adrenal cortex is nearly completed by birth, and the X zone (fetal zone) does not form until P14–21 and regresses by P35 in males. Abbreviations: Dax1, dosagesensitive sex reversal–adrenal hypoplasia congenita critical region on the X chromosome 1; E, embryonic day; m, month; P, postnatal day; Sf1, steroidogenic factor 1; w, week; y, year.

Molecular interactions

The details of how the urogenital ridge specifies formation of the adrenogonadal primordium have remained elusive. However, recent studies indicate that molecules that specify global urogenital ridge fate (WT-1 and WNT-4) interact directly with molecules specifying adrenal and gonadal development, such as SF-1 and DAX-1. Previous studies have shown that WT-1 and SF-1 interact physically to activate synergistically the AMH promoter [55]. DAX-1 represses this synergistic activation, thereby inhibiting the male-specific pathway. In addition, there is growing evidence that SF-1 and DAX-1 are intimately involved in the WNT signaling pathway. β-Catenin, a downstream effector of WNT signaling, activates SF-1 transcriptional programs in the adrenal gland [56]. Furthermore, WNT-4 upregulates DAX-1 in testicular cells, thereby antagonizing SRY [21]. This is the hypothesized mechanism by which sex reversal occurs with altered gene dosage of WNT4. Future studies will clarify the role of these crucially important molecules in urogenital ridge and adrenogonadal development. http://tem.trends.com

Following the migration of primordial germ cells and the separation of the adrenogonadal primordium, the human adrenal cortex becomes encapsulated at nine weeks, followed by migration of neural crest cells to form the adrenal medulla (Fig. 3a) [2]. Both the fetal and definitive zones are composed of cells with ultrastructural characteristics of steroid-producing cells. During the second trimester, the fetal zone enlarges and secretes DHEAS [2]. After birth, significant remodeling of the adrenal cortex occurs, including apoptosis of the fetal zone by the third postnatal month and zonation of the definitive cortex by puberty (Fig. 3a) [2]. Adrenal development in the mouse differs somewhat from that of humans and primates, although the basic developmental patterning is probably programmed by the same genes. The mouse adrenal gland develops from the urogenital ridge, and neural crest cells migrate centrally during development followed by encapsulation (Fig. 3b). However, the X zone in mice becomes evident histologically at 10–14 days of age and enlarges until three weeks of age. It subsequently begins to degenerate in males, coinciding with sexual maturity, and is gone completely by 38 days of age (Fig. 3b) [57]. In females, the X zone undergoes apoptosis during the first pregnancy [58]. Only after degeneration of the X zone does the medulla become separated from the cortical cells by a fibrous connective tissue band [57]. Although the timing of development of the fetal zone in humans and the X zone in mice differs, several lines of evidence suggest that these two zones are analogous structures. They are in the same position – adjacent to the adrenal medulla – and they both have ultrastructural features of steroid-producing cells. Furthermore, loss of function of the DAX1 gene in humans and mice results in lack of regression of the fetal and X zones, respectively [7,8,43]. Although several studies have attempted to delineate the origin and function of the adrenocortical X zone [3,4], its function remains elusive. Mice with a targeted mutation of the gene encoding caspase 2 recapitulate the histological phenotype of Dax-1-knockout mice [59], suggesting a role for proapoptotic molecules in X-zone regression. These and other existing models of aberrant X-zone formation and/or regression should assist in understanding the function of this intriguing developmental zone (Table 2). Adrenocortical differentiation

The three classic differentiated adrenocortical cell types are believed to derive from the definitive zone and are defined by their ability to synthesize and secrete specific steroid hormones in response to specific peptide hormone stimulation. However, the process by which lineage determination of adrenal cortical cells occurs is still a mystery. The migration theory is based upon two key observations: (1) tritiated thymidine studies reveal centripetal

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migration of adrenocortical cells from outer to inner layers [60]; and (2) enucleation of the adrenal cortex results in centripetal regrowth and zonation from the remaining adrenocortical capsule and underlying cells [61]. These findings suggest a pluripotent stem cell zone able to differentiate into three phenotypically distinct steroid-secreting cells. Immunohistochemical studies in rats with zonal specific markers aldosterone synthase (CYP11B2, ZG) and 11β1-hydroxylase (CYP11B1, ZF) reveal an undifferentiated zone between the ZG and ZF that expresses no differentiated markers [62,63]. This zone surprisingly expresses genes encoding the preadipocyte factor 1 and the proliferation marker, proliferating cell nuclear antigen, both of which are upregulated following enucleation [62]. It is plausible that cells migrate both outward from this zone to populate the ZG and inward to populate the ZF and ZR. Confirmation of this theory will require detailed lineage-determination studies. Expression of a Cyp21 transgene was found in linear stripes along the adrenal cortex, suggesting a common lineage for all three zones, and supporting centripetal migration of adrenocortical stem cells [64].

bolstered by the recent discovery of adrenal secretory protease (ASP), a serine protease that is synthesized specifically in the outer adrenal cortex (i.e. stem cell zone), is upregulated following unilateral adrenalectomy and is capable of cleaving POMC to the active N-terminal fragment (1–52, adrenoproliferin) [72]. Examination of the synthesis and regulation of ASP and adrenoproliferin during adrenal development should show whether their tropic actions are crucial to early growth of the adrenal cortex. Because innervation of the capsule and gland is required for compensatory adrenal growth following unilateral adrenalectomy [73], communication between the capsule and stem cell zone could be important for adrenocortical growth and differentiation. There are several single gene defects that result in adrenal hyperplasia, cytomegaly and/or cancer, including those encoding inhibin, IGF-II, p57Kip2, p53, protein kinase A regulatory subunit, and G-protein-stimulatory subunit Gs [74–80]. Given that several molecules that are dysregulated in neoplastic processes have important roles in development, they might also have a role in early adrenocortical growth and/or differentiation.

Adrenocortical growth and/or growth maintenance

The major tropic factor regulating the growth of the adrenal cortex is ACTH. ACTH is a cleavage product of the precursor peptide proopiomelanocortin (POMC). The adrenal glands of mice that carry a deletion of the Pomc gene are significantly reduced and show altered cellular composition at pre- and early postnatal stages [65]. These mice also develop obesity, as do human patients with mutations in POMC, because of a lack of α-melanocytestimulating hormone in the arcuate nucleus [66,67]. In adult mice with a deletion of the Pomc gene, the adrenal glands have completely atrophied [67], demonstrating that ACTH is important for continued postnatal growth maintenance of the adrenal cortex. In addition, several growth factors modulate the stimulatory effect of ACTH, including basic fibroblast growth factor, transforming growth factorβ, and insulin-like growth factors I and II (IGF-I and IGF-II) [2]. Further evaluation of the phenotype of mice with defects in these various peptides will help to answer important questions regarding the interface between membrane-initiated signaling and transcriptional mechanisms that regulate adrenal growth maintenance. The compensatory adrenal growth model, in particular, has been used by several groups to examine factors that are important for growth maintenance of the adrenal cortex [68–70]. In this model, following unilateral adrenalectomy, the remaining adrenal gland grows to compensate for the presumed reduction in steroid output. Several early studies have suggested that an N-terminal fragment of POMC (1–52), unique from ACTH, is a specific adrenal mitogen [70,71]. This hypothesis has been http://tem.trends.com

Prospects

The complex details of the cascade leading to the development and differentiation of the adrenal cortex are beginning to fit together. A recently described mouse knockout of the Cited2 gene, a coactivator of the transcription factor AP-2, has the interesting phenotype of complete adrenal aplasia in addition to cardiac and brain defects [81]. Furthermore, there are three uncharacterized spontaneous mouse mutations, Ex, Ezg and acd, which have isolated defects in adrenocortical development (Table 2) [82–84]. Although Ex and Ezg have no effect on the lifespan of the mouse, the adrenocortical dysplasia (acd) mutation is associated with reduced survival. This mouse carries an autosomal recessive mutation that arose spontaneously on the inbred DW/J strain, and has defects in all three of the organ systems derived from the urogenital ridge [84]. acd-mutant mice are born with adrenal dysplasia and hypofunction (primary adrenal insufficiency with elevated ACTH and low corticosterone), hypogonadism and focal hypertrophy of the ureteral epithelium, leading to hydronephrosis. Interestingly, the adrenocortical X zone fails to develop in acd mice, but medullary cells are unaffected. The lack of X-zone development and abnormal adrenal cortex in these mice is reminiscent of human miniature adult congenital adrenal hypoplasia. Although the gene responsible for the acd phenotype has not yet been identified, it is probably a crucial component of the adrenal developmental cascade. Several complex questions remain unanswered. For example, what are the early genes and/or

Review

Acknowledgements The authors gratefully acknowledge Felix Beuschlein for critically reading this article.

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signals required for condensation of the urogenital ridge and initiation of steroidogenic program before SF-1? Although WT-1 and WNT-4 are clearly important developmental regulators for the urogenital ridge, what other factors are crucial for the derivation of urogenital ridge structures? How does the separation of the adrenogonadal primordium occur following contact with the primordial germ cells, and what molecular signals are required for this process to occur? What genes are necessary for X-zone formation, and what is the function of the X zone? What factors are downstream of ACTH and N-terminal POMC in the

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