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Molecular Genetics and Metabolism 80 (2003) 81120 www.elsevier.

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DAX1 and its network partners: exploring complexity in development


Robert Clipshama and Edward R.B. McCabea,b,*
b a UCLA Molecular Biology Institute, Los Angeles, CA, USA Departments of Pediatrics and Human Genetics, 22-412 MDCC, David Geen School of Medicine at UCLA, Mattel Childrens Hospital at UCLA, UCLA Center for Society, the Individual and Genetics, 10833 Le Conte Avenue, Los Angeles, CA 90095-1752, USA

Received 8 August 2003; accepted 12 August 2003

Abstract DAX1 encoded by NR0B1, when mutated, is responsible for X-linked adrenal hypoplasia congenita (AHC). AHC is due to failure of the adrenal cortex to develop normally and is fatal if untreated. When duplicated, this gene is associated with an XY sexreversed phenotype. DAX1 expression is present during development of the steroidogenic hypothalamicpituitaryadrenalgonadal (HPAG) axis and persists into adult life. Despite recognition of the crucial role for DAX1, its function remains largely undened. The phenotypes of patients and animal models are complex and not always in agreement. Investigations using cell lines have proved dicult to interpret, possibly reecting cell line choices and their limited characterization. We will review the eorts of our group and others to identify appropriate cell lines for optimizing ex vivo analysis of NR0B1 function throughout development. We will examine the role of DAX1 and its network partners in development of the hypothalamicpituitaryadrenal/gonadal axis (HPAG) using a variety of dierent types of investigations, including those in model organisms. This network analysis will help us to understand normal and abnormal development of the HPAG. In addition, these studies permit identication of candidate genes for human inborn errors of HPAG development. 2003 Elsevier Inc. All rights reserved.
Keywords: Adrenal; Adrenal cortex; Development, steroidogenic axis; Hypothalamicpituitaryadrenal/gonadal axis; DAX1, adrenal hypoplasia congenita; Nuclear receptor, NR0B1; Steroidogenic factor 1

Introduction The DAX1 gene, recently designated NR0B1 under the uniform nomenclature system for nuclear receptors (http://www.receptors.org/NR/), was cloned using a positional approach and mutations in this gene cause Adrenal Hypoplasia Congenita (AHC) (OMIM # 240200 and 300200) [1]. NR0B1 is situated in the Xp21.3 region of the X chromosome and was located by CpG island identication [2,3] or identication of genomic sequences expressed in adrenal cortex [4] within a critical region. NR0B1 maps telomeric to the glycerol kinase (GKD) and Duchenne muscular dystrophy (DMD) loci, which collectively belong to a contiguous gene syndrome (CGS) region on the X chromosome [2]. Patients affected by this CGS can present with mixed phenotypes,

* Corresponding author. Fax: 1-310-206-4584. E-mail address: emccabe@mednet.ucla.edu (E.R.B. McCabe).

including AHC, depending on the unique rearrangements. When the 160 kb critical region containing NR0B1 is duplicated with an intact SRY gene present, an XY sex-reversed female phenotype results [5]. Therefore, this critical region was designated as the dosage sensitive sex-reversal (DSS) region (OMIM 300018) [5]. Subsequently, the original DAX1 nomenclature was derived as an acronym for DSS AHC Xlinked. Due to the X-linked inheritance pattern, males are nearly exclusively aected, as reports have included only one female homozygous for NR0B1 mutation and one other heterozygous female [6]. Age of onset is bimodal, with predominance either before two months or between 2 and 9 years of age [7] and occasionally later [8], and median age at presentation is three months [9]. The incidence of AHC is reported at 1:12,500 births [10], but this may be an overestimation [9]. Aected patients exhibit symptoms of adrenal cortical insuciency, including weakness, nausea, vomiting, diarrhea, and hypotension [8,10]. Hypoglycemia,

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hyponatremia and hyperkalemia result from lack of glucocorticoid and mineralocorticoid synthesis. The condition is generally lethal without replacement therapy. The clinical signs of adrenal insuciency are the end result of failure by the adrenal gland to develop a permanent, adult cortex with dened functional zones [8,11]. Normal adult adrenal glands produce mineralocorticoids (e.g., aldosterone) from the outer zona glomerulosa (ZG), glucocorticoids (e.g., cortisone) from the medial zona fasiculata (ZF) and cortisol plus androgens (testosterone) from the innermost zona fasiculata (ZF) [12,13]. The structurally disorganized fetal zone (FZ) in X-linked AHC is retained, though it is smaller than normal, and lacks sucient steroidogenic capacity independent from maternal and placental contributions [14]. The undeveloped cortical denitive zone (DZ) persists as a very thin layer peripheral to the FZ and is unresponsive to adrenocorticotropic hormone (ACTH) stimulation. Skin hyperpigmentation may be present due to excessive plasma ACTH levels and associated propiomelanocortin (POMC) cleavage products, including melanocyte stimulating hormone (MSH), due to lack of negative feedback from adrenal cortical steroid production. The POMC precursor molecule is produced by the pituitary [15,16]. The nonresponsive adrenal cortex also fails to undergo adrenarche due to the lack of DZ development. Adrenarche is dened as the increased production of C19 corticosteroids, especially dihydroepiandrosterone (DHEA and DHEA-sulfate), which results in androgen production by the ZR, just prior to onset of puberty [17,18]. This event is a recent evolutionary event, being restricted to humans and chimps [19], and may require a drop in ZR 3b-hydroxysteroid dehydrogenase (3bHSD) levels for initiation [2022]. Additionally, a pituitary connection for ZR function in adrenarche has been suggested for patients defective for PROP1, a gene expressed in both the pituitary and adrenal cortex [23]. Patients with PROP1 mutations may develop adrenal insuciency (see below). Hypogonadotropic hypogonadism (HH) with absent or delayed puberty is seen in those aected with DAX1 mutations [9] and is due to impaired or absent production of gonadotropin releasing hormone (GnRH) from the hypothalamus and/or gonadotropins (FSH, LH) from the anterior pituitary [7,8,24]. Gonadal development can be aected with cryptorchidism, low testosterone levels and/or defective spermatogenesis [8,2527]. Abnormalities of the urogenital system may also include hypospadius, micropenis, ureteral reux, and urethral stenosis [1]. Variable success in supporting pubertal development is reported with testosterone, human chorionic gonadotropin (HCG, an LH mimetic), DHEA or pulsatile GnRH therapy [25,2832]. Normal minipuberty of infancy has been documented among infant males subsequently diagnosed with HH [8]. The female

patient homozygous for the NR0B1 mutation due to gene conversion was reported to have isolated HH without adrenal insuciency [6]. NR0B1 is expressed throughout the hypothalamic pituitaryadrenal/gonadal (HPAG) axis, including the ventromedial nucleus (VMN) of the hypothalamus, anterior pituitary, adrenal cortex, testicular Leydig and Sertoli cells, and the ovary [9,33,34]. Expression is also demonstrated in prostate cells [35] and skin structures [36]. NR0B1 initially appears in humans at 33 dpc in the urogenital ridge and its successor, the adrenal cortical anlage [37,38]. Expression patterns in the mouse parallel those of humans [33,37,3941], with additional expression reported in embryonic stem (ES) cells and preimplantation embryoblasts [4244]. Reports of subcellular localization(s) of NR0B1 transcripts and DAX1 protein to the cytoplasm and/or nucleus are variable [2,34,36,45]. The phenotypic spectrum in patients with NR0B1 mutations can have signicant variability [8,26,27,30,32,4650] and the correlation between genotype and phenotype is not well dened [45,5154]. Not all patients who present with classic AHC have identiable mutations in the NR0B1 promoter and/or coding sequence [8,5558].

NR0B1 gene structure The NR0B1/Nr0b1 gene is composed of two exons, with the majority of the coding sequence contained in exon 1 [1,8,59,60]. Structurally, this gene encodes an atypical member of the nuclear receptor superfamily. Sequence analysis reveals a unique amino (N) region composed of 3.5 repeat motifs, which may constitute a novel DNA binding domain (DBD) or a protein sequence with an as yet unknown function. The carboxy (C) terminal region is simlar to other nuclear receptor superfamily members. Nuclear receptor protein structures are classically divided into regions AF, according to the functional domains contained within each region [1]. A/B is the 30 most region and can have transactivational (TA) domains. Region C is generally the most highly conserved and typically incorporates a DBD with two zinc ngers and conserved cysteine placement, also known as Region I [61]. Region D is a hinge domain that connects the DBD with the ligand binding domain (LBD). This region is poorly characterized and may also contain TA and transcriptional silencing domains [6265]. Region E consists of the LBD and is typically the second most conserved region among family members. Dimerization and TA domains may also be present in Region E. Two functional regions within E are more highly conserved and are designated Regions II and III [61]. The most distal amino acids of region E are identied as a functional AF-2 TA domain.

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NR0B1/Nr0b1 lacks regions AD. The putative DBD and canonical zinc ngers motifs are instead replaced by the 3.5 repeat sequence, consisting of 6567 amino acids each, with cysteines positioned to form possible novel zinc ngers [1,4,8,66]. Recently, functional studies showed a nuclear localization signal (NLS) within the N terminal 3.5 repeats that required the presence of a bipartite C terminal domain to prevent cytoplasmic retention of the translated protein [45,53]. The crystal structure of the gene product has not been reported to date and all structurally dependent functions have been extrapolated from other family members, such as the thyroid hormone and retinoid-X receptors [45,52]. No ligand has been conrmed to date, classifying DAX1 as an orphan receptor.

phenotypes, as dened by anatomical, molecular, and/or biochemical parameters [7]. Miniature adult form of AHC A miniature adult form of AHC (OMIM 240200) is sporadic or autosomal recessive and has FZ:DZ volume ratio closer to normal values than the cytomegalic form. Although the total gland mass is signicantly less than normal [10,68], all three zones are present. This form is also termed anencephalic or secondary, and can be the result of an absent or defective hypothalamus and/or pituitary gland [1,8,68]. Steroidogenic factor 1 mutations SF1, recently reclassied as NR5A1, is located on chromosome 9q33. Mutations are reported for both heterozygotic females with AHC and XY sex-reversal in recessive [71] and dominant [72] inheritance modes (OMIM 184757). Patient phenotypes have some similarities and dierences compared with those with NR0B1 mutations (Table 1). Intragenic mutation variants are reported to aect the clinical phenotype [27,7274]. Isolated AHC was also reported in an XX,46 female [73]. NR5A1 is generally coexpressed with NR0B1 in the HPAG [9,33,34,75] and in the skin [36]. WNT4 duplication WNT4 duplications are reported to result in XY sexreversal [76,77] or ambiguous genitalia and are accompanied by AHC in one case report [78]. Coding sequence mutations and deletions of WNT4 have not been reported for AHC to date. IMAGe This recently described syndrome was reported in three male infants with intrauterine growth retardation, metaphyseal dysplasia, aHC and genital abnormalities without NR0B1 mutations [56]. Clinical signs of AHC adrenocortical insuciency, without evidence of GKD or DMD, were present.

Forms of adrenal insuciency Here we will consider various forms of AHC and adrenal insuciency. We have proposed previously that gene products involved in pathogenesis of a phenotype are members of a single network or related networks [67]. Therefore, exploring these phenotypes may help us to understand more fully the networks involved in normal HPAG development. AHC due to NR0B1 mutations AHC due to NR0B1 mutations is dened as the Xlinked cytomegalic form (OMIM 300200) with large often vacuolated FZ cells with characteristic eosinophilic staining [68]. Cytomegalic AHC with autosomal recessive inheritance is seen in both sexes (OMIM 202155), but is not associated with CNS defects known to cause adrenal insuciency, including anencephaly or pituitary abnormalities [8,69,70]. Other forms of AHC and human adrenal insuciency Numerous other forms of congenital adrenal hypoplasia not associated with DAX1 mutations are documented to result in adrenocortical insucient
Table 1 Mouse and human DAX1 and SF1 mutation phenotype comparison Phenotypic trait Adrenal cortex Hypothalamus Pituitary Sex-dierentiation M llerian structures u Testis Sperm Puberty NR0B1 mutation FZ retained, zones GnRH impaired FSH/LH impaired Normal Absent HH possible impaired impaired (males) Nr0b1Y = Mouse

NR5A1 mutation FZ retained, zones GnRH impaired FSH/LH impaired XY sex-reversed Present Dysmorphic Absent Absent

Nr5a1= mouse Agenesis, Star and # Nr0b1 expressed in HPAG GnRH impaired VMH absent FSH/LH absent XY sex-reversed Present Absent Absent Absent

XZ retention in male # Sf1 Normal Normal Normal Absent Hypogonadal, " Cyp19 and estradiol Defective N/A

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Adrenoleukodystrophy (ALD) X-ALD is an X-linked disease that maps to Xq28, involves mutations in a peroxisomal membrane protein with similarity to ATP-binding cassette (ABC) transporters, and aects hemizygous males and some heterozygous females at variable ages of onset (OMIM 300100) [7,79]. Clinical manifestations are highly variable with seven separate neurological phenotypes, depending on specic neuropathy. AHC is not always present [80,81]. Neonatal ALD (NALD) is an autosomal recessive disorder (OMIM 202370) associated with deciency of multiple peroxisomal b-oxidation enzymes [82]. Clinical features are in the Zellweger spectrum, but milder cortisol response to ACTH stimulation may be impaired in NALD, but clinical adrenal insuciency is infrequent [83,84] (OMIM 202370, 300100, and 214100). ACTH deciency ACTH deciency can be due to defective enzymatic cleavage of POMC precursor. This form of AHC has been documented with mutations in the coding sequence in two out of many aected patients [85]. Most have isolated familial ACTH deciency [8,86]. ACTH acts primarily on the ZF and ZR for glucocorticoid production, with transient eects in the ZG producing aldosterone. Angiotensin II and serum potassium concentrations have greater regulatory impact on the ZG for aldosterone secretion [87]. Angiotensin receptors are expressed predominantly in the ZG and medulla and are functional late in gestation in the rat [88]. Melelanocortin-2 receptor mutations The MCR-2 or ACTH-R gene is located on chromosome 18p11.2 and mutations are inherited in an autosomal recessive manner [89,90]. Mutation of this receptor expressed in adrenal cortex results in familial glucocorticoid deciency (FGD) (OMIM 202200, 607397). However, 40% of aected individuals do not have MCR-2 mutations (FGD type I) and this phenotype is genetically heterogeneous [9193]. FGD type I patient presentation includes AHC, cortical disorganization and an absence of ZF and ZR. Aldosterone levels are normal to moderately decreased. POMC mutations POMC mutations present as severe adrenal insuciency with hypocortisolism, lack of ZF and ZR with normal ZG, absence of ACTH and a-MSH, marked obesity from the rst year of life and failed adrenarche [85]. All patients are hyperphagic, but gain weight even on calorie-restricted diets, have elevated leptin levels, are prone to sepsis and succumb without replacement hormone therapy. Patients also have orange-red hair and pale skin due to the concomitant lack of melanocyte stimulation in the dermis. The N-terminal fragment (amino acids 152) of the POMC gene product

(N-POMC) acts as an adrenal-specic mitogen, whose receptor (MCR-2) exists at the ZG/ZF boundary, the proliferative TZ. Adrenal serine protease (AsP or Aprotinin) cleaves this product into the active N-POMC peptide form in the adrenal cortex. TPIT mutations This transcription factor is involved in activation of the POMC gene via interaction with PITX1, another regulatory factor [94,95]. PITX1 is a frequent regulator of pituitary-specic expression, including aGSU [94]. Two patients with adrenal insuciency non-responsive to ACTH stimulation were diagnosed with separate mutations [95,96]. The aected individuals had isolated forms of pituitary-dependent adrenal insuciency. Familial hypoadrenocorticism Aected patients share AHC clinical signs (OMIM 240200) with the miniature adult form, but lack hypoaldosteronism [8]. There is no known genetic etiology currently identied. Triple A syndrome Also known as Allgroves or AAA syndrome, this disease is dened by a triad of signs, including ACTH resistance, Achalasia and Alacrima [89,92]. It usually presents during the rst decade with variable onset and severity. Approximately 15% of aected individuals will have mineralocorticoid deciency. Multiple neuropathies, including mental retardation (MR), often accompany this triad, as well as palmar/plantar hyperkeratosis. The gene (aladin or AAAS) responsible maps to chromosome 12q13 and the disorder is inherited in an autosomal recessive fashion [93]. No MCR-2 mutations have been found to be associated with this condition. Holoprosencephaly I, alobar type This condition has been reported in two brothers with AHC, micropenis, and pituitary gland agenesis (OMIM 236100). This may be an additional form of miniature adult AHC [8]. LH deciency Absence of pituitary leutinizing hormone due to defective gonadotropic cells has been reported with AHC and probable autosomal inheritance that excludes Xp21.3 [8,97]. Congenital adrenal hyperplasia Congenital adrenal hyperplasia (CAH) is a group of recessively inherited diseases caused by congenital defects in enzymatic synthesis of steroid hormones [87] from cholesterol since the adrenal cortex is functionally unresponsive to ACTH stimulation because of lack of production of steroidogenic end-products, the feedback loop is interrupted, and ACTH production is increased,

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resulting in adrenocortical hyperplasia. The enzymatic pathways are extensively reviewed [12,70,87,89,98] and individual genetic defects produce decient hormones or excessive steroids, often in a tissue, cell type or developmentally temporal manner. Specic adrenal-related forms are described as follows: Steroidogenic acute regulator (StAR) Mutations of the StAR gene, located on chromosome 8p11.2, result in the lipoid form of CAH [99,100]. This gene facilitates the transport of cholesterol from the outer to inner mitochondrial membranes [101,102] for conversion to pregnenolone and has a primary regulatory role in steroid synthesis. Homozygous mutations lead to severe impairment of steroidogenesis and XY sex-reversal (OMIM 600617). These patients survive due to basal levels of StAR activity [103]. P450 side chain cleavage (P450scc or CYP11A1) gene mutations on chromosome 15q23q24 were previously considered the cause of lipoid CAH. Homozygosity for CYP11A1 P450scc mutations is predicted to be embryonic lethal

[87]. Cases previously suspected to have CYP11A1 mutations currently are generally considered to be caused by StAR [87]. P450 enzymes. Cytochrome oxidase P450 enzymes are present in all layers of the adult cortex and steroidproducing gonadal cells (Fig. 1). The fate of pregnenolone metabolism is determined by the expression patterns of P450 17a hydroxylase/17,20 lyase (P450c17) and 3b-hydroxysteroid dehydrogenase/D isomerase (3bHSD). The P450 genes are highly conserved among species, but some signicant dierences in expression patterns exist [98]. If both are present, glucocorticoids are produced [12] while 3bHSD expression alone directs steroidogenesis toward mineralocorticoids. P450c17 alone limits steroidogenesis to DHEA synthesis, which permits androgenesis, primarily occurring in the ZR [87]. The following steroidogenic enzymes in these pathways are known to aect humans. P450scc. Recently, a heterozygous mutation of this rate-limiting enzyme was diagnosed in a patient resulting in enzymatic inactivation with adrenal insuciency

Fig. 1. Human steroid biosynthesis pathway. Schematic owchart of HPAG endocrine inuences on steroid producing cells and the pathways for mineralocorticoid, glucocorticoid and androgen/estrogen synthesis. Regulatory genes for the adrenal cortex are listed. This Figure applies primarily to the humans.

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and XY sex-reversal [104]. ACTH and aldosterone were elevated and cortisol levels were reduced. 21-Hydroxylase. CYP21 mutations are the most common cause of CAH (95%), with adrenal insuciency that aects all three zones and/or results in XX virilization [7,87]. An expressed pseudogene, CYP21P [105], also located on chromosome 6p21, is responsible for unequal crossover and gene conversion events in the majority of patients [87]. 11b-Hydroxylase. Mutations for these two enzymes, also known as CYP11b2 or aldosterone synthase, and CYP11b1, are the second most frequently aected genes, seen in about 5% of CAH cases [18]. Both map to 8q22 and CYP11b1 mutations result in ZF/ZR cortisol production deciencies with XX partial sex-reversal (ambiguous genitalia). CYP11b2 defects are located in the ZG and cause autosomal recessive aldosteronism and hypertension without CAH [12,106]. P450c17. P450c17a-OH or P45017,20 lyase defects are very rare and are caused by mutations in the same gene on chromosome 10q24q25 [18,107]. The protein catalyzes conversions of multiple substrates, and affected individuals exhibit a spectrum of phenotypes. Patients can present with excess mineralocorticoids and absence of androgens from the ZF/ZR zones, leading to hypertension and XY sex-reversal or ambiguous genitalia [87]. This enzyme also is involved in testosterone production from Leydig cells [98]. Cortisol levels can vary depending on which gene region is aected and the partial agonistic actions that excess precursor substrates can have on various receptors [87]. Patients with isolated CYP17-17,20 lyase deciency that retain P450c17a-OH activity have been identied and are associated with specic intragenic mutations [17,107]. A gain-of-function disorder is associated with premature adrenarche and polycystic ovary syndrome [17]. 3bHSD. Deciency of this prevalent enzyme, which maps to 1p13.1, is very rare. 3bHSD deciency results in CAH and adrenal insuciency aecting all three cortical zones with accumulations of 17a hydroxypregnenolone and DHEA, leading to virilization of some female, but not male, patients [7]. 46,XY patients may present with pseudohermaphroditism, hyponatremia and inadequate puberty, due to gonadal involvement [18]. A phenotypic spectrum exists, including mild or late-onset, premature adrenarche and hirsutism. A drop in the production of this enzyme in the ZR relative to normal levels in the ZG and ZF is thought to be required for normal adrenarche [18,20,22].

key members in the network. Most orthologs maintain a high degree of sequence identity. Due to dual contributory roles played by many of these genes in development and sex-determination/dierentiation of the HPAG, knowledge of animal models is critical for molecular analysis of human patient phenotypes. Nr0b1 Deletion model An Nr0b1 disrupted mouse model was created using a conditional Cre-lox system due to the inability to delete successfully Nr0b1 by homologous recombination [42,108]. This approach has been required due to the proposed lethality in Ahch deleted ES cells [42,108]. The resultant phenotype consists of X-zone (XZ) persistence in postpubertal males and reduced P450scc expression from a poorly developed ZF [108]. No mention of ZR presence is made [108,109], and this may be due to its exceedingly narrow width or routine absence in some strains [110,111]. Serum corticosterone levels are normal [108], but are elevated above wildtype (wt) levels following stress testing [112]. Males are hypogonadal with reduced testis mass, at approximately 50% of wt littermates but do not sustain cryptorchidism. Defective spermatogenesis leading to complete germ cell degeneration is due to germinal epithelium dysgenesis after 14 weeks of age. Leydig cell hyperplasia and hypertrophy are present, possibly due to aromatase (P450c19) upregulation [113]. Sertoli cell cytological appearance is apparently unaected. Testis cord aplasia or dysplasia may be the result of cell signaling defects between perimyeloid and Sertoli cells [114]. Testicular defects can be seen as early as 13.5 dpc [114]. Sf1 and P450scc are both expressed [112], but some mouse strains for this model exhibit reduced Sf1 and P450scc expression [114]. Changes in Nr5a1 transcription or translation levels are more obvious in the Dax1Y= mouse model after the eects of induced stress [112]. Estradiol and aromatase proteins are both upregulated in the Leydig cells of Nr0b1 deleted mice, while Sf1, Star, Cyp17, Cyp11a, and 3bHSD (Type II) mRNA are similar to wt mice [115]. Pituitary gland integrity, ovarian steroidogenesis and gonadotrophin production are not aected [108]. Homozygous disrupted females are fertile and have normal gonads with minor foci of pathology [108]. Scattered focal areas of abnormally proliferative granulosa cells with multiple oocytes surrounded by a single layer of thecal cells are noted in ovarian tissue of mature females. This may be analogous to the proposed Sertoli cell anomaly in males [116]. Sexual dimorphic Nr0b1 expression patterns in the developing adrenal cortex [117] and gonads may support a sex-dependent mechanism responsible for normal female fertility in this model. However, the absence of overt adrenal cortical AHC pathology (hypoplasia) in either sex raises some

Mouse models for adrenal insuciency and related genes Genetically engineered mutations in the steroidogenic genes of mice are critical for dissecting the complex developmental and regulatory mechanistic functions of

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concerns about the relationship of this model to human NR0B1 mutations and AHC. The persistence of truncated Nr0b1 mRNA due to the controlled excision of only the diminutive exon 2 may not produce a completely null allele, but rather a hypomorph. If this is the case, its presence could aect the phenotypic interpretation of this model. Alternatively, the networks in humans and mice may dier. Nr0b1 disrupted mice bred onto a Poschiavinus strain of Mus domesticus (known for spontaneous XY sex-reversal due to a weak Sry allele) produced 100% Nr0b1Y = sex-reversed ospring [118]. No testis-specic markers were expressed for Sertoli (Mis) or Leydig (P450scc) cells and Sox9 expression was markedly reduced by 12.5 dpc in the hemizygous null XY ospring. These ndings dier from null mice produced in a 129SV/j background [112,114]. Nr0b1 null mice crossbred with a haploinsucient Sf1= model allows for evaluation of Nr0b1 impact on steroidogenesis when compared to wt, Dax1Y = , and Sf1= mice [112]. Specication of the Nr0b1 and Sf1 genotype is obligatory for evaluating adrenal phenotypes in an Nr0b1 loss-of-function model in the presence of diminished Nr5a1 activity due to adrenal and gonadal agenesis in Sf1 null mice. The Dax1Y = :Sf1= compound knockout (KO) model has a phenotype that includes adrenal glands of normal mass, less hypoplastic ZF and moderately reduced Sf1 mRNA and protein expression when compared with Sf1= mice. Dax1Y = mice exhibit lower ACTH levels and ACTH:corticosterone ratios than Sf1-targeted heterozygotes. Compound KO mice have intermediate levels falling between both these values, indicating an adrenal cortex-based, rather than a CNS, defect. These ndings are proposed to be due to an enhanced ACTH responsiveness by the adrenal cortex and an inhibitory role for Nr0b1 in adrenal steroidogenesis [119]. This is reinforced by comparison of adrenal gland functional analysis after stress testing these models. Sf1= mice produce normal P450scc and increased Star protein levels compared to wt mice [112,120]. Following stress testing, Star protein is elevated in the Sf1= and compound Nr0b1/Sf1= KO but not Dax1Y = mice, while no change is seen for P450scc levels [112]. In contrast, Mcr-2 (Acth-R) protein expression is upregulated by Dax1Y = and compound knockouts, compared to no change in Sf1= and wt subjects. P450scc levels remained static in adrenals among all three models and wt mice following stress testing. This is in contrast to the dierences of lower basal and higher post stress plasma coticosterone levels in Dax1Y = mice relative to the wt and Sf1-deleted genotypes [112]. Transgenic (Tg) overexpression model Nr0b1 Tg expression in mice with a high number of cDNA copies [510] driven by an 11 kb Nr0b1 upstream

genomic regulatory sequence mated onto the Poschiavinus (POS) genetic background produced both sexreversed and hermaphroditic XYPOS ospring [121]. Sex-reversed XY mice had normal female reproductive tracts, while XYPOS Tg males had reduced testis mass. When these XYPOS Tg Nr0b1 mice were crossbred with a Nr0b1 promoter-driven Sry Tg line, all XX pup ospring developed as females. The same crossbreeding with an XX transgenic Nr0b1 line only produced 50% XX females and a third mating of Nr0b1 transgenic line expressing Ahch at less than wt levels resulted in all XX male phenotypic pups due to the presence of the Sry transgene. These ndings suggested antagonistic activity by Nr0b1 against Sry, in a dose-dependent manner. No Nr0b1 Tg mice with a discernable phenotype have been reported on any alternative genetic background. A Sertoli cell-specic human NR0B1 Tg partially rescues spermatogenesis in Nr0b1Y = KO mice, indicating that all testicular pathology is not necessarily derived from Sertoli cell-based Nr0b1 mechanisms [116]. Hypophysectomy The hypophysis releases GnRH and corticotropin releasing hormone (CRH), which then act on the gonadotropes and corticotropes of the pituitary adenohypophysis, respectively. Subsequently, LH, FSH and ACTH are secreted for stimulation of their respective target organs. Hypophysectomized rats show hypoplastic adrenal cortices aecting the ZF and ZR only when compared with sham-operated controls [122]. Adrenal cortical cell volume is also reduced, especially in the ZF. Sf1 persists at normal levels, but P450scc was signicantly reduced in the ZF and ZR. This suggests that adrenal and gonadal Sf1 expression have at least some pituitary-independent mechanisms. Gonadal expression of Sf1 in the ovary was unchanged, but upregulated in testis and spermatogenesis was suppressed. P450scc was reduced in the gonads of both sexes. Sf1 Targeted disruption of Nr5a1, which codes for Sf1, in mice produces null ospring with adrenal cortical insuciency and elevated ACTH due to lack of responsiveness with adrenal agenesis [123,124]. Nr5a1 expression is noted very early in normal embryogenesis as a marker for the adrenogenital primordium (AGP) cells [44,125]. This is the rst stage of tissue-specic dierentiation toward adrenal cortical development. The homozygously mutated mice also lack gonads in both sexes, are XY sex-reversed, and have both excessive genital ridge and adrenocortical primordia apoptosis by 12.0 dpc, misdirection of gonadal germ cell migration and absence of the hypothalamic ventromedial nucleus (VMH) [123,126131]. The functional

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importance for the developing hypothalamus and pituitary is conrmed in a pituitary-specic Nr5a1 KO mouse model [132]. Pituitary gonadotrophin production is aected by Sf1 expression [133], with absence of LH, FSH and GnRH-R in knockouts [131,134]. KO mice have normal [112] to markedly impaired levels of Nr0b1 mRNA and protein in the urogenital ridge, but expression is less aected in the pituitary, VMH and more developed gonads [33,135]. Sf1 KO mice fail to express Star [136], however, null embryos persist in producing extraembryonic P450scc and P450c17 in the placenta and maintain normal corticosterone levels in utero via maternal-fetal unit enzymatic pathways [124]. Adrenal medullary development also fails in this model [123,124,126], possibly due to induction/migration signal deprivation for neural cells. Defective splenic formation and function is seen as well and is associated with vascular deformities [137]. Homozygously disrupted mice rescued with adrenal transplantation eventually exhibit late-onset obesity due to VMH pathology, including loss of expression of a hypothalamic-specic gene for obesity (tubby) [130]. Leptin and insulin levels are elevated and reect inuences on metabolic pathway mechanisms. The complexity of the role of Sf1 in HPAG development is highlighted by a dose-dependent phenotype in Sf1 heterozygote KO mice [112,120,138]. While externally undistinguishable from wt mice, heterozygotes have hypoplastic adrenals with visible cortical zonation, disorganized cortices with vascular anomalies, adrenal medullary dysgenesis in a sex-dependent manner and immune system defects, and have other abnormalities, including apoptotic deciency [120,139]. The medulla is organized eccentrically in the cortical tissues in males, while females have normal central positioning. However, not all Sf1= models show cortical and medullary defects and this phenotype may be strain-dependent [112]. Unlike Nr0b1 null mice, this mouse model has normal XZ regression [112]. Corticosterone levels are lower than normal, while Nr0b1, P450scc, and Mcr-2 mRNA expression is normal [120,139]. Star mRNA and protein expression are substantially elevated, possibly as a compensatory mechanism. Sf1 heterozygous KO mice demonstrate an inability to compensate for unilateral adrenalectomy with hypertrophy of the contralateral cortex [140]. These mice also express upregulated AsP (cleaves POMC into active an polypeptide for Mcr-2) and Pcna, but not Pref-1, in the adrenal cortex [141]. These ndings indicate Sf1 participates in cortical cell proliferation and/or replacement, but these events are not directly dependent on Pref-1. Further considerations regarding genotype/phenotype correlations are demonstrated by functional dierences among dierent Sf1 alleles from separate mouse strains and their capacity to aect expression of other steroidogenic axis genes [142].

Wilms tumor (Wt1) Wt1 homozygous targeted mutant mice for both primary alternative splice variants (+KTS and )KTS) fail to develop adrenals, kidneys or gonads [106,143]. These transcripts are highly conserved among vertebrates [144,145]. The WT1 ()KTS) isoform is considered functionally to be a transcription factor [146148]. All of these structures are derived from a common progenitor cell type, and Wt1 is expressed very early in embryogenesis [70,111,143,149,150]. Additionally, splenic and cardiac defects are present [106,151]. Neither Nr0b1 nor Sf1 is expressed in these Wt1 null mice [152]. Ablation of +KTS represents a Frasier Syndrome model [153,154] that expresses only the )KTS isoform, and results in a female expression pattern for both Nr0b1 and Sox9 in XX and XY embryos at 12.5 dpc with XY sex-reversal. Sox9 and Mis are not expressed in wt XX gonads. This may be due to a proposed increased activity by Nr0b1 [154]. No adrenal phenotype is present. A mouse model homozygously engineered to ablate the )KTS isoform revealed reduced Nr0b1 expression and urogenital duct dysgenesis with reduced gonadal mass and no adrenal pathology [153]. The Wt1 complete KO model can be partially rescued with a human Tg containing WT1. Null Tg complemented mice survive 48 h postpartum and have varying degrees of adrenal, renal, and cardiac development [149], conrming a critical functional contribution by Wt1 to adrenal cortical ontogenesis. It is considered that WT1 contributions to adrenal cortical development are temporally limited to the adrenal primordium (AP), as expression is quickly restricted to the gonad in the embryonic and postpartum mouse [149]. Wt1 also appears to play a critical role in early embryogenesis, as Wt1= embryoblasts fail to achieve uterine implantation [155]. Star Star KO mice exhibit lethal adrenal cortical insuciency and XY sex-reversal by 10 days postpartum (dpp) [156158], at which time there is 90% mortality. The testes are histologically normal and ovaries are unaffected. Adrenal gland size, and both corticosterone and aldosterone production, are reduced. The ZF is disrupted, with excessive lipid accumulations in the cortex and within fetal Leydig cells of XY testes. CRH and ACTH secretions are elevated, while gonadotrophic hormone levels are normal. The adrenal medulla is normal [99]. Star is required for cholesterol transport from the outer to the inner mitochondrial membrane for processing by P450scc [159,160]. Adrenocortical dsyplasia Also known as Acd, this murine disorder is due to an inherited autosomal recessive mutation on Chromosome

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8 [161]. Both sexes have reduced survival, as well as growth retardation, hyperpigmentation, and urogenital defects [162]. Adrenal cortical XZ fails to develop with homozygous mutations and elevated ACTH, but aldosterone levels are normal in both sexes. Females exhibit low corticosterone levels. Glucocorticoid serum levels in heterozygotes and homozygotes were equivalent when adjusted for relative adrenal gland mass. Cortex histology reveals hypertrophic cell character with enlarged nuclei, possibly driven by elevated ACTH action. This may be an under-appreciated model for AHC with a high degree of utility, given the adrenal phenotype without sex-reversal. There is a hypogonadal component to this phenotype. POMC POMC models are deleted for all POMC-derived peptides and show adrenal dysgenesis with no recognizable cortical architecture, hyperpigmentation and obesity, but without presence of diabetes [163,164]. Adrenal hormonal functions are aected in a dosagesensitive fashion, including extremely low corticosterone and aldosterone levels. Adrenal medullary function is also aected. A high incidence of non-functional pituitary tumors is noted in both hetero- and homozygous mutants by seven months of age. 21-Hydroxylase 21-OHA and B deletions in a mouse model show 100% lethality by two weeks of age [165]. Aected mice have small, irregular adrenals and showing disorganized, hypertrophic, and hyperplastic ZF cells. Heterozygotes are unaected phenotypically. Conrmation was achieved by rescue of homozygotes with a human CYP21 transgene [166]. CYP11A1 Gene disruption by neomycin gene insertion results in Cyp11a1 null mice with absence of corticosterone production [167]. These mice die shortly after birth due to adrenal insuciency. This model exhibits elevated ACTH levels, XY sex-reversal and lipid accumulation in the adrenal cortex. Nieman-Pick C1 NPC-1 null mice have adrenals indistinguishable from wt mice at eight weeks of age. However, homozygous null mice sustain ZF hypoplasia, a signicantly widened XZ and increased lipid accumulations in all zones over time [168]. Aected mice have adrenals with at least 50% reduction in mass. No report of adrenal corticosteroidal insuciency is made. A Tg-reporter

investigation showed expression in transfected adrenal Y-1 and Leydig cells [168]. This gene is involved in cholesterol metabolism, including steroidogenesis in the adrenal cortex, testis ovary, and placenta. Cited2 This mouse model was created by homologously targeted recombination for this CREB Binding Protein/ P300-Interacting Transactivator with ED-Rich Tail-2 gene, which binds CREB and EP300 proteins [169]. CBP/P300 is a well-known transcriptional activator of WT1 [146]. The trait is an autosomal recessive with embryonic defects, including adrenal agenesis, an increase in neural apoptosis at 9.5 dpc and embryonic lethality starting at 10.5 dpc. Neural tube, cardiac and aortic arch defects are also present. Transcriptional regulation of P450scc by SFI is reportedly mediated by CBP/P300 in humans [170]. Gli-3 Gli-3 homozygous deleted mice have complete adrenal agenesis and severe kidney dysgenesis, among other developmental defects [171]. This model is typically employed in investigating Pallister-Hall syndrome, an autosomal dominant malformation disease in humans. Ex Earlier X-zone degeneration locus is a mouse strain with fatty degeneration of the XZ in females by 8 weeks of age with no impact by ovarectomy [161]. No biochemical or gene characterization is provided. Ezg Extent of Zona Glomerulosa locus is a mouse model with a poorly dened ZG, hyponatremia and normal aldosterone levels [161]. The trait is autosomal dominant, but no gene is reported to have been cloned for this model, to date. Caspase Deletion models for some of the numerous caspase family members produce adrenal cortical developmental defects with improper zonation. Caspase family members are critical components in apoptotic remodeling and proinammatory cytokine processes [172,173]. Caspase-11 null female mice have reduced ZF and ZR thickness [174]. Caspase-2 null females have a wider-than-normal XZ with a reduction in normal cellular apoptosis [175]. Ovarian apoptosis is also signicantly reduced in Caspase-2 KO females [173,175]. ZG and external appearance is normal in both caspase KO models.

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Gata4 Gata4 null fetal mice die before 9 dpc, but chimeric ospring derived from ES cells deleted for this gene are available [176]. GATA4 is functionally linked to regulation of MIS and StAR and is upregulated by FSH and LH in Sertoli, Leydig and granulosa cells. Gata4 is expressed at 14.0 dpc in mice throughout the developing cortex and GATA4 is present in the human adrenal, primarily in the FZ. Expression is markedly downregulated after birth. Therefore, the Gata4 null mouse model is a potentially powerful tool for elucidating early adrenal cortex ontogenesis. The closely related Gata6/ GATA6 is also strongly expressed in the developing adrenocortex of both species [176]. P450scc CAH is inherited as a natural occurring deletion in an autosomal recessive mode with normal adrenal cortex and gonadal appearance, lethal adrenal insuciency and XY sex-reversal in rabbits [177,178]. Corticosterone levels are nonexistent, 21-OH levels are normal and P450c17a is increased. Additional models involving steroidogenic related genes without adrenal abnormalities Wnt4 Targeted deletion of Wnt4 in mice, which is normally expressed in the adrenal cortex, results in masculinization of XX females, with aberrant expression of Sertoli cell markers Mis and Desert Hedgehog (Dhh), in mutant ovaries [179]. Testosterone is produced via P450c17 expression in XX gonads of these mice. Wt1 is also normally expressed suggesting that it is developmentally upstream of Wnt4. No adrenal pathology is reported. Nr0b1 expression is present in the ovary of null mice. These mice die of renal agenesis postpartum [180,181]. A Tg mouse with two Wnt4 copies showed incomplete XY sex-reversal, abnormal testicular vasculature, decreased testosterone levels and seminal vesicle dysgenesis [182]. No adrenal developmental defects are noted, as are reported with duplication in humans [78]. X-linked adrenoleukodystrophy Mice deleted for the X-linked Ald gene develop swollen adrenocortical cells with decreased lipid accumulations, seminiferous tubule atrophy, gonadal interstitial cell hyperplasia in both sexes, and reduced fertility by six months of age [183]. Spermatogenesis can be affected in hemizygous mice. These mice suer from a very-long chain fatty acid tissue accumulation after six months of age but adrenal insuciency is not reported. Sox9 Sox9 loss-of function due to gonadal-specic regulatory region deletion result in XX sex-reversed mice [184].

XX testis have Sertoli cells and seminiferous tubules present. Heterozygotic XX gonads are indistinguishable from XY littermates. XY heterozygotic males are fertile with normal testicular histology [184]. A Sox9 Tg insertion disrupts a gonadal-specic regulatory region and produces XX sterile sex-reversed males with normal Sertoli and Leydig cell appearance but without adrenal defects [154] XX Tg testes are histologically identical when compared to XY transgenic testis, including Sertoli and Leydig cell development [185]. No adrenal histopathogenic changes are reported.

Adrenocortical ontogenesis The paired adrenal, or suprarenal, glands are paired organs located at the cranial poles of each kidney and are crucial for stress adaptation and maintaining physiological homeostasis, especially glucose and salt metabolism [8,12,13,87,186]. The adrenals are derived from two distinct germ layer tissues. The cortex is derived from mesoderm, in common with the gonads and kidneys, while the medulla is of neuroectoderm origin and is created by neural crest cell migration into the mesodermal portion [12]. Dierences exist in adrenal cortical structural organization and biochemical capacities between species due to a slightly divergent series of developmental events. For example, rodents lack signicant P450c17aOH expression and, therefore, do not synthesize androgens at signicant levels in the ZR, as produced by primates [187]. Rodents utilize an alternative pathway in the ZR to form the androstenendione that they do make [186]. It is relevant to note that adrenal production of androgens in sexually mature humans only accounts for about 5% of total body synthesis. These disparities do not preclude the use of lab animals as investigational tools for AHC, since evidence indicates that the basic patterns of development are likely to be genetically programmed by a conserved network. However, some molecular mechanisms may require contextual interpretation in a speciesspecic manner. A comparison of mouse and human adrenal development is shown in Table 2. Adrenal gland ontogeny produces architectural stratication of the cortex termed zonation with zonespecic steroidogenic end products in higher vertebrates. A primary dierence between rodents and humans is the presence of a wide inner FZ comprising 7090% of the fetal cortex in the human fetus [11,188]. The FZ starts to disappear postpartum and is completely lost at the time of adrenarche. The thin peripheral DZ is synchronously activated to dierentiate into the functional adult zones. Rodents possess a disorganized cortex early in embryogenesis that subsequently surrounds the XZ postpartum. The XZ regresses in a sex-dependent fashion, either at puberty in males or during approximately the rst half of pregnancy in females [110,189]. Experimental evidence

R. Clipsham, E.R.B. McCabe / Molecular Genetics and Metabolism 80 (2003) 81120 Table 2 Comparison of human and mouse adrenal gland development Embryonic event PGC migration from yolk sac Urogenital ridge formation Adrenogenital primordium formation Adrenal/gonadal primordial dierentiation Gonadal sex dierentiation Symphoblast migration DZ cell migration into cortex Adrenal cortical capsule formation Adrenal cortical cell dierentiation begins FZ maximal size (20 to rapid growth) TZ appearance Postpartum event Cortical rapid growth phase FZ collapse-rapid phase FZ collapse-slow phase ZG/ZF established ZR established (strain-dependent) Medullary capsule formed XZ formation XZ collapse Mouse 7.510.0 dpc 9.0 dpc 1010.5 dpc 12.012.5 dpc 12.013.5 dpc 12.014.5 dpc N/A 14.0 dpc 14.0 dpc N/A 13.0 dpc 130 dpp N/A N/A 16 dpc-birth 21 dpp 35 dpp 10 dpp (1) 38 dpp in males (puberty onset) (2) by mid-gestation in females Human <3 wpc 34 wpc 33 dpc 45 wpc 4144 dpc 79 wpc 9 wpc 9 wpc 912 wpc 1624 wpc 2030 wpc N/A birth-2 wpp 2 wpp12 mpp 24 mpp1 yr 14 yr 1214 mpp N/A N/A

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from numerous genetic mouse models for adrenal development, including Nr0b1, suggests the FZ and XZ may be analogous structures. A historical note of interest is the original designation of the FZ in human adrenals as an X-zone by some authors [188,190], which is confusing, since the murine X zone is the equivalent of the human fetal zone; therefore, we will use X zone to refer to the murine zone. Other anatomical features, such as accessory adrenal tissue in mice [110], which can have strain-dependent variable distribution, must be incorporated into the designs for specic investigations, such as those involving adrenalectomy and transplantation. The following discussion details the comparative progression of adrenal ontogeny between primates and rodents on a histological, genetic, and functional basis. Collectively, the numerous rodent models and human mutation patients with adrenocortical developmental disorders manifesting as adrenal insuciency illustrate the pivotal roles that known genes play in adrenal ontogeny and the incomplete existing knowledge. Comparisons drawn between similar phenotypes in dierent species often augment data deciencies for a single species. However, these comparative genetics eorts require numerous analyses before any credible hypothesis can be proposed due to inherent species-dependent idiosyncrasies. The events of adrenal and gonadal development in rodents are summarized in Fig. 2. Urogenital ridge and adrenal primordium The appearance of the urogenital ridge is initiated by a consolidation of the coelomic epithelium or mesen-

chyme to form epithelium [12,75,191]. This occurs at 34 weeks of gestation in humans [11,12,87,111] and at 9.0 dpc in the mouse [189,192]. By week 5 of gestation (10.511.0 dpc in mice) a population of these cells have condensed and migrated medially and forward of the mesonepheros to form the adrenogenital primordium (AGP) [12,125,193]. Following sex determination at 7.5 dpc (mouse), primordial germ cells (PGC) migrate from the yolk sac by way of the hindgut [193] into the rostral aspects of the AGP by around 10.0 dpc [125]. PGCs become widely distributed throughout the AGP and increase in number during the next 1.01.5 dpc [75]. In general, portions medial to the mesonepheros form the adrenal primordium (AP) that will become adrenal cortex, while cells ventrally will form the gonadal primordium [12,125,191]. The adrenal anlage is identiable from the undierentiated gonads around 12.0 dpc [110,125,189] and by 12.5 dpc the cortex becomes a distinctly identiable structure [194]. Sex dierentiation of the bipotential gonad is visibly evident at 4144 days in humans [195] and around 12.0 dpc in mice [193]. This event is marked by a rapidly progressive wave of Sry expression appearing in the male genital ridge starting at 10.511.0 dpc and disappearing by 12.5 dpc [196]. In comparison, SRY mRNA is present in human XY gonads from day 41, peaks at day 44 and then persists forward throughout gestation [196]. Adrenal dierentiation By day 12.0 dpc, adrenal primordial cells are pale in comparison to chroman cell precursors (symphoblasts) derived from the neural crest. This is the initiation of

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Fig. 2. Adrenal gland and gonadal ontogenesis in rodents. Larger arrows indicate more signicant developmental eects and dashed arrows indicate less signicant developmental eects. Progressive events during gestation are noted as days post-coitum (dpc). Comparative time-related events are noted for mouse and rat. PGCs, Primordial germ cells (yellow); Symphoblasts, neural crest adrenal medullary progenitor cells (green); DZ, denitive zone; TZ, transitional zone; AGP, adrenogenital primordium; AP, adrenal primordium; GP, gonadal primordium; Sf1, Steroidogenic factor 1; Nr0b1, DAX1 ortholog; Wt1, Wilms tumor 1.

medullary cell invasion. Symphoblast migration is complete in mice by 14.5 dpc. Neural cell migration for medullary formation is reported to occur as early as 7 weeks [11,197] and up to 9 weeks [189] in humans. This discrepancy may be due to the disseminated nature of rudimentary symphoblast islands that cluster in the central region by 15 wpc [197] and may not form a centrally located, denitive medulla until after birth [12]. In humans, by the eighth week of gestation, the adrenal shows early chord (stroma) development compatible with steroidogenic capabilities [197]. These cells will continue to develop as the embryonic FZ. By 9 wpc of gestation, a second wave of epithelial cell migration into the adrenal occurs to envelope the primitive FZ as the future DZ [11,12]. Conicting opinions exist as to whether these two cortical components originate from the same [198], or separate [199], coelomic progenitor cells. A cortical capsule forms at this time in humans [12,189] from mesenchymal cells that migrate from Bowmans capsule of the kidney [200]. This same event occurs at 14.0 dpc in mice. Interaction of the capsule and cortical cells may stimulate cortical cell dierentiation

for the formation of the embryonic DZ. By 13.0 dpc preliminary capillary invasion can be seen [201]. Sinusoidal capillaries proliferate between the stromal cords and increase in complexity throughout gestation [202]. Adrenal cortical ontogeny becomes relatively less active at the histological level after 1012 weeks post coitum (wpc) and 14.0 dpc, in humans and mice, respectively. The adrenal now consists of an FZ having strong CYP17 expression, an outer circumference of tightly packed cells and the inner regions showing a loose reticular pattern, interspersed with clusters of symphoblasts in humans. The FZ reaches maximal width relative to the DZ between 16 and 24 wpc [11,197,203] and the adrenal is relatively larger than the developing kidney. The FZ now comprises 8090% of the glandular mass, primarily due to hypertrophic expansion [8]. The DZ appearance at this time is a narrow band of compacted cells with ultrastructural characteristics typical of proliferation [12] and the DZ lacks CYP17 expression [111]. During the nal trimester, the ZG and ZF become identiable and establish steroidogenic capabilities independent of maternal physiology [87].

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Recently, a very thin zone located between the inner edge of the DZ and the external margin of the FZ has been identied as an intermediate or transitional zone (TZ) [12,187,204206]. This unique cortical zone is characterized by expression of 3bHSD by 2030 wpc [187], extremely narrow dimensions (56 cells wide) and lack of functional corticosteroid production [38,88,204], and is a site of cellular proliferation [88]. This TZ is dependent on ACTH stimulation for development to a greater extent than the earlier requirements of the FZ in primates [207]. A similar band of proliferative cells is noted to express Pref-1 at 14.5 dpc in the cortex of rats [208], which is equivalent to 13.0 dpc in mice. This zone does not express zone-specic markers for either ZG (P450aldos) or ZF (CYP11B1) [88,141]. A-inhibin expression is also present, but at levels lower than the FZ [209]. It is proposed that the TZ is populated by an adrenal-specic stem cell that is a source of initial cells for dierentiation, as well as cortical renewal during adult life. These assumptions are strongly supported by observations and experimental data, including cell-labeling studies [110] and adrenocortical regeneration from the residual capsule following enucleation [210,211] or transplantation [212,213]. These data have given rise to two competing theories for adrenocortical zone renewal from the TZ. The rst supports a centripetal migration from the ZG/TZ inward [119,205,214216] and the other favors a bi-directional movement simultaneously from the TZ outward to the ZG and inward toward the ZF [194,204,217]. Both theories show evidence of increasing incidence of cortical cell apoptosis in the inner zones, with the highest incidence reported in the ZR [88,206,214]. It is of some signicance that the original publications by Wylie and Kerr describing initiating the investigations in apoptosis were performed using the developing adrenal cortex [119,218,219]. Postpartum dierentiation Critical events in adrenal cortex development continue into the postpartum period. The FZ undergoes a collapse starting at birth via apoptosis [12,119,206,217] and is rapidly reduced in volume within two weeks [220]. This process continues at a reduced rate over the next 6 12 months postpartum (mpp) [12,220222]. Adult cortical functional zonation is initiated from the DZ during the rst 24 months with the establishment of the ZG and ZF [223] and is concurrent with the initial phase of FZ involution. Zonation is well-established histologically by one year of age, but the ZR is not clearly evident until 14 years and is likely to be derived from dierentiated ZF cells [11,12]. A medullary capsule is intact by 1214 mpp in humans [70,222]. Zone dierentiation is complete between eight [222] to twenty [70] years of age, which correlates with the age range for adrenarche and pubertal onset. DZ zonation is achieved

by hyperplasia, rather than by hypertrophy as seen during FZ growth [190]. By comparison, preliminary cortical dierentiation in mice is partially acquired at 1618 dpc [70] with some reports indicating that structural dierentiation is well dened in rodents at birth [88,224]. Evidence supports strain-based dierences for adrenal cortical zonation in mice and may be the source of this confusion [225]. The adrenal medulla is encapsulated by 35 days after birth [70] and loses its brous connections to the cortex only after XZ collapse [226]. The infant cortical cell zone of mice has been termed an interlocking layer [110] and lacks the FZ of primates. This adrenal region widens up to 2130 days postpartum in males and continues to expand in females until approximately four months of age. The interlocking layer starts to dierentiate to form the XZ at around 10 days [110]. The greater width of the female XZ accounts for their larger adrenal size over males. XZ maintenance is also hormonally dependent, as castration prevents normal pubertal collapse at 38 days in males [70] leading to nal zone width equal to that in females, regardless of ovarectomy [110,227]. XZ disappearance in females is coincident during the rst pregnancy [226]. However, XZ volume and rate of regression are both strain dependent [110,228]. An association with the agouti locus and XZ morphology has been suggested [229]. The proposed analogy between the FZ and XZ is due to similarity in intraglandular location, ultrastructural architecture characteristic for steroid production and lack of involution with loss of NR0B1/Nr0b1 [230]. Recent data strongly suggest that activins are directly involved with XZ collapse via apoptotic mechanisms [231]. Functions of the XZ remain elusive, but Caspase-2 KO mice have a phenotype with signicant similarities to the Nr0b1 null model in regard to adrenal development [175]. This comparison further suggests that an apoptotic mechanism is required in the developmental process for nal zonal dierentiation [174]. The XZ is situated between the ZG/ZF zones and the miniscule ZR [110]. The ZR forms beginning at 21 days postpartum [186] but is only a few cells wide, at most [110,204]. The ZR is not present in all mouse strains and its identity and function are controversial. It may be for this reason that some investigators place the XZ between a combined ZG/ZR zone and the medulla [111]. The mouse, due to an inherently low production of Cyp17 [111], does not produce any substantial amounts of androgens or cortisol from the ZF/ZR and, therefore, produces corticosterone as the major cortical glucocorticoid. The process of murine adrenal cortical dierentiation to become histologically mature is dependent on clonal expansion of precursor cells and their migration, similar to humans [204,216,224,232,233]. The proposed site of cellular proliferation is supported by mouse models with a Cyp21-LacZ reporter transgenic showing clonal

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expansion from the inner margin of the ZG from 11.5 dpc [224] up to 14.0 dpc [232]. The proliferative transgeneexpressing cells of this model rapidly form radial stripes extending to the medullary border. The TZ is unique as it does not secrete corticosterone/cortisol, aldosterone or DHEA, but is known to have protein expression for P450scc, 3bHSD, Pref-1, and proliferating cell nuclear antigen (PCNA), a marker of cellular replication [204]. However, these observations do not resolve any mechanistic distinctions between adrenal cortex development, cortical cellular renewal or precursor cell type(s). In addition to the genetically related events noted above, HPAG development is also critically dependent on paracrine and endocrine factors extrinsic to the adrenal cortex. These include innervations of the adrenal gland [203,234236], cortical vasculature formation [114,182], the renin-angiotensin system [237], and placental factors [238,239].

synthesize androgens and is required for the onset of spermatogenesis. Testosterone appears at 7 wpc in human testis and precedes the onset of cytodierentiation, contrary to events seen in rodents [245]. There is also a third wave of increased Leydig cell population in humans by the onset puberty. Female gonad dierentiation is delayed relative to testis and proceeds after 15.0 dpc (10 wpc) with limited germ cell meiosis and reorganization into recognizable follicles. Granulosa cells are evident by 20 wpc under the inuence of adjacent meiotic oocytes [196,241]. Wolan duct dierentiation occurs at the last third of gestation (25 wpc in humans) in males with formation of epididymis, vas deferens and seminal vesicles. These Wolan structures degenerate earlier in normal females.

Pituitary gland ontogenesis Pituitary development is initiated by an ectodermal placode formation at 8.0 dpc (3 wpc in humans [96,246]. At 8.5 dpc, an invagination of this oral ectodermal structure forms the earliest pituitary primordium, the rudimentary pouch [16,246248]. This structure will dierentiate into glandular epithelium, including the future adenohypophysis. The second component of the future pituitary is derived from an extension of the diencephalon (neuroectoderm) and will form the future infundibulum, which comprises the exocrine (posterior) portion of the pituitary. These two elements achieve contact at 9.5 dpc (5 wpc) and move caudally to their nal position by 14.5 dpc (13 wpc). The rudimentary pouch closes to form the denitive (Rathkes) pouch and proceeds to fold over itself (6 wpc), while the diencephalon migrates ventrally along the posterior aspect of the pouch to form the infundibulum. The closer the contact between these two tissue origins, the more destined the cell fate is toward epithelium (anterior pituitary), while less contact directs dierentiation toward an exocrine fate [248]. The stalk connecting the oral cavity and denitive pouch is lost by 12.5 dpc (13 wpc). The anterior wall of the pouch will dierentiate into the anterior lobe, while the posterior portions form the middle (pars intermedia) lobe. The infundibulum gives rise to the posterior (pars nervosa or neurohypophysis) lobe. The adult arrangement is essentially complete by 19.5 dpc (21 wpc). The anterior lobe (adenohypophysis) is the major contributor (intermediate lobe presence is speciesdependent) to the HPAG axis. The pars intermedia is vestigial and of undetermined function in humans. Functionally, an a-glycoprotein (aGSU) subunit, which is a shared molecular component of FSH, LH and TSH, is expressed at 12.5 dpc [246,247]. This marks a commitment to thyrotrope and gonadotrope cell lineages of the future adenohypophysis. Full thyrotrope (TSH) dierentiation is seen at this time, followed by

Gonadal ontogenesis Disassociation of the embryonic bipotential gonad from the AP occurs at 10.511.5 dpc (4144 dpc in humans). Sex dierentiation is initiated subsequent to Sox9 (9.0 dpc) and Sry (10.512.5 dpc) expression at around 12.0 dpc (67 wpc in humans), with distinguishable ultrastructural commitment to Sertoli cell lineage in XY gonads at 12.5 dpc [110,125,189,193]. Expression of Mis/ MIS is a genetic cell marker for this event [240]. This hormone is responsible for repression of the Mllerian u duct by apoptosis in males and induction of derivative structures (oviduct, uterus and vagina) in females [241]. Gonadal dierentiation aects three cell lineages, not including the primordial germ cells: (1) supporting cells that surround the germ cells, with Sertoli (XY) and granulosa (follicular) (XX) cells being equivalent cell types; (2) interstitial cells that produce steroids, with Leydig (XY) and thecal (XX) cells acting as equivalent cell types; and (3) connective or stromal cells that provide structural characteristics to each gonad type. The peritubular myloid (XY) cells that surround Sertoli cells lay down basal lamina and create the testicular cords. Stromal cells of the ovary possess no myoid properties and are not testicular equivalents. Migration of mesonepheric cells is proposed to occur during 11.516.5 dpc into the XY, but not XX, developing gonads under the inuence of Fgr9 [242,243]. These cells are believed to undergo dierentiation to give rise to both endothelial and myoid cells and are required for the testis cords [244]. Precursor Leydig cells begin to appear at 13.5 dpc (8 wpc in humans) subsequent to undetermined paracrine signals, probably from nearby Sertoli cells. These cells multiply until midgestation and are well dierentiated by 15.0 dpc [245]. A second wave of Leydig cell hyperplasia occurs during puberty when these cells

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hormone production from melanotropes (MSH) at 13.0 dpc, somatotropes (GH) at 17.0 dpc, gonadotropes (LH, FSH) and lactotropes (PRL) at 18.0 dpc [94,247]. Gonadotropes selectively express Sf1 around 14.0 dpc in mice. ACTH synthesis is a marker for corticotropes and is present at 13.0 dpc. This maturation pattern progresses in a ventral to dorsal fashion as a result of a temporally and spatially restricted set of transcription factors.

HPAG developmental gene expression patterns The earliest structure destined to form the denitive adrenal gland is the urogenital ridge, a mesodermal condensation of the coelomic epithelium, and is the origin of the primordia for both gonads and adrenal cortex. At approximately the same time, the diencephalon, a prosencephalon-derived structure, starts initiating neural dierentiation for formation of the future hypothalamus and pituitary gland. Genes expressed during these developmental transformations to create the HPAG possess inherently time-, tissue-, and compartmentally sensitive patterns. The mechanistic role played by Nr0b1/NR0B1 is dictated by its presence or absence within these expression patterns and is dependent upon contextual network dynamics [249]. Knowledge of specic cell-type and timing of gene expression proles is crucial for targeting potential gene interactions for in vivo investigation and assisting in interpretation of results from cell modeling experiments or patient proles. Nr0b1/NR0B1 Mouse Ahch appears in the developing mouse urogenital ridge and Rathkes pouch (the future pituitary gland) at 9.0 dpc and is a molecular marker of this primary commitment to HPAG organ dierentiation [33,34,41,70,108,121]. The AP, indierent gonads and developing CNS positively express Nr0b1 by 10.5 dpc [9,33,41,121]. Nuclear expression at both the mRNA and protein levels persists throughout the development of HPAG structures. Nr0b1 is expressed in the primitive Rathkes pouch at 9.5 dpc and becomes progressively limited to its dorsal regions by 11.5 dpc [34]. Expression here is followed by expression in the prosencephalon at 11.5 dpc [9] and subsequently in the diencephalon and tongue at 14.5 dpc [34,41]. Expression is weak by 13.5 dpc and ceases by 14.5 dpc in the denitive pouch. Nr0b1 subcellular localization has been noted to be both nuclear and cytoplasmic in selective CNS and gonadal tissues in mice [45,184], as well as other cell types [35]. Numerous additional CNS structures are also noted to express Dax1 protein between 14.5 and 18.5 dpc [33,41,121].

Gonadal transcription is documented in the primordial gonad at 11.5 dpc [41]. Simultaneous expression within the surrounding coelomic epthelium is proposed to be from pre-Sertoli precursor cells that will rapidly migrate into the gonadal somatic structure [34]. Nr0b1/ NR0B1 is also detected by immunohistochemistry (IHC) at 12.5 dpc in the interstitial (Leydig) cells, where it colocalizes with Wt1 [250], Sry [41,193], Sox9 [75,193], Wnt4 [179,194], and Mis [193]. The onset of a sexually dimorphic Nr0b1 expression pattern occurs around 13.5 dpc [33,41,121], with a restriction to Sertoli [41,250] and/or Leydig [34,41,45,121,135] cell lineages of the XY gonad. The general consensus of reported results show decreasing levels of Nr0b1 expression throughout the remainder of gestation and into the postpartum period. Levels are maximal in Sertoli cells with impending onset of puberty and are upregulated during spermatogenesis under FSH inuence [251]. Ovarian expression is restricted to thecal [41] and granulosa [135] cells, with a decrease after 14.5 dpc [33,41], although ovarian persistence is reported [9]. Sexually dimorphic patterns are also noted within the developing adrenal cortex by RTPCR in mice [117]. Equal RNA adrenal cortical expression is evident by 14.5 dpc in both sexes, but decreases from 16.5 dpc to 84 dpp to 10% of original levels in males, while increasing by 23-fold in the ovary. The debate over XX vs. XY and/or Leydig vs. Sertoli cell relative RNA expression [33,34,41,45,252] may be based on technical issues. This is supported by immunohistochemical (IHC) analysis that shows little or no dierences at the protein level between sexually dimorphic gonads during development. All cortical regions are positive for Nr0b1 expression in both sexes until 14.5 dpc. By birth, females retain whole cortex expression, except the XZ, which is negative. Males show ZG expression only. XZ analysis is not reported for males due to early degeneration of the XZ at puberty. However, radioactive in situ hybridization showed positive signal for the ZF region and negative results for the medulla at 11.5 dpc [41]. IHC studies of adult rats revealed strong ZG expression, weak levels in the ZF/ZR and an absence of Nr0b1 in the medulla [135]. In addition to the hypothalamus, gonadotropes of the anterior pituitary and gonads, transcripts in lung, heart, spleen, kidney cerebral cortex and spinal cord of adult rodents has been demonstrated by RT-PCR [40]. Human Expression patterns essentially parallel mouse results with onset in the urogenital ridge at 33 dpc by both mRNA [38] and protein [37] analyses. Subsequent expression in the AP is greater than in its AGP precursor. RNA levels maximize at 52 dpc in the AP and then decrease until 18 wpc, but persist throughout gestation [37,38]. DAX1 protein expression coincides throughout development of the cortex, including both the FZ and

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DZ [37]. Nuclear expression is seen in adult adrenal cortex, gonads, anterior pituitary, hypothalamus and uterus [2,10,253,254], as well as cytoplasmic localization in adrenocortical cells [184]. The testis experiences a downregulation of NR0B1 expression during development relative to the ovary. NR0B1 is colocalized with NR5A1 in skin cells, including keratinocytes, preadipocytes and papillary cells [36]. Its presence in skin is associated with CYP11A1 (P450scc), CYP17, CYP21, and CYP19 (arom), which may be subject to regulation by these nuclear receptors. Nr5a1/NR5A1 Mouse NR5A1 is expressed at 9.0 dpc (11.0 dpc in rats) in the urogenital ridge [9,70,125,126,255,256] as a genetic marker for early adrenal cortical cell lineage commitment, along with Nr0b1. Expression persists in the AGP and both derivatives, the AP and indierent gonads at 10.511.5 dpc [9,125,194]. Nr5a1 is consistently coexpressed with Ahch throughout the adrenal cortex, including ZG, ZF/ZR [257], and TZ [204]. Adrenal cortical cell expression persists into adult life and is equivalent in all three zones of the denitive cortex [258]. No medullary expression for Nr5a1 is reported. Sf1 is nuclear [133,258] and exceeds Ahch expression at all ontogenetic stages, with testicular levels greater than those seen in the ovary [121,126,255]. Testis levels of Nr5a1 increase after 12.5 dpc while ovarian expression disappears around 13.5, with reappearance at 18.5 dpc [33,255]. Leydig cell expression exceeds Sertoli levels, which are positive for Nr5a1 by 15.0 dpc [9,135,259]. Species dierences may exist, as expression may not occur in rat Sertoli cells until birth [257,258]. Upon its reappearance at 18.5 dpc, Nr5a1 expression in the ovary is demonstrated in both granulosa and thecal cells in late gestation [135,257,258]. It later appears during folliculogenesis at 1 week postpartum (wpp) and in the subsequent corpus lutea [9,122,259]. Expression colocalizes with P450c19 in granulosa cells and P450scc in thecal and corpus luteal cells [257]. Splenic expression is also noted in the nucleus by 14.5 dpc [137,260]. The alternatively spliced forms of Nr5a1 are designated Elp1-3 and were originally cloned from embryonic carcinoma cells [261,262]. These transcripts are reported by one group to be endogenously expressed by all steroidogenic tissues in the rat [257]. However, another group, using Elp1-3 transcript-specic primers and RT-PCR analysis did not nd endogenous expression in mouse adrenal, ovary, testis or placenta [124]. Similarly, we were unable to demonstrate expression of the Elp1-3 transcipts in nontumorogenic murine ES cells by RT-PCR [43]. CNS evaluation reveals colocalization with Nr0b1 at 11.5 dpc in the prosencephalon [255] and the diencephalon at 12.5 dpc [126,128,263], where it is restricted to

postmeiotic cells [129]. These structures eventually form the hypothalamus and adenohypophysis. Expression is conned to the VMH of the hypothalamus by around 17.0 dpc [9,33,127]. Pituitary presence is weak at 13.5 dpc, but increases steadily [134] throughout gestation. Nr5a1 expression in the pituitary [131] is restricted to gonadotropes of the adenohypophysis and precedes FSH and LH [9,33,134]. Nr5a1 expression has also been documented in mouse placenta [124] at 14.0 dpc, as well as in pigs [264]. This may not be species-wide and some controversy exists as to the mechanistic capabilities of placental steroidogenesis, as other results for mice are negative [109]. Human Expression patterns in humans are essentially the same as rodents, with the exception of more extensive expression pattern in CNS structures [195]. Fetal adrenal IHC analysis reveals initial appearance at 33 dpc in the urogenital ridge and strong expression by FZ, TZ, and DZ [265] in the subsequent AP [37,38,265]. Nr5a1 does not persist in the developing kidney component of the urogenital ridge [195]. Ninety percent of adrenocortical cells produce high levels of NR5A1 but is not produced by the medullary chroman cells [266,267]. Gonadal expression is evident from the indeterminant stage [37,265] throughout gestation and into adulthood [38] in both the interstitial and steroidogenic cells of both sexes [259,266]. Increased testicular expression appears to follow testis cord development at 18 wpc [265]. Positively expressing cells of the pituitary are limited to gonadotropes [133], as seen in mice. Placental expression for NR5A1 has been shown positive for tropho-, cyto- and syncytiotrophoblasts in humans [259,268,269]. Curiously, NR5A1 is expressed in dermal keratinocytes and preadipocytes [36]. Wt1/WT1 Mouse This gene is expressed at 9.0 dpc in the urogenital ridge [106,149,150,250,252] with persistence in both the indeterminant gonad and metanepheric derivatives. At 12.0 dpc, a sexually dimorphic expression prole emerges with restriction in XY gonads to the Sertoli cells, rete testis and tunica albuginea. XX gonads express Wt1 in granulosa cells and ovarian epithelium. Fetal kidneys and testis have an additional unique transcript of unknown functional signicance when compared to adult tissue mRNA expression [106]. Expression in adult tissues persists in kidney, testis (Sertoli cells) ovary (granulosa cells), and uterus. Expression peaks from 17.0 dpc to 3 dpp, with a 20-fold decrease in levels at maturity [106]. All proteins are localized to the nucleus [270,271]. No expression is present in the developing adrenal cortex after the AGP stage, including adult

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adrenocortical cells. Wt1 is absent in the hypothalamic and pituitary components of the HPAG. Human Expression of WT1 is rst detected at 2832 dpc in the urogenital ridge and mesonepheros [195]. It persists in the indierent gonad at 49 dpc with identical expression patterns as in mice [106,272]. Expression is signicantly stronger in kidney than is seen in gonads [32,195]. XY gonadal expression becomes conned to the cords, but remains diuse in the less developed ovary, where it is noted to persist through at least midgestation (18 wpc). Eventually, testicular expression is limited solely to Sertoli cells after 18 wpc [195]. Placental [273] and uterine [254] expression is also reported. Star/StAR Mouse Star appears at 10.5 dpc in the urogenital ridge and continues to be expressed in the AP with persistence throughout the developing adrenal cortex [274]. Symphoblast and chroman cells of the migratory and dened adrenal medulla are negative for Star expression. However, adrenal medullary expression is reported in rats and cow [275] indicating potential species and/or testing sensitivity dierences. The indeterminate gonad expresses Star with eventual restriction to the testicular Leydig and ovarian thecal (interstitial) cells where it colocalizes with Sf1. Corpus lutea of the active ovary later also express Star, in keeping with its steroidogenic function. Ovarian and adrenal cortex levels are greater than testes. Star protein is present in the hypothalamus, where it colocalizes with P450scc [276]. Adult expression is conrmed in the testes, ovary, ZG, ZF, and ZR zones of mice and rats [158,275,277279]. Despite its absence in the medulla, adrenal cortical expression of Star may be regulated to some degree by the adjacent chroman cells. Co-culture experiments report increased steroid and Star expression up to 10-fold over cortical cells alone [280282]. CNS expression is relatively widespread in mice and includes the hypothalamus [276]. Expression in the placenta is noted for mice at 9.5 dpc [283] and rat trophoblasts from 11.0 dpc to birth [155]. Placental expression is reported in pigs [264] and cows, as well [284]. Human Fetal adrenal gland expression of StAR is present in the FZ and TZ from 14 to 40 wpc, in the DZ at 24 wpc [285] and then persists for life in the same tissues as mice. StAR is located on chromosome 8p11.2 and a pseudogene on chromosome 13 has low expression levels [286]. No StAR is reported in human placenta or brain, indicating a species-specic expression dierence exists [286,287]. StAR is reported to be unnecessary for placental steroidogenesis in humans [225].

Wnt4 Mouse Wnt4 initially appears at 9.5 dpc in the urogenital ridge and then persists in the genital ridge at 11.0 dpc, along with the related Wnt6. Expression from the urogenital ridge extends into the developing AP and is noted to colocalize with Sf1 at 12.5 dpc [179,194]. XX gonadal expression is continued while dierences in gonad expression are obvious between 11.5 and 12.5 dpc when male gonads undergo a decrease in expression but female levels remain unchanged [179,194,288]. Wnt4 also has a strong presence in the developing meso- and metanepheric kidney. In the developing CNS, Wnt4 expression is evident in Rathkes pouch and infundibulum at 12.5 dpc, where it provides critical signaling for pituitary development through 14.5 dpc [194], but rapidly disappears around 17.0 dpc. Sox9/SOX9 Mouse Expression of Sox9 is evident at 9.0 dpc in the urogenital ridge and continues expression into the primitive gonad where it assumes a sex-specic prole by 11.5 dpc [193,288290]. Sox9 is not expressed by PGCs [70]. Nuclear expression in testis, where it becomes restricted to Sertoli cells between 12.5 and 13.0 dpc, is higher than in ovary [290,291]. Levels then decrease dramatically by 14.5 dpc [288]. Sox9 can be detected in the adult mouse [291] and rat Sertoli cells, but not in mature ovary [290]. No expression is detectable in any zone of the adrenal cortex. Humans Early development emulates mouse expression patterns. Sertoli cell levels drop at or around 18 wpc after gonadal dierentiation has occurred [245,292]. SOX9 pattern suggests a transfer from cytosolic to nuclear compartmentalization during sex dierentiation around 67 wpc prior to onset of MIS expression in Sertoli cells, but remains cytosolic in the XX gonad [272]. No expression is found in adult adrenal cortices. Couptf/COUPTF Mouse Couptf1 or Ear3 expression is considered to be generally constitutive in early embryos and expressed at signicantly lower levels than Couptf2 or Arp1 [44].In vivo expression is reported only for the placenta [293], but may be more a reection of tissue-specic expression levels and sensitivity of techniques for proteins versus mRNA detection. Couptf2 expression by IHC documents its presence at 11.5 dpc in the developing CNS and urogenital ridge at high levels, followed by

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hypothalamic expression at 14.5 dpc [294,295]. RNA expression is reported at 14.5 dpc in the primitive adrenal cortex and dierentiated gonads of both sexes at 15.5 dpc [296]. A closely related gene, mRIP140, is expressed at 12.5 dpc in mouse placenta [297] and appears to be crucial for successful embryoblast implantation [298]. Human COUPTF1 expression is noted in the adrenal cortex [299,300]. COUPTF2 analysis by IHC is reported as a nuclear protein within both FZ and TZ cells at 16 wpc to 8 wpp [301]. Levels in the ZG increase over those in the ZF/ZR from 7 months to 8 years of age and are then are dramatically reduced at or around adrenarche. Adrenal medullary expression is positive [259,301], in addition to testicular (moderate Leydig and low Sertoli), ovarian (moderate thecal and low granulosa), uterine and syncytiotrophoblast (placental) cells [293,302]. Alien/ALIEN (also known as Cops2, TRIP15) This gene is ubiquitously expressed as a subunit of the highly conserved COP9 Signalsome (CSN), which is involved in signal transduction and phosphorylation [303 305]. It is expressed in both mouse and humans [249,306]. The Cops2 subunit functions as a nuclear corepressor in numerous tissues, including during development [303,307]. Alien is known to interact with the NR0B1 protein and has an adrenal-specic transcript [308]. NCOR (Nuclear co-receptor), SMRT (Silencing mediator of retinoic and thyroid receptors) This set of alternatively spliced genes is expressed in mice and humans [249,306]. These are co-regulatory genes and are reported to be expressed by RNA analysis of the adrenal gland [299,300,309]. Pref-1 (also known as pre-adipocyte factor-1) Mouse/rat This gene was originally cloned from adipocytes and has a rat homologue termed ZOG (Zona Glomerulosa protein). This protein is expressed in the rat AP at 14.5 dpc where it is restricted to the ZGZF interface (TZ) [208]. It is coexpressed with Cyp11b1 until 20.5 dpc, when Cyp11b2 (P450aldos) expression is initiated in the ZG. Expression persists after birth in the TZ and throughout the medulla [194]. Adult expression is documented in the adrenal cortex in adult rats [310,311]. Pomc-derived peptide (N-Pomc) This is the N terminal fragment (amino acids 152) derived from the Pomc precursor polypeptide and acts

as the ligand for the Acth-R (Mcr-2) located in the adrenal cortex [312315]. Pomc is expressed at 10.5 dpc in the developing murine diencephalon and appears in the derivative adenohypophysis at 12.5 dpc [256]. Pomc is synthesized in corticotropes of the anterior, and melanotropes of the intermediate, pituitary lobes in rodents [15]. Expression is primarily in the anterior pituitary in species that have vestigial or no intermediate lobes, including humans [15]. This fragment is highly conserved between species [314] and is produced from Pomc by AsP enzymatic action in adrenocortical cells. AsP protein is limited to the TZ [313,316] where it removes the c-Msh sequence from N-Pomc [317]. Interaction with its receptor stimulates cortical mitogenesis and aldosterone production, as shown in vivo by adrenal enucleation and hypophysectomy experiments [15,236,317]. Pomc expression is upregulated fteen times normal following adrenalectomy, but not after hypothalamic destruction [15]. P450 enzymes These enzymes are considered to be the downstream targets and functional products of the steroidogenic network in the HPAG [9,70,75,87,98,189,196]. A normal expression prole is mandatory for homeostasis and is often adversely impacted when Nr0b1/NR0B1 and/or its network partners are defective [27,32,54,73,106, 108,112,146,194,318]. Functionally, the end result of ontogenetically directed zonation and expression levels of P450s are intricately linked. These enzymes are the direct eectors of adrenal suciency/insuciency and/or aberrant masculinization/feminization [71,75,87,104, 118,121,123,157,179,189,245,319]. Figs. 1 and 3 indicate the functional relationship and zonal restriction of several critical cytochrome oxidase enzymes. P450scc (also known as P450 side chain cleavage enzyme, CYP11A, P45011A1) Mouse This enzyme converts cholesterol to pregnenolone in the mitochondria [17,87,319]. No expression is noted for mice and rats in the AGP, but is produced by the AP at

Fig. 3. P450 enzymes and zone-specic end products. Legend. +, positive expression; , negative expression; ZG, zona glomerulosa; ZF, zona fasiculata; ZR, zona reticularis.

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10.5 dpc and in the undierentiated gonad at 11.0 dpc [255]. It is quickly restricted to the testis in 12.5 dpc in mice [274,320] and by 14.5 dpc in rats [258]. Ovarian expression is initiated at 14.5 dpc [274]. Adrenocortical expression persists throughout gestation, but becomes more restricted to the periphery over time [320]. P450scc is produced in all zones of the adult cortex [98,109], including the TZ [112,204] in addition to the sex steroid producing (Leydig and thecal), plus the granulosa and corpus luteal cells. Expression is reported to be more abundant in the ZF/ZR region than ZG [257]. Expression also occurs in the placental trophoblasts and uterine deciduas in mice at 4.58.5 dpc [287] and in rat placental trophoblasts [287,321]. Human FZ expression may be present as early as 7 wpc [38]. Strong protein expression is noted from the FZ and TZ from 14 to 40 wpc, but DZ production does not start until 23 wpc (the last trimester) [187,285]. This pattern persists through birth [257] and on into the postpartum period. Comparable results are seen in rhesus monkeys, but with an earlier onset of DZ secretion [187]. Placental expression of P450scc by syncytiotrophoblasts is noted in the last half of gestation in humans [322,323] and in baboons [239]. P450c17 (also known as CYP17) Mouse This gene encodes the 17a-OH/17, 20 lyase enzyme, which converts pregnenolone and progesterone to DHEA and androstenedione in the ER [87,263,319]. This enzyme is generally considered repressed in the rodent adrenal cortex and, therefore, not a primary participant in cortical steroidogenesis [256]. Expression is well documented in gonadal steroidogenic (Leydig and thecal) cells [98,109,194,256]. However, these investigations also report adrenal cortical transcript expression as early as 12.5 dpc and in testis at 16.5 dpc, with postpartum persistence in the ZF/ZR. Since gene product analysis results are unavailable, the functional signicance of these ndings is unknown [194,256]. Species dierences are known, as no P450c17 could be demonstrated in guinea pig adrenals [257]. This enzyme is produced by trophoblasts in rat placenta at 10.5 12.5 dpc [287,324], as well as in pigs [325]. Human FZ expression is shown by IHC as early as 41 dpc, but functionality has not been demonstrated at this early stage [37]. FZ expression is well established at 52 dpc [37,38] and followed by TZ expression at 18 wpc. P450c17 is not produced by the fetal DZ [12] and adult cortical expression is limited to the ZF and ZR [87,187,319].

3bHSD (also known as 3b-hydroxysteroid dehydrogenase) Mouse/rat Onset of endogenous expression is late in rats, with no presence in the ZG at birth but increasing to high levels in the ZF at 14 dpp and appearance in the ZR at 20 dpp [326]. ZG levels drop signicantly after puberty but ZR levels continue to rise until at least 90 dpp. Type IV isoenzyme is expressed in the mouse and rat placenta [327]. Substantial confusion exists in isoenzyme nomenclature designation between species due to the sequence of their cloning. The adrenal-specic form in mouse, rat, hamster, monkey and cow is identied as Type I in these species and is equivalent to Type II in man. A novel Type VI form has been recently cloned in mouse placenta [283,287]. Human The type II isoenzyme (human) is adrenal-specic and is present in all adrenocortical cells within the SER and mitochondria where it converts pregnenolone to progesterone and DHEA to androstenedione [18,263,319]. 3bHSD protein may be present in the FZ in small amounts at 50 dpc [38] but is clearly evident in the FZ, TZ, and DZ by 24 wpc [12,285] with a corresponding pattern in rhesus monkeys [187]. Fetal DZ levels exceed TZ amounts [187] and are 35X greater than FZ expression by mid- [13] to late gestation [12]. Decreasing levels in the ZR are associated with initiation of adrenarche after 8 years of age (early puberty) [20]. Examination of adult cortex reveals high ZG and ZF and low ZR expression [21,326]. Other isoenzyme forms are present, including Type I isoenzyme which is expressed by the placenta [159,239,327], as well as in other nonsteroidogenic tissues [87]. P450c21 (also known as 21-hydroxylase, CYP21) Mouse This enzyme is required for conversion of progesterone to deoxycorticosterone (DOC), precursor for mineralocorticoids in the ER [87,263,319]. The onset of expression is dened at 16.5 dpc as multifocal patches in the developing adrenal cortex and progresses throughout development of all three adult zones as radiating stripes [109,224,232]. Expression is absent in testis, ovary and pituitary, but is reported in other areas of the CNS [131]. Placental presence is reported in rats [324] and pigs [325]. Human Developmental studies show positive expression in the FZ and TZ at 14 wpc, with additional expression in the DZ starting at 24 wpc [285] through gestation.

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Placental expression is also documented [328]. DZ and TZ levels exceed FZ amounts by midgestation [12]. P450c19 (also known as Aromatase, P450arom, CYP19) Mouse This enzyme converts C19 steroids DHEA and DHEA-S to estrogens in cytoplasmic microsomes [98], and is expressed in fetal rat AP at 11.5 dpc and fetal cortex at 18.5 dpc [125,328]. Adult expression persists in the ZF/ZR, as well as steroidogenic gonadal (Leydig and thecal) cells [109,143]. Aromatase converts testosterone to estradiol. Reports documenting P450arom transcription in fetal cortex and testis of mice do not demonstrate concurrent enzymatic activity [75,328]. CNS expression is seen, but does not include the pituitary gland [131]. Endometrial and placental presence is reported for rats [324] and pigs [325]. Human Endometrial and placental presence is reported for humans [328]. Aromatase deciency is reported and allows fetal- and placental-derived androgen accumulation to virilize mother and/or fetus [329,330]. P450c11B1 (also known as 11b-Hydroxylase, P450c11) Mouse This enzyme is expressed as early as 9.0 dpc in the urogenital ridge by RNase protection [331]. Expression in the undened adrenal cortex is positively documented by IHC at 16.0 dpc and by RT-PCR in the testis and ovary at 18.5 dpc [75,125]. Adult expression patterns limit the presence to the ZF/ZR of mice and rats [98,109,263,332]. Species dierences are noted, as low level ZG expression is present in the bovine adrenal cortex [263]. Human Expression is limited to the TZ by midgestation, but has moderate expression in the DZ and FZ by late gestation [12]. Adult expression is relegated to the ZF and ZR. P450c11B2 (also known as P450aldos, Cyp11B2, aldosterone synthase) Mouse This enzyme converts corticosterone to aldosterone in the ZG mitochondria, and is not detectable at the transcriptional level until after 14.5 dpc in the adrenal cortex. Expression is evident after zonation and is solely restricted to the ZG [70,98,109,331] and continues to increase until before puberty in rats [88]. Interspecies variations are known, as the bovine adrenal gland utilizes P450c11B2 for synthesis of both aldosterone and cortisol from ZG and ZF, respectively [263].

Human Expression is evident in the ZG after zonation [70,109]. Any defect impairing mineralocorticoid production does not aect ZF/ZR glucocorticoid synthesis [87]. Therefore, ACTH regulation is maintained and no adrenal hyperplasia occurs. Moderate to severe hyponatremia is present in aected individuals. Expression in the placenta is also reported [323]. Other adrenal cortical expressed genes Midkine Rat. Expressed at 13.014.0 dpc in developing rat adrenal cortex by Northern blot and in situ hybridization and then declines signicantly between 15.0 and 19.0 dpc [333]. Expression is absent in the newborn. Positive expression is noted between 7.0 and 11.0 dpc in the adenohypophysis and other nonsteroidogenic tissues. Only kidney has signicant expression after 15.0 dpc. Gata4/GATA4 Mouse. Mouse adrenals show expression at 15.0 dpc to 60 dpp by RT-PCR, but not by Northern blot [176]. The related Gata6 is coexpressed in adrenal cortex, as evidenced by Northern blotting, RNA in situ and IHC studies [176]. Mouse adrenals demonstrate positive expression by IHC from 15.0 dpc to 2 dpp in the nucleus. Gata4 is also expressed early in gonadal development and possesses a sexually dimorphic prole, with testis levels greater than ovary [334]. Human. GATA4 appears in Sertoli cells after SOX9 localization to the nucleus of XX gonads [272]. RNA expression in the developing cortex is reported by 19 wpc in the FZ [176] but ceases to exist after birth. Gata6 expression is greater in the DZ than the FZ at 19 wpc and ZR levels exceed those of both the ZG and ZF in the adult zones. WNT11. This eukaryotic paralog of one of the many genes in the Drosophila Wingless family is expressed in the urogenital ridge and its successors, the AP and primitive kidney from 33 to 52 dpc in humans [335]. Wnt11 is not present in the adrenal primordial of mice and no mutations are associated with adrenal insuciency. The proposed role for this gene is proliferation and survival of cells during development. Adrenodoxin (also known as Adx) Mouse/Rat. Adx is present in rat adrenals between 12.5 and 18.0 dpc by RNA in situ and IHC [320]. This gene is required for conversion of cholesterol to pregnenolone within the mitochondria for further P450 activity, along with adrenodoxin reductase [306]. Placental expression is known in trophoblasts after 8.5 dpc, and is accompanied by adrenodoxin reductase and Star expression [287].

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MSharp-1 (also known as mDec2) Mouse. Expression is noted at 16.5 dpc in the adrenal cortex after an earlier appearance in the developing brain, eye, heart, limb buds, and other structures [336]. Mcr-2 (also known as ACTH-R, melanocortin receptor2) Mouse. This is a unique form belonging to ve related receptors and is expressed solely in the adrenal cortex, along with Mcr-5 which has expression in additional tissues, including gonads [337,338]. The related Mcr-3 is expressed in the placenta during gestation. Activins and inhibins. These are TGFb superfamily members and are related dimers of a highly conserved group. Activin proteins (A and B) are composed of homodimers (bAbA, bBbB), while inhibins are heterodimers (plus a recently discovered bBbB homodimer) [339,340]. During development, both a and b monomers are expressed in the FZ and DZ by 20 wpc in human adrenals, but technical limitations prevent designating specic activin or inhibin dimer characteristics. They are speculated to regulate growth dynamics of the FZ [209]. Recently, monomeric functional activity is thought to be possible. Activins are considered to be involved in adrenal cortical growth through an apoptotic mechanism [206,322]. Inhibin null mice develop adrenal tumors of the XZ and activin has been shown to induce XZ apoptosis [231]. AHC patients can present with low inhibin protein levels [49]. All subunit types are expressed in adult rat adrenal cortex in the ZG and ZF, but not in the medulla. ACTH regulates the expression of all monomeric subunits and activin may enhance the shift of DHEA-S to cortisol synthesis at birth when the maternal-fetal axis is severed. ACTH actions on adrenocortical cells have also been associated with apoptosis during development [119,205,214,219,341]. Expression in testicular Sertoli and Leydig cells is established [245]. Additional expression is seen in ovaries, placental trophoblasts and uterine deciduas during gestation of humans, monkeys, mouse, and rats [340]. Gonadotropic, corticotropic, and somatotropic activity is thought to be regulated by these molecules, as well as FSH and LH synthesis in both sexes. CIP-2 (also known as Couptf interacting protein) Human. Northern blotting results reveal expression in the adrenal gland, testis and ovary and experimental results show interaction with COUPTF1, COUPTF2, SF1, and NR0B1 [342]. The action is thought to enhance the co-repressor eects of COUPTF1. A related gene, CIP-1 [343] is expressed in adrenocortical tumor tissue with similar capabilities. PCNA (Proliferative cell nuclear antigen) Rat. PCNA is present in the nucleus of rat adrenal cortical cells of the ZG and TZ during S-phase of

replication, but absent in the ZF and ZR [344]. This gene is associated with cortical cell replication and/or replacement. Human. Human adrenal developmental analysis reveals positive expression by IHC at 6 wpc in the central and inner regions of the FZ [345]. Later, expression appears in the DZ at 7 wpc at levels less than the FZ, but increases to equivalent levels by 8 wpc. Prop-1 (also known as Prophet of Pit-1). This gene is expressed in the adrenal cortex and pituitary gland [24] and promotes maturation of cells that synthesize adrenal androgen stimulating hormone (AASH) [23]. This protein directly aects the ZR status. The highest expression levels are during gestation. This gene is impaired in the Ames dwarf (df) mouse model, where homozygous females and most males are infertile. Deciencies in humans can reduce adrenal glucocorticoid [94,96] and DHEA-S [23] production.

Molecular interactions in steroidogenic development The reported interactions for NR0B1 and other members in this network tend to be focused toward two interrelated aspects of HPAG development: steroidogenesis, especially P450 gene regulation, and sex dierentiation. This is largely due to the availability of relevant primary or immortalized cell lines. Pure cell cultures retrieved from embryos prior to mid-gestation, such as AGP cells, are technically dicult to obtain and/or maintain in an undierentiated state. Adult cell lines are fully dierentiated making them less than ideal model systems for developmental investigations. The majority of lines currently in use originate from steroidogenic axis neoplasms. An increasing number of animal models are currently assisting in clarication of cell-specic mechanisms, but are time and labor intensive. Nr0b1/NR0B1 The functional role ascribed for DAX1 is usually as a transcriptional repressor. This is due, in part, to its proposed structure, based on sequence homology, and comparisons with other nuclear receptors [45,346]. Additionally, analysis of AHC mutations supports a lossof-function hypothesis for its repressor actions [8,30,37,45,46,347349]. Transcriptional repression is achieved through the C terminus, which tends to be the commonly aected region among AHC mutations, regardless of whether they are frameshift, missense or nonsense mutations. Target genes for DAX1 are diverse within the HPAG (Table 3). This variety of targets portrays DAX1 as a global regulator of HPAG ontogenesis and steroidogenesis.

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Table 3 Dax1/DAX1 regulatory targets Target gene DAX1 [346,347] SF1 [427,428] AR, ER [429] StAR [136,346,350,376,382,383,430432] P450scc [350] P45011B [37] P450c19 [115,254] P450c17 [37] P450c21 [37] LHb [355,384] HDL-R [351] 3bHSD [350] MIS [252,352] Cofactor(s) SF1[346] (autoregulation) Ncor [347], RIP140 [376] SMRT [429], AR ligand SF1 + RIP140 [376]SF1 [382]

SF1 SF1 [115,254] SF1 SF1 SF1 + Egr-1 [355] SF1 or SREBP-1a SF1 + WT1 + GATA4 + SOX9

The negative regulatory function of DAX1 is accomplished through several dierent mechanisms, including: 1. Direct binding of promoter transcription factor recognition elements (RE) of other genes. This is the case for transcriptional repression or activation for StAR and HDL-R [350,351]. An alternative binding strategy is caused by steric interference with other DNA-bound transactivational proteins [252,352 355]. This mechanism is thought to be eected through an LXXLL motif in the C terminus of NR0B1 [356]. The negative inuences by DAX1 on gene activation can be dose-dependent [254,347, 351,357]. 2. Binding of DAX1 to its own promoter to prevent SF1- And WT1-mediated transactivation using their respective RE sites [250,346]. This suggests a negative feedback autoregulatory loop. However, this is a simplistic explanation, as many cells only express DAX1 or SF1 and WT1 is not expressed in the denitive adrenal cortex or neural HPAG components [249]. 3. DAX1 may serve as a nucleocytoplasmic shuttling mechanism in post-translational regulation of transcriptional factors or other signal transducing gene targets. This proposed action is suggested to occur by sequestration of targets in the cytoplasmic compartment to prevent gene transcription [35,53,184]. A bipartite NLS is dependent on a C terminal sequence, which is aected in AHC patients [53]. Androgen receptor (AR) mediated repression is due to localization by DAX1 to the cytoplasm and is AR-ligand dependent. DAX1 may also function as a regulator of other nuclear receptors [2,358,359]. 4. DAX1 may perform as an RNA-binding protein in the nucleus and cytoplasm with a role in mRNA metabolism and/or processing of target genes [184]. Other transcriptional repressors are known to play critical roles in development, including COUP-TF1 [360], COUP-TF2 [361], NCoR [362], and Alien

[303,307]. Couptf1 and 2 have been shown to repress Sf1 mediated transcription of the Nr0b1 promoter in vitro, with Couptf2 binding more readily than Couptf1 [363]. Both COUP-TF1 and 2 have also been demonstrated to co-repress P450 gene expression [364,365] and these actions may be assisted by NCoR [366]. These co-regulators of DAX1 targets are also likely to be assisted by a complex set of protein co-factors, as noted for other transcriptional regulatory mechanisms [367]. Recently, COUP-TF-Interacting Proteins 1 and 2 were identied and functional binding occurs with COUP-TF1 and 2, SF1 and DAX1 [342,343]. AHC mutations are able to abrogate binding with the co-repressor Alien [308]. These co-regulators are ubiquitously expressed in development and throughout the adult tissues. Their coding regions produce multiple isoforms through alternative splicing mechanisms [367370]. The interactive and repressor domains are generally separate and distributed through the length of the coding sequence [371374]. Distinct isoforms may possess signicantly dierent binding capabilities for interacting with DAX1. For example, the NCoR variant RIP13D1 is known to bind with COUP-TF2 by proteinprotein interactions, but not with NCoR or RIP13 [375]. Furthermore, these co-regulators can alter their repressive roles to become co-activators, dependent on expression levels, cell-specic environments, cell compartmentalization and genespecic targets [366,376378]. An example of reversal of co-factor inuence on Nr0b1 expression is seen when Couptf1 and 2 alone can act as activators, but become repressors in the presence of Sf1 [363]. Collectively, these variables inuence the stoichiometry of individual regulatory complexes. The commonly described role for DAX1 is as a steroidogenic gene antagonist and targets include SF1, StAR, and MIS. The fact that Sf1 protein levels decrease more profoundly in Nr0b1 null than in compound Dax1Y = :Sf1= mice supports these ex vivo based conclusions [112]. Furthermore, P450c21 protein levels are decreased in Sf1= and increased in Dax1Y = following stress testing conrms this assessment in vivo [112]. However, in some experiments, DAX1 alone may be able to act as an agonist for genes that include SF1 [376]. It is assumed that in the context of cellular compartments that there are highly diverse interactions shaped by physiological status. In early adrenal ontogenesis, several key genes are co-expressed, or are in close temporal proximity. DAX1 is shown to be upregulated by Wnt4, and may be assisted by SF1 [379]. WT1 )KTS is a transcriptional activator of NR0B1 via two RE sites in its promoter [250,380]. WT1 is a positive regulator of WNT4 transcription [380]. Wt1 )KTS and SF1 together have the capacity to transactivate the MIS gene, but are antagonized by DAX1 via its disruption of their synergistic binding of MIS promoter DNA [252]. DAX1 similarly can disrupt the synergism between

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GATA4 and SF1 activation of MIS expression in Sertoli cells [352]. SF1 and SOX9 also competitively bind the MIS promoter as transcriptional regulators [381]. Other genes regulated by DAX1 are presented in Table 4. Additionally, DAX1 expression is subject to hormonal aects, including upregulation by PGF2a [382] and decreased by FSH [251], angiotensin II [383] and, possibly, by MIS [34]. Factors that target Nr0b1/NR0B1 for regulation are listed in Table 4. DAX1 is capable of gonadotrope-specic gene regulation in the pituitary for LH [355,384] and is co-expressed with Sf1 during pituitary and hypothalamic development [33]. The negative inuences of DAX1 may be restricted to the HPAG, as a cardioactive steroid gene expression by adrenocortical cells is unaected by overexpression of DAX1 [385]. This assumption is further supported by a lack of dermal pathology in DSS or AHC patients, where DAX1 and SF1 are also co-expressed. Sf1/SF1 NR5A1 and NR0B1 are intricately linked in HPAG development demonstrated by NR5A1 loss-of-function models and mutation patients that exhibit adrenal insuciency. Co-expression of these genes in the AGP and their persistence in both adrenal and gonadal derivatives support their interactive roles. SF1 function is most often as a transcriptional activator, whose target genes include DAX1, StAR, P450s, 3bHSD, and MIS (Table 5). SF1 is more widely distributed throughout the HPAG than DAX1 or WT1, both spatially and temporally. Temporally, it is co-expressed in the AGP with WT1, SOX9, and WNT4, in addition to DAX1. SOX9 is known to upregulate NR5A1 expression [291]. Signicantly, SF1 has the capability to autoregulate its own transcriptional activity [386]. Targets of SF1 include the orphan nuclear receptor SHP1, which is closely related to DAX1 and is also co-expressed in the adrenal cortex [387,388]. Other target genes in the HPAG include those coding for oxytocin [389,390], MC2-R (ACTH-R) [337,391] and the gonadotropin LHb [392,393]. Just as for DAX1, co-factors are involved in SF1-mediated gene regulation. Known co-factors associated with SF1
Table 4 Regulatory factors for Nr0b1/NR0B1 Regulatory protein DAX1 [346,347] SF1 [135,405,433,434] [317,363,379,435] Wnt4 [379] WT1 )KTS [250,380] COUPTfI [363] FSH [251] PGF2a [382] Angiotensin II [383] MIS [34] Cofactor(s)

Table 5 Nr5a1/NR5A1 gene regulatory targets Target gene DAX1 [135,317,363,379,405,433435] WT1 [252] SF1 [386] StAR [136,395,431,436438] P450scc [439442] P45011B [440443] CYP19 [444446] CYP17 [394,447] P450aldos [442,448] CYP21 [449] 3bHSD [327,430] MIS [450] MIS-R [451] SF1 [386] (autoregulation) GnRH-R [133] MC2-R [452,453] LHb [392,393] aGSU [454] Oxytocin [389,390] Prolactin-R [455] Inhibin a [456] HDL-R [457]

include COUPTF1 for oxytocin expression [389] and CYP17 [394]. CBP/p300 potentiates transcription of P450scc by SF1 [170] and C/EBP is an SF1 coactivator for StAR [395]. TCF-4, a transcription factor in developing and preadipocyte, synergizes with b-catenin to activate NR5A1 expression [396]. Coincidentally, CBP/ p300 is also a transcriptional activator of WT1 [146]. Post-translational modication of SF1 is an additional regulatory mechanism and is accomplished through phosphorylation of specic residues [138,397]. This modication of SF1 protein can reduce its transactivational capacity [364]. StAR, which is also a direct target gene for SF1 and GATA4, is also subject to phosphorylation during mitochondrial membrane cholesterol transfer [103,279]. Wt1/WT1 The role of WT1 is intercalated with those of SF1 and DAX1 during adrenocortical development. WT1 targets

Action on Nr0b1/NR0B1 Decrease Increase Increase Increase Decrease Decrease Increase Decrease Decrease

SF1 (autoreg.) Wnt4 [379], b-catenin [435]

SF1 [363]

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relevant to HPAG dierentiation include the genes encoding WNT4 [380], inhibin a [380,398], MIS, and AR [399]. WT1 expression is not noted in neuroectodermalderived cells of the HPAG. WT1 presence does not continue beyond AGP dierentiation in the adrenal cortex, but expression does persist in the gonadal Sertoli cells [195]. WT1 targets itself for autoregulation, both in a positive [380,400,401] and negative [148,400] manner. The cell model ndings are supported by isoform in situ expression data [401]. These reported ndings must be viewed with the caveat that cell culture results are ex vivo and only representative of selective aspects of the endogenous cellular machinery. Phenotypic data from patients and animal models are crucial for comparison and interpretation. It is important to consider that the outcome of these interactions is often aected by the extent and strength of expression for at least one component. Gene dosage is also important for many of these interactions, as sex-reversal is well documented for hemizygous mutations in DAX1 (DSS) [5], or heterozygous mutations in WT1 (Denys-Drash and Frasier Syndromes) [146,402,403], WNT4 [76,77], SOX9 (campomelic dysplasia) [292,404], P450scc [104], and SF1 [71,72]. Additionally, not all cells that express DAX1 co-express SF1 and vice-versa [34,45]. In vivo interactions Transgenic mouse models are an important resource for evidence of likely in vivo regulatory mechanisms for these genes. An 11 kb Nr0b1 promoter reporter transgenic indicated Sf1 mediated expression is located 4 kb upstream of the transcriptional start site [405]. Essentially all Nr0b1/NR0B1 RE sites described for mechanistic investigations in cell lines are located within a few hundred base pairs of the start codon. Mutational analysis of this transgenic reporter conrmed that the RE sites previously characterized ex vivo are required for full strength expression, but are not sucient and adequate alone in vivo. An Nr0b1 transgene reporter required at least 11 kb of upstream genomic promoter for gonadal expression, but failed to produce expression in adrenal, hypothalamus or adenohypophysis [121]. These results are concordant with our own eorts to express a Nr0b1-GFP reporter constructs using up to 6 kb of promoter sequence in mice and murine ES cells (Clipsham et al., unpublished). A 50 kb Sf1 promoter and partial coding region GFP fusion reporter resulted in uorescence only in the adrenal cortex and VMH. GFP was not expressed in gonadotropes, granulosa cells or corpora lutea [406]. An approximately 600 bp Sf1 promoter transgene was able to express b-galactosidase only in gonads and was not expressed in adrenals or hypothalamus [152]. Together, these transgenic models emphasize the complex regulation of target genes and

the potential hazards of relying on minimal promoter construct results. Despite a lack of complete understanding of the in vivo regulatory elements in genes of the steroidogenic network, these animal models do provide key insights that these genes share common, critical pathways. However, substantial evidence suggests that they may function in parallel, rather than in series, during HPAG ontogeny. Nr0b1 KO mice express Sf1 and do not have adrenal hypoplasia [112], while Nr5a1 KO mice express Nr0b1 [33,112,135]. Sf1= mice fail to show compensatory adrenal cortical hypertrophy after unilateral adrenalectomy and do not show increased PCNA expression compared to wt mice [140], further indicating a crucial role for Sf1 in adrenocortical cell proliferation. Wnt4 KO mice express Wt1 identical to wt mice [179]. Wt1 KO mice fail to express either Nr0b1 or Nr5a1 indicating that Wt1 is likely to function directly upstream of them, but also upstream of Wnt4. Interestingly, a Wt)KTS KO mouse model does not express Sox9 or Mis in the developing gonad, does possess a female Nr0b1 expression prole in XY gonad and does exhibit increased apoptosis in the primordial gonad [154]. The recent application of physiological testing of complex animal models is also contributing greatly to this premise of parallel and interconnected pathways. For example, the enhanced ACTH responsiveness of the adrenal cortex in Nr0b1 KO over that of Sf1 decient mice is documented by Mcr-2 upregulation and increased corticosterone levels in the face of relatively lower ACTH plasma levels [112]. These data give substantial reason to consider approaching these developmental mechanisms as a dynamically coupled complex network with exible pathways between HPAG genes. This is elegantly demonstrated by two observations in mouse models for Sf1: (1) both P450scc and P450c17 are expressed by the placenta of Sf1 null and Sf1= mice which is a blastocyst derived, extraembryonic tissue [124]; (2) both p450scc and Star, which are well-documented targets of both NR0B1 and SF1, are not downregulated by reduced Sf1 dosage or deletion of Nr0b1 in mice, indicating that they may function through a compensatory mechanisms independent of both transcription factors [112]. Conversely, hypophysectomized rodents have reduced P450scc, but normal Sf1 adrenocortical expression levels, suggesting that other gene products are positioned between Nr0b1 plus Sf1 and their P450 targets and have signicant impact on their regulation. Finding these secondary and/or compensatory pathway(s) will help dene the composition and complexity of the network(s). Further considerations for mechanistic interpretation of HPAG Nr0b1/NR0B1 interactions are their inuence on MIS in gonadal dierentiation where considerable eorts have been focused. Sertoli cell dierentiation is considered a hallmark in sex dierentiation and marks a

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departure from the female pathway [193,241,407409]. Since SF1 and DAX1 regulate MIS and its receptor, these genes are considered crucial for pre-Sertoli cell and Mllerian duct dierentiation. The perception that u DAX1 might be an anti-testis gene is reinforced by observation of XY sex-reversal by overexpression of Nr0b1 in mice [121]. This possibility is supported by observations of reduced Sox9 levels present in the Nr0b1Y = mouse model by 12.5 dpc [118]. The fact that Nr0b1 is expressed in a sexually dimorphic pattern in both the AGP [33,252] and developing adrenal cortex [117] further this argument. While Nr0b1 null mice sustain defective spermatogenesis, Leydig cell hyperplasia and vascular anomalies [108,114,118] that mirror the HH patient phenotype [47,49,245], the requirement of Nr0b1/NR0B1 for Sertoli-based testis determination is directly refuted by the presence of dierentiation of all three basic testicular cell-types. Mis expression is only absent when the Nr0b1Y = genotype is crossed onto the unique M. poschiavinus genetic background [118]. Additional evidence contradicting the anti-tesis hypothesis is presented by a Mis null mouse that develops fully descended testes with viable sperm and whose lack of reproductive capacity is due to misdirected sperm into persistent Mllerian ducts [409]. It is noteworthy that u both of these KO models share a common phenotype of Leydig cell hyperplasia. This suggests that both genes might be regulators of Leydig cell replication rather than Sertoli cell development.

Network complexity, robustness, and vulnerabilities In a comprehensive assessment of the separate issues involved in HPAG and adrenal cortical development addressed in the preceding sections, a common connection is noted between the ex vivo and in vivo ndings, regardless of technique or cell origin. HPAG development rests on both the composition and complexity of its developmental gene network. These networks are complex and inherently robust, providing resistance to many perturbations [44,249,410]. Mutations constitute a system attack and the robustness of the system seeks to prevent a failure [411,412]. System attack may be internal, such as by gene mutation or allelic dierences, or external via environmental changes. These networks are dynamically coupled between DNA, RNA, and protein components. Transcription factors (proteins) act as connectors between evolutionarily conserved RE motifs (DNA) to dictate specic gene transcription (RNA). These interactions at a local level produce a binary output, regardless of individual composition or complexity, resulting in activation or repression of transcription (an outcome that is plus or minus). These local events are dened by the cell-type, stage of dierentiation, cell compartment, and genotype. Therefore, it is

critical for the output that all contributors (network components) be present and at the correct concentrations for testing of the process to prevent aberrant signaling. Signal transducers, including nuclear receptors, can traverse cell compartments, and local output signals will seek to join other output signals downstream to regulate subsequent regional and time-sensitive interactions. As seen by the inuence of endocrine substances on the developing HPAG, local output signals may impact far distant network nodes [249]. This is the coupling aspect of the network that crosses cell and tissue types in a time-dependent manner in ontogenetics. The result of numerous local interactions, termed tactics by Burstein [413], is to amplify their inuence from within the network to become more overarching strategies, which possess the inherent quality of adaptability. The developmental inuence of these strategies is based on the dynamic component of dynamically coupled networks. One of the fundamental properties that allow this combined power of tactics to exceed their own sum is the incorporation of feedback loops within complex, or scale-free, networks [414]. This situation is documented for DAX1 [250,415], SF1 [386], and WT1 [148,380,400,401]. This phenomenon creates threshold ranges, as each individual interaction proceeds forward based on the specic coalition of components of a specic tactic. Each of the protein components binds either their target DNA and/or each other with a componentspecic binding capacity. Feedback loops at some point either maintain, upregulate or diminish this state by altering the composition of the assembled complex and the binding status of its own proteins. Ultimately, this eect either maintains, upregulates or diminishes its regulatory inuence, changing the output signal. This self-regulating ability sets threshold levels for gene activity within limits established by transcriptional complex stoichiometry. Self-regulation fosters network adaptability, but if self-regulation cannot compensate for the perturbation, for example by a mutation, a phenotype results [410,416,417]. The dierence between the two states, i.e., the normal or mutant phenotype, is not an absolute, but rather a continuum. Identical mutations in individuals in the same family may result in a normal or a disease phenotype showing that the primary mutation is inuenced by genetic and environmental modiers [416]. Disease phenotypes are often dicult to reconcile with their corresponding genotypes, as the contributing susceptible component(s) responsible for modifying some tactic or strategy may be located at any step. A critical tactic may not be an obvious network component or a minor tactical change may reverse a strategy. Evolution has selected for adaptation or robustness for survivability [410]. This is necessary for organisms since input from the environment is unpredictable and

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inuences network output at all levels [413]. An example of a remote, environmental input with a signicant impact on the steroidogenic network is demonstrated by the connections that extend to the extraembryonic tissues during gestation. Due to developmentally normal fetal adrenal enzymatic deciencies, the placenta metabolizes DHEA-S and other fetal zone steroids until the last trimester [197]. At term, the fetal adrenal cortex only produces 5075% of the fetal plasma cortisol, and the maternal and placental environmental inputs are critical for survival. Loss of this input becomes life threatening for infants with NR0B1 mutations at birth. Insights into the robustness of any network, including the one responsible for HPAG development, are gained not only from identifying the potentially critical nodes, but also by understanding network behavior [410]. Network behavior is predicated by the network topology (scale-free, random, small-world, etc.), diameter (largest number of nodes across the systems shortest route), direction of each connection (vector value), weight of each vector and feedback loops (Fig. 4). It is well known that DAX1, SF1, WT1, StAR, and WNT4 are highly vulnerable nodes in the developmental network for the HPAG based on patients and animal models with loss-of-function mutations. The inuences of the various network partners on these critical nodes is demonstrated by the dierent phenotypes that result when animal models are created in dissimilar genetic backgrounds. This is evident when evaluating the Nr0b1Y = mouse sex-dierentiation characteristics in 129SV/j vs. M. poschainus strains [108,118]. Likewise, adrenal phenotypes vary for medullary migrational position in Nr0b1Y = vs. Nr0b1Y = :Sf1= mice [108,112]. The perplexing questions underlying the mysteries of adrenal cortical commitment and zonal lineage determination have not been dened. The use of totipotent and dierentiated ES cells for cell modeling and for creation of genetically manipulated mouse models, in conjunction with the better characterization of classic cell lines will aid in this goal. ES cells with their capacity for dierentiation (plasticity) without sustaining critical component failures (adaptability) makes them an extremely powerful tool for dissecting the composition, complexity and behavior of HPAG ontogenic networks. ES cells have the potential to provide a model system to test dynamic network(s) at each progressive developmental stage. ES cells may possibly reveal yet undesignated pleiotrophic functions for Nr0b1 that are completely independent of those currently identied in dierentiated cell lines. The observations that mice with targeted deletion of Sf1 unexpectedly retain expression of Nr0b1 [123], and the coexistence of Sf1 and Ahch in the ICM of embryos [44] are an intriguing consideration for the use of ES cells to study these phenomena. This warrants consideration for the use of ES cells to study these phenomena. The need for a better model system,

such as ES cells, for investigations into HPAG development, is further supported by several reports that question the existence of steroidogenesis in early, wildtype mouse embryos [418420], while other evidence suggests this is a bona de embryonic phenomena [321,421,422]. Interestingly, several reports document de novo expression of murine steroidogenic genes in extraembryonic and placental tissues induced by embryo implantation starting at 4.5 dpc [283,287,321]. Future investigations of Ahch using ES cells in various states of dierentiation could have profound benets for understanding novel functions, as well as genotypephenotype correlations in AHC or DSS. The functional understanding of network topology is expanding. It is observed that common modules occur repeatedly between widely dierent species [410]. The organization of these modules possess a great deal of uniformity, whether it is in, for example, metabolism or sex determination, allows for similar types of information transfer between nodes using conserved motifs, tactics and strategies [423]. Scale-free systems incorporate these conserved nodes into strategies through hierarchical connections and are well recognized for their application in modeling for complex genetic traits [410,417,424]. More recently, there has been a new appreciation of the role that other network types, such as small world networks [425], can have in this information transfer process. It is now recognized that the inhomogeneous connections of scale-free networks may also possess clustering properties [426]. A rigorous examination of scale-free networks resulted in discovery of small world subsystems within the larger topology of scale-free networks [425,426]. This is not incompatible with complex network functionality because the small clusters, with their more homogeneous and tightly linked nodes, are connected into larger, less cohesive groups. Overall, the network remains free of scale for its pathways and uses a hub-and-spoke global topology. A prime example of this small world arrangement is seen for the P450 cytochrome oxidase genes, which comprise a small cluster in the potential steroidogenic network (Figs. 1 and 5) [44]. If the metabolic pathways are traced for steroidogenesis in the adrenal cortical zones and gonadal cells, there is a distinct small world connectivity pattern (Fig. 1). It is accepted that perturbations in the critical nodes of HPAG histogenesis (e.g., AHC) will aect some of these pathways. The phenotypes for Nr0b1Y = and Nr0b1Y = : Sf1= KO mice have some altered enzyme levels, but not to the same extent for most nodes as a direct attack on the P450 gene cluster (e.g., P450c21 deciency). This is not true of all nodes, as the topology of small world systems allows for some adaptability through higher connectivity for some nodes, but is not as robust overall as for scale-free networks. The behavior resulting from this type of connectivity is seen by the expression of

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Fig. 4. Proposed network for ontogeny in steroidogenic tissues. Node connections are shown as weighted vectors with feedback loop vectors in color (adapted from Clipsham et al. [249] with new information).

Fig. 5. Small-world network topology of cytochrome P450 enzymes and related proteins within scale-free steroidogenic gene network.

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P450scc and P450c17 in the placental tissues of Sf1= and null mice and a lack of downregulation of Star in reduced Sf1-dosage or Nr0b1-deleted mice, even though they both have reduced corticosterone levels. It is the behavior of these clusters relative to the overall coupled dynamics of the system that produce the phenotype as the nal output for the organism. Despite the obvious pathogenesis, the fact that the organism retains the majority of recognizable characteristics and metabolic pathways is a tremendous display of global network adaptation at the level of the organism. This discussion highlights the dialectic for DAX1 vs. Ahch mutations. Numerous cases are conrmed for complete genomic deletion in humans and none have been found spontaneously, or articially generated, in mice possibly due to embryonic lethality. This poses the question; Why do humans appear to have an adaptable network and mice do not? The answer is obviously extremely complex, and undoubtedly includes the fact that the dierences in ES culture environment vs. the input contributions of the maternal and placental environment on the developmental network(s) may be underappreciated. The ability to pose this question and the possibility through systems biology to begin to formulate approaches to answer this question are intriguing and exciting.

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Acknowledgments This research was supported by P30 HD34610 and R01 HD39322.
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