Table Of ContentC H A P T E R
1
Introduction
Maria I. New
Department of Pediatric Endocrinology, Mount Sinai School of Medicine, New York, NY, USA
The history of steroid disorders is very old. The first family with possible non-classical steroid 21-hydroxy-
published report of a cadaver with ambiguous genitalia lase deficiency, as the features of impaired fertility and
whose sex was changed from female to male was given consanguinity are frequently observed in families with
to me by one of my mentors, Alfred Bongiovanni [1]. The this deficiency.
cadaver was a male who was found at autopsy to have As time went on, steroid endocrinology made fre-
ovaries, uterus, and Fallopian tubes, and the adrenals quent and important advances (Fig. 1.2). Early studies
were extremely large. These findings were considered of steroid disorders investigated steroid metabolites in
by the dissector, de Crecchio, to be wondrous and mys- the urine, and later used serum hormone levels to iden-
terious. This publication by de Crecchio is considered by tify the disorder. Thereafter, steroid disorders benefited
many to be the first report of a female with congenital greatly from the advent of molecular biology.
adrenal hyperplasia raised as a male. Indeed, this book demonstrates that each steroid dis-
In a later publication [2], I speculated that the story order causing both clinical and biochemical abnormali-
of a female pope [3,4], Pope Joan, referred to in various ties in patients now has a genetic basis. The genes for
publications [5,6], could have been a female virilized by each step in steroidogenesis have been mapped and
congenital adrenal hyperplasia who presented herself as cloned, and the mutations in the gene causing the dis-
a male and became a pope. However, after much read- order have been described. In addition, the structural
ing I concluded that the story of Pope Joan was a leg- biology of the protein resulting from the mutation in the
end and not history because the case was reported only gene has been reported for many of the disorders.
400 years after her presumed death in 800 A.D. While it The authors of the chapters herein are pioneers and
was clearly possible to have a written report in 800 A.D., experts in the various genetic disorders presented in this
nothing about Pope Joan appeared until 400 years after book. They are not the sole contributors to this field, but
her death (she was killed by a crowd who witnessed the they are my teachers and I owe much of what I have
birth of her child while in procession from St. Peters to learned to them. I wish to thank all the great scholarly
the Lateran). This subject has been treated by several scientists who made this book possible.
authors as an interesting fact [7]. Indeed, one of the tarot I wish to thank the NIH and the Genesis Foundation
cards is of Pope Joan. for the support of my research.
However, when I studied the Old Testament and was Finally, I owe a great debt of gratitude to my primary
taught the history of the Jews by the great historian, mentor, Dr. Ralph Peterson, who is an unsung hero of
Salo Wittmayer Baron, I realized that the ancient pedi- steroid endocrinology and who inspired me to develop
gree demonstrated consanguinity (Fig. 1.1). Congenital this book.
adrenal hyperplasia is an autosomal recessive disorder,
which occurs more frequently in consanguineous fami-
lies. Abraham’s wife, Sarah, was his niece. She was the
daughter of his dead younger brother, Haran. Sarah
was infertile and did not bear Abraham a son until she Maria I. New MD
was 99 years old. This history could be construed as a Editor-in-Chief
1
Genetic Steroid Disorders. http://dx.doi.org/10.1016/B978-0-12-416006-4.00001-6 Copyright © 2014 Elsevier Inc. All rights reserved.
2 1. INTRODUCTION
I Terach
Reuman
Nachor Haran
II
Hagar Abraham Sarah Keturah
Lot
III 6 4
Ishmael Isaac Milcha Sarah
IV Bethuel 7
Machaluth
V
Rebecca Laban
Moab Ammon
Zilpan
Bilhan
VI
Esau Jacob Leah
Rachel
VII
Gad Asher Dan Naphtali Reuben Simeon Levi Judah Issaschar Zebulun Dinan Joseph Benjamin
FIGURE 1.1 Pedigree of Abraham and Sarah. Source: the Old Testament of the Bible.
1. INTRODUCTION 3
RIA’s for Levine, Dupont, &
serum New: HLA linkage New et al.:
hormones to 21OH gene led
no statistical difference in
Simpson & Tait: developed to the mapping of
CAH children in gender
Isolate and synthesize the gene on
bbeehhaavviioorr, ccooggnniittiioonn,
aldosterone Childs et al.: 21OHD chromosome 6p career, marital status,
autosomal recessive White et al.: memory, or medical
Hench & Kendell: Cortisone inheritance outcomes in those treated
Mutations in
treatment for rheumatoid arthritis and not treated prenatally
21OH gene
with dexamethasone
New et al.:
SSaarrrreetttt:: low aldosterone New et al.: Van Wyck & New:
cortsisyonnthee astiz Mede rke excretion in SW CAH firdsita gpnreonsaistal AdrenaCleAcHtomy for Lin-Su et al.:
growth
CAH is an Bongiovanni & Bongiovanni: Urban et al.: hormone in
autosomal Clayton: measure discovered short stature in CAH patients even treatment of
rreecceessssiivveettrraaiitt uurriinneepp’ttrriiooll 33ββ-HHSSDD wwhheennttrreeaatteedd CAH
1930 1940 1950 1960 1970 1980 1990 2000 2010
Reichstein: Jailer: SV CAH Eberlein & Forest et al.: Speiser & New:
DDOOCCssyynntthheessiizzeedd ccaauusseeddbbyyeennzzyymmee BBoonnggiioovvaannnnii:: FFiirrssttpprreennaattaall ggeennoottyyppeeooffYYuuppiikk
Nobel Prize defect of 21OH Discovered 21OHD treatment of CAH Eskimos
defect
Wilkins & Bartter: Speiser & New:
started treatment of Prader & Sieberman: high frequency of non-classical
CAH with cortisone discovered lipoid adrenal 21OHD CAH
hhyperpllasiia
White et al.: Hirvikoski et al.:
Crigler: clones gene and describes
short-term
treatment of SW CAH with salt, Pang: spot test for structure of 21OH
memory loss in
DOC, pellets, and IM cortisone 17OHP on paper later used for
screening New et al.: CAH children
prenatally treated
nnoonn-ccllaassssiicc2211OOHHDDCCAAHH
wiitthh
New & Blizzard: describe Rosler: Pollack et al.: dexamethasone
(later refuted
urinary aldosterone need for Florinef even prenatal diagnosis of CAH
see above)
excretion in SV CAH by HLA from amniocentesis
FIGURE 1.2 Advances in steroid endocrinology.
References [4] N ew MI, Kitzinger ES. Pope Joan: A recognizable syndrome. J Clin
Endo Metab 1993;76:3–13.
[1] d e Crecchio, Luigi. Sopra un caso di apparenzi virili in una donna. [5] d e Mailly, Jean. Chronica Universalis Mettensis (1254) the fable of
Morgagni 1865;7:154–88. Pope Joan first appears in written form. In: Monumenta Germaniae
[2] N ew MI. Ancient History of Congenital Adrenal Hyperplasia. In: Historica: Scriptores, vol. 24. Hannover; 1879. p. 502–26.
Ghizzoni L, Cappa M, Chrousos G, Loche S, Maghnie M, editors. [6] P olanus Fr Martin. Chronicon Pontificum et Imperatum, AD 1265.
Pediatric Adrenal Diseases, Endocr Dev. Basel: Karger; 2011. In: Pardoe R, Pardoe D, editors. The Female Pope. Wellingborough,
p. 202–11. England. Crucible: The Mystery of Pope Joan; 1988.
[3] D ’Onofrio Cesare. La papessa Giovanna: Roma e papato tra storia e [7] B occaccio Giovanni. De Claris Mulieribus 1350. Litterarischer
leggenda. Romana Società Editrice 1979:286. Verein; 1995. p. 341.
C H A P T E R
2
Adrenal Development
Yewei Xing*, John C. Achermann†, Gary D. Hammer**
*Department of Internal Medicine, University of Michigan, 109 Zina Ptcher Pl, Ann Arbor, MI 48109, USA,
†Clinical and Molecular Genetics Unit, UCL Institute of Child Health, 30 Guilford Street, London, WC1N 1EH, UK,
**University of Michigan, Millie Schembechler Professor of Adrenal Cancer, Director, Endocrine Oncology Program,
Director, Center for Organogenesis, 109 Zina Pitcher Place, 1528 BSRB, Ann Arbor, MI 48109-2200, USA
INTRODUCTION (E) 10.0 in mice), the adrenogonadal primordium (AGP)
is first distinguished by expression of the essential tran-
The adrenal glands comprise two distinct endocrine scription factor SF1/Sf1 (Ad4BP, NR5A1) [2–5]. By 44 dpc
organs: the inner medulla and the outer cortex. The inner in humans (E10.5 in mice), the AGP separates into two
medulla is made up of neuroectodermal cells derived distinct tissues, the adrenal primordial and the gonadal
from the neural crest and produces the catecholamine hor- primordial tissues. This process is accompanied by migra-
mones norepinephrine and epinephrine, which are cru- tion of neural crest cells through the fetal cortex to estab-
cial for stress responses. The outer cortex is derived from lish the medulla and formation of a mesenchymal capsule
the mesoderm and synthesizes steroid hormones that are around the fetal cortex (48–52 dpc in humans, E11.5–E12.5
essential to maintain fluid and electrolyte balance, modu- in mice), which represents the formation of fetal adrenal
late intermediary metabolism and regulate inflammatory gland [6] (Fig. 2.1). In the second phase, as encapsula-
processes. Steroidogenesis in the adrenal cortex is mainly tion progresses (beginning the 20th week of gestation in
regulated by trophic hormones controlled by the hypothal- humans), the formation of the adult cortex (or so-called
amus–pituitary endocrine axes [1]. Adrenal organogenesis definitive zone) is initiated [7,8]. The human fetal zone
and development of adult steroidogenesis are carefully histologically regresses at birth, while the mouse fetal
orchestrated by action of a number of gene products. zone (X zone) regresses during puberty in males or at the
Although the pattern of development differs somewhat in time of first pregnancy in females [9–11]. The third phase
diverse primates, the same genes appear to regulate the represents the homeostatic phase during adult life, when
basic developmental program in all mammalian species. the adrenal gland is maintained by stem/progenitor cell
Most basic laboratory research is done in mice, in which repopulation throughout the lifespan. Each of the three
prenatal development occurs within a compressed period phases will be detailed individually as below (Fig. 2.1).
of approximately 19 days and in which adrenals at birth
are considerably less developed than in their human coun-
Fetal Adrenal Gland
terparts. This chapter describes the contributions of genes
responsible for the proper development of the adrenal The early precursor population of both the adrenal
cortex, as well as how an understanding of adrenal gland cortex and the gonads comprises the AGP, a population
disease provides novel fundamental insights into the regu- of cells located in the coelomic epithelium. The AGP can
lation of adrenal development and steroidogenesis. be detected as early as embryonic day 11.5 (E11.5) in rats,
E10.0 in mice or 28–30 dpc in humans, by expression of
steroidogenic factor 1 or adrenal 4 binding protein (Sf1,
ADRENAL ORGANOGENESIS Ad4BP, NR5A1; hereafter Sf1) [2], which is essential for
adrenal development and a key regulator of steroidogenic
Adrenal gland organogenesis can be divided into three pathway gene expression [3,4,12]. The AGP first appears as
discrete histological phases. In the initial phase (28–30 a thickening of the coelomic epithelium between the uro-
days past conception [dpc] in humans, embryonic day genital ridge and the dorsal mesentery. Each AGP contains
5
Genetic Steroid Disorders. http://dx.doi.org/10.1016/B978-0-12-416006-4.00002-8 Copyright © 2014 Elsevier Inc. All rights reserved.
6 2. ADRENAL DEVELOPMENT
FIGURE 2.1 Overview of developmental stages of the adrenal gland. (A) Different stages and ages for mice adrenal organ development.
(B) Representative symbols for different tissues in the adrenal gland. (C) Lineage of different types of cells in the adrenal gland during develop-
ment. Modified with permission from Wood MA, et al. Fetal adrenal capsular cells serve as a progenitor cell niche for steroidogenic and stromal adrenocortical
cell lineages in mice. Development.
a mixed population of adrenocortical and somatic gonad is mainly caused by the enlargement of the fetal zone
progenitor cells. Sf1-positive AGP cells delaminate from (FZ), while the outer neocortex zone does not change
the epithelium and invade the underlying mesenchyme significantly in size. At this stage, the human fetal adre-
of the intermediate mesoderm. The majority of these nal produces large amounts of the steroid dehydroepi-
cells migrate dorsolaterally to form the gonadal anlagen, androsterone (DHEA), which is then converted by the
whereas a subset of the AGP that expresses higher levels placenta to estrogens that are necessary for the mainte-
of Sf1 migrates dorsomedially to form the adrenal anlagen, nance of normal pregnancy [10,15]. By the 20th week of
settling ventrolateral to the dorsal aorta [2,13]. gestation, a new functional zone referred to as the defin-
At about 48 dpc in humans (E11.5–E12.5 in mice), itive zone (which later develops into adult zona glo-
neural crest cells migrate from the dorsal midline just merulosa (ZG) and zona fasciculata (ZF)) is identified.
lateral to the neural tube to the area where the adrenal Throughout the fetal period, the size of the definitive
cortex is developing [6,14] and differentiate to form the zone remains constant, and the fetal zone constitutes
catecholamine-producing chromaffin cells of the adre- the majority of the gland. According to Johannisson, the
nal medulla, which persist as discrete islands scattered fetal adrenal is one of the largest organs in humans at
throughout the adrenals until birth. Meanwhile, the term (0.2% of the total body weight, almost one-third of
adrenal gland starts to separate from surrounding mes- the size of the kidney), with 80% of the gland composed
enchyme and becomes encapsulated by the formation of of fetal zone cells [16].
a fibrous layer overlying the developing cortical cells. In the mouse, proliferating cells are observed in a
The whole process is largely complete by about 52 dpc scattered pattern throughout the adrenal gland up to
(E14.5 in mice) [7]. day E13.5 [17,18]. At later time points, these proliferat-
ing cells assemble in the periphery of the adrenal gland
[19]. The prenatal adrenal cortex is composed of fetal
Transition from a Fetal to a Definitive Adrenal
adrenal cells surrounded by a second group of cells that
Cortex
develop to form a densely packed structure, the defini-
Rapid growth of the fetal adrenal cortex begins from tive (adult) cortex. The hypothesis that adrenal precur-
the first developmental stage. For the human fetal sor cells of the fetal zone give rise to the definitive/
adrenal gland, the increase in the weight of the gland adult cortex is supported by experiments performed by
ADREnAl ORgAnOgEnEsis 7
Zubair et al. using a FAdE-cre mice model [20]. As men- cells, called the X-zone [23,24] that degenerates by apo-
tioned before, Sf1 is the critical factor for proper adrenal ptosis at puberty in males and after the first pregnancy
organogenesis and is required for steroidogenic func- in females [25,26]. Recent studies suggest that the X-zone
tion in both the fetal and adult adrenal cortex. Zubair is the fetal adrenal zone [20].
et al. identified a fetal adrenal enhancer (FAdE) that
directs Sf1 expression solely in the fetal cortex. Dur-
Adrenal Homeostasis and Stem Cells
ing fetal adrenal development, a transcription complex
containing the homeobox protein PKNOX1 (Prep1), After the functional adult zones are established, they
homeobox gene 9b (Hox) and pre B cell leukemia tran- are maintained throughout life by stem cells or pro-
scription factor 1 (Pbx1) initiates fetal zone expression genitor cells located in the adrenal gland (reviewed
of SF1 by binding to the FAdE region, which is later in references [27–29]). Ever since the 1950s, numer-
maintained through autoregulation by Sf1 itself [20]. ous studies have provided evidence that cells from the
This enhancer is not utilized to activate Sf1 expression capsule/ subcapsular region of the adrenal gland grow
in the definitive cortex. However, by breeding a Rosa26 centripetally inward to repopulate the adrenal cortex
mouse with a transgenic mouse harboring a Cre-recom- (reviewed in Kim et al., 2009 [28]). Several studies show
binase gene driven by a basal Sf1 promoter and FAdE that most proliferating cells are located in the outer layer
enhancer, investigators were able to lineage-trace the of the mature adrenal gland [19,30,31], suggesting the
fate of fetal adrenal cells during the development pro- stem/progenitor cells reside in or directly under the
cess. This study shows that control of Sf1 expression capsule of the cortex. However, all the above studies
through the FAdE is only active before E14.5, at which have been primarily conducted using histology and pro-
time the fetal cortex begins to regress; however, all the liferation markers. In 2010, new genetic data emerged
adult cortex cells are derived from FAdE-expressing to support this hypothesis. Three laboratories inde-
cells of the fetal adrenal. On the contrary, the adrenal pendently provided evidence that the sonic hedgehog
capsule and the medulla were not reported as contain- (Shh) signaling pathway is essential for adrenal gland
ing FAdE-derived cells. Moreover, using tamoxifen- development and maintenance [32–34]. An established
inducible FAdE-cre mice, definitive cortex staining is factor that is involved in the development of vertebral
only observed when tamoxifen is administered early in organ systems and regulation of both embryonic stem
embryogenesis (E11.5–E12.5). Sequentially later admin- cells and adult tissue stem cells, Shh was shown to be
istration of tamoxifen results in only fetal zone inner present in the adrenal gland at E11.5, primarily in the
expression of LacZ. If given after E14.5 or after birth, subcapsular region of the adrenal cortex. It co-localizes
no LacZ-positive cells can be observed, which is con- with Sf1 in cortical cells of the subcapsular region but
sistent with the absence of FAdE activity at later stages. not in differentiated ZG or ZF cells, which express
These results suggest that the fetal cortex gives rise to both Sf1 and markers of fully differentiated steroido-
the definitive/adult cortex. genic cells (i.e. Cyp11b1, Cyp11b2). Mice in which Shh
Following birth, substantial remodeling of the adre- is ablated ( specifically in Sf1-expressing cells) revealed
nal gland occurs: the chromaffin cell islands coalesce to marked adrenal hypoplasia, decreased proliferation,
form a rudimentary medulla; fetal cortical cells regress; and a depleted capsule. On the other hand, observa-
and the adult cortex begins to differentiate into zones. tions in a tissue-specific Shh-knockout mouse show that,
Studies in humans show that the fetal zone regresses despite a decrease in size, the adrenal glands maintain
by cell apoptosis; the number of apoptotic nuclei in proper zonation, which suggests that Shh does not have
the inner fetal zone increases with advancing gestation a role in the initiation of differentiation. Together, these
and is maximal during the first postnatal month. The data imply that the Shh pathway is actively involved
fetal zone completely disappears by the third postna- in proliferation and maintenance of the adrenal cortex.
tal month in humans [11,21]. In conjunction with fetal Lineage-tracing studies show that descendents of Shh-
zone regression, the definitive zone of the adrenal cortex expressing cells do express adrenocortical differentia-
forms discrete functional compartments (the outer ZG tion markers (Cyp11b1 and Cyp11b2), suggesting that
and the inner ZF). The most inner zona reticularis (ZR) Shh-positive cells may serve as progenitor cells for the
arises around the age of 6–8 years and growth accelerates adrenal cortex. Further studies provide evidence that
until puberty. Like the FZ, the ZG produces DHEAs and the adrenal capsule could be the adrenocortical stem/
is thus hypothesized by some to originate from residual progenitor cell niche by focusing on a downstream acti-
fetal zone cells after birth. Zonation is only completed vator of the hedgehog pathway, Gli1 [32–34]. In contrast
around age 12 years, with the final differentiation of ZG to Shh-expressing cells, Gli1-expressing cells locate spe-
to ZF and ZR of the gland [22]. While a similar devel- cifically in the adrenal capsule and do not express Sf1.
opmental process occurs in rodents, the mouse adre- This subpopulation of cells is capable of giving rise
nal cortex does not contain a ZR and does not produce to Sf1-expressing, differentiated adrenocortical cells.
DHEAs. It does contain an inner region of eosinophilic Whether capsular Gli1-positive, Sf1-negative cells or
8 2. ADRENAL DEVELOPMENT
subcapsular Shh-positive, Sf1-positive cells (or both During development, as the outer definitive zone of the
populations) serve as adrenal stem/progenitor cells and cortex begins to emerge, ACTH participates in the regu-
what the specifics of the relationship between those two lation of steroidogenesis, cell differentiation, and cell
cell population is remains unclear [29]. Further studies growth [43–45].
are needed to determine the exact mechanism of Shh sig-
naling to the Gli1-positive cells and what factors might Insulin-like Growth Factor 2 (IGF2)
regulate Shh signaling in the adrenal gland. These stud- Both IGF1 and IGF2 are mitogens expressed in
ies have been in agreement with cell migration models the adrenal gland. Upon binding to the dimeric/
of adrenocortical zonation, which propose that a pre- heterodimeric cell surface receptors (insulin receptor
cursor population differentiates first into ZG cells and [IR] or IGF receptor 1 [IGF1R]), IGFs can induce auto-
then changes its phenotype as it migrates centripetally phosphorylation of the intracellular part of the receptor,
into the ZF and the ZR. Studies using chimeric ani- which leads to activation of two downstream signaling
mals and analyses of the expression pattern of a steroid pathways, Ras/MEK/ERK and PI3K/AKT [46,47]. Tar-
21-hydroxylase (CYP21)-β-galactosidase transgene have get genes participate in many cellular processes, includ-
been interpreted to support this cell migration model ing cell cycle activation. Although both IGF1 and IGF2
that is important for adrenal homeostasis [35,36]. As are present during the process of adrenal development,
cells reach the medullary boundary (ZR) an increased IGF2 is generally considered to play a key role in early
frequency of cell death is observed [21,37–39]. Whether fetal development [48–50]. In support of the above state-
these two different experimental observations that cells ment, infusion of IGF2 does cause a significant increase
of both the Sf1-positive fetal zone and cells of the Sf1- in adrenocortical growth during embryogenesis. In
negative capsule give rise to Sf1-positive adult adreno- adulthood, a switch in expression levels makes IGF1
cortical cells reflect two temporally distinct lineages of the dominant IGF in the adrenal where it functions as
the definitive cortex or reflect a singular developmen- a regulator for postnatal growth maintenance. Although
tal and homeostatic mechanism of adrenal growth is IGF2 levels are much lower in adults compared to the
unclear. fetal adrenal cortex, its expression is restricted to the cap-
sular region, which coincides with the stem/progenitor
location in the gland [48]. Furthermore, IGFs, along with
MOLECULAR MECHANISMS THAT
FGF, have been shown to be essential factors for stem
REGULATE ADRENAL DEVELOPMENT
cell niche [51,52], supporting a potential role of IGF2 in
adrenal stem/progenitor cell maintenance as well.
As mentioned above, adrenal development is a highly
orchestrated process, controlled by numbers of auto-
Adrenal Steroids
crine, paracrine and endocrine factors at different loca-
As the major products from the adrenal glands,
tions and different stages. The following section will
adrenal steroids are generally considered to exert their
focus on talking about the function of known factors that
endocrine effects by working on other organs. How-
interplay in adrenal organogenesis and homeostasis.
ever, a study by Gummow et al. suggested that gluco-
corticoids can also stimulate an intra-adrenal negative
Hormonal Regulation of Adrenal Development feedback loop via activation of the dosage-sensitive sex
reversal, adrenal hypoplasia critical region, on chromo-
Adrenocorticotropic Hormone (ACTH)
some X, gene 1 (Dax-1) expression [53]. A glucocorticoid-
As a major component in the hypothalamic–pituitary–
dependent synergy between Sf1 and the glucocorticoid
adrenal axis, ACTH is a 39-amino acid peptide secreted
receptor (GR) leads to activation of Dax-1, while ACTH
from the anterior pituitary gland under the control of
stimulation disrupts the formation of this complex by
corticotropin-releasing hormone (CRH) [40]. By binding
abrogating SF1 binding to the Dax-1 promoter. These
to the transmembrane receptor MC2R, ACTH exerts its
data indicate that, instead of the being considered solely
effect mainly by activating downstream Ras/MEK/ERK
as a product, steroids may also play an important role in
signaling pathways [41]. It has been established that,
adrenal regulation.
although during the first trimester of human pregnancy
adrenal growth does occur independently of ACTH,
after about week 15 of gestation ACTH starts to play an Transcriptional Regulation of Adrenal
essential role in the morphological and functional devel- Development
opment of the adrenal gland [42]. Part of its functions is
Sf1
suggested to be through the stimulation of locally pro-
duced growth factors such as insulin-like growth factor Sf1 (Nr5a1) is a 462-amino acid orphan nuclear
2 (IGF2) and fibroblast growth factor beta (FGFβ) [10,15]. receptor, which is required for adrenal and gonadal
MOlECulAR MECHAnisMs THAT REgulATE ADREnAl DEvElOPMEnT 9
development and regulates a large group of adrenal and before hypoplasia, which may be explained by the fact
gonadal target genes. In the mouse, Sf1 is first expressed that Dax-1 represses adrenocortical steroidogenesis (or
in the urogenital ridge at E9 and subsequently in the differentiation) by blocking Sf1 activity [72]. Research
adrenal primordium at E11, and adrenocortical cell at results from the Morohashi and Parker groups further
E13 [54,55]. Homozygous deletion of Sf1(–/–) in mice indicate that Dax-1 may act as the repressor for FAdE
results in adrenal agenesis and death shortly after birth activity in the adrenal gland during fetal-to-adult transi-
owing to steroid deficiency, while heterozygous mice tion [20]. All the above data support the hypothesis that
(Sf1+/–) exhibit smaller adrenal glands and signifi- loss of adrenal function in DAX-1-deficient patients is
cantly decreased steroid production and steroidogenic caused by a depletion of an aging adrenocortical progen-
gene expression, suggesting a dose-dependent effect itor reserve [73–75]. Combined with the recent finding
of Sf1 on adrenal development and differentiation that Dax-1 is highly expressed in the mouse embryonic
(steroidogenesis) [56,57]. Similar phenotypes are reported stem cells, while knockdown of this gene results in
in human patients bearing mutations in the SF1 gene increased differentiation, it is reasonable to propose that
[58,59]. Dax-1 plays an important role in maintenance of stem/
As discussed above, different enhancers can regulate progenitor cell pluripotency.
the activity of Sf1 at individual developmental stages, Accordingly, the regulation of Dax-1 expression is
providing a switch mechanism in organogenesis and predicted to be a dynamic process balancing progeni-
zonation. FAdE directs Sf1 expression solely in the fetal tor renewal and adrenocortical differentiation/ste-
cortex. During fetal adrenal development, Prep1, Hox roidogenesis. Dax-1 transcription in the adrenal gland
and Pbx1 initiate fetal zone expression of Sf1 by binding is activated by Sf1 in cooperation with paracrine Wnt
to the FAdE region, which is later maintained through signaling together with glucocorticoids synthesized in
Sf1 autoregulation [20]. Lineage-tracing studies using the differentiated adult cortex [53]. Conversely, ACTH,
the FAdE enhancer show that control of Sf1 expression the well-established glucocorticoid stimulator, has
through the FAdE is only active before E14.5. After that, been shown to remove Sf1 complexes completely from
Sf1 is maintained by the action of a proposed definitive the Dax-1 promoter, thus leading to effective shut-
adult adrenal enhancer (DAdE) and definitive zones down of Dax-1 transcription. This process would be
start to form. However, all the adult cortex cells are predicted to be permissive to the response of the Sf1-
derived from FAdE-expressing cells of the fetal adrenal, positive progenitor cells to ACTH and the subsequent
while the capsule and the medulla do not show a FAdE initiation of steroidogenesis [53]. In mice embryonic
origin. Sf1 regulates the transcription of a vast array of stem cells, Dax-1 has also been proven to be activated
genes involved in sex determination and differentiation by luteinizing hormone releasing hormone (LRH) effi-
(WT1, DAX1, AMH, AMHR), reproduction (GNRHR, ciently [76].
GSUA, LHB, FSHR, oxytocin, PRLR, INSL3, inhibin
alpha, Oct3/4), steroidogenesis (ACTHR, STAR, CYP11, Wnt/β-catenin
CYP19, Akr1b7, etc.), and metabolism (HDLR, SHP, The Wnt/β-catenin signaling pathway has been
SRB1, SCP2) by direct/indirect binding to their promoters. intensively studied for its role in embryonic develop-
Depending on the co-activators/co-repressors with which ment, stem cell maintenance, and cell fate determina-
it associates, Sf1 can exert a diverse range of effects on ste- tion in many tissues [77–79]. As in a number of cancers,
roidogenesis and development. Additionally, Sf1 activity β-catenin activating mutations have been identified in
can also be regulated by several forms of post-translational a subset of sporadic adrenocortical adenomas and car-
modification, such as phosphorylation [60–64], acetyla- cinomas [80–82]. Wnt ligands signal by binding to the
tion, and SUMOylation [65–69]. All of these factors make Frizzled cell surface receptor, which, upon activation,
Sf1 a fascinating yet complicated mediator of adrenal will disrupt the cytoplasmic complex composed by
development and homeostatic maintenance. adrenomatous polyposis coli (APC), Axin, GSK3β, and
β-catenin. Once released from the complex, β-catenin
Dax-1
(instead of being degraded by ubiquitin) will move into
The orphan nuclear receptor Dax-1 (Nr0b1) was first the nucleus in the non-phosphorylated form and acti-
cloned as the gene responsible for X-linked cytomegalic vate downstream target genes as a transcription factor.
adrenal hypoplasia congenita. Its expression in the adre- Studies performed in the mouse adrenal revealed that
nal gland is enriched in the subcapsular region, suggest- β-catenin expression and activity is present early in the
ing a potential function of Dax-1 in adrenal maintenance. fetal cortex. However, by E18.5, with the emergence of
As detailed below, DAX-1-deficient patients classically new definitive cortex, β-catenin is restricted to the sub-
exhibit histologic adrenal hypoplasia and resultant adre- capsular region [83]. Studies performed by Kim et al.
nal insufficiency [70,71]. However, some studies also employed cre-lox technology to ablate β-catenin specifi-
demonstrate the presence of a hyperfunctional period cally in Sf1-expressing cells of the adrenal cortex [84].
10 2. ADRENAL DEVELOPMENT
In mice expressing a high level of the Sf1-cre transgene, Considering the fact that Shh signals to Gli-positive
adrenal aplasia is found and mice are embryonic lethal. cells, it has been speculated that Shh in the subcapsu-
Careful examination of these mice showed that normal lar region activates Gli signaling in the capsule cells,
adrenal development continued until E12.5, when adre- that serve to maintain an adrenal progenitor pool in
nal failure became evident precisely when few defini- or under the capsule, thereby regulating the process of
tive/adult cortical cells emerged between the coalescing adrenocortical homeostasis.
capsule and the fetal zone. Intriguingly, in mice bearing
Others
a low expressing Sf1-cre transgene, which continued to
express β-catenin in half of the adrenocortical cells, adre- Wilm’s tumor1 (Wt1) and Cited2 play an important
nal development progressed normally. However, as the role by upregulating Sf1 expression at the stage of sepa-
mice aged (i.e. 30 weeks), a progressive cortical thinning ration of the AGP into gonadal and adrenal regions.
and a decreased steroidogenic capacity was identified While both genes are involved in development of adre-
[83]. This progressive failure of the cortex is hypoth- nals and gonads, they are critical factors for adrenal
esized to be because of the loss of adrenocortical pro- specification [14].
genitor cells. In support of the role of the Wnt/β-catenin Pbx. In the fetal adrenal gland, Sf1 expression is
signaling pathway in maintenance of stem/progenitor regulated by a fetal adrenal-specific enhancer (FAdE)
cells in the adrenal gland, overactivation of this path- located in the fourth intron of Sf1. Transgenic assays
way is frequently observed in adrenocortical carcino- revealed that the activation of FAdE requires binding
mas [80–82,85,86]. Although the exact mechanism of of a Hox–Pbx1–Prep1 complex to a site in the FAdE and
Wnt/β-catenin signaling remains unknown, the fact that that maintenance of FAdE-dependent Sf1 expression
β-catenin and Sf1 can directly activate Dax-1 suggests over time is effected in an autoregulatory manner by
that Dax-1 may be a critical mediator of Wnt action in Sf1 itself [20].
the adrenal cortex. Inhibin α is an atypical member of the TGFβ family
of signaling ligands. Under physiological conditions,
Shh/Gli
it is present in the cortex but not the medulla, with an
Sonic hedgehog (Shh), along with Desert hedgehog inner zone-specific pattern. Inhibin α is expressed in
(Dhh) and Indian hedgehog (Ihh), in mammals are the both adrenocortical carcinomas and benign adrenocorti-
three members of an evolutionarily conserved protein cal adenomas. Following gonadectomy, the adrenal cor-
family. Shh has been shown to play important roles in tex of inhibin-null (Inha–/–) mice undergoes profound
embryonic development, adult stem cell maintenance, remodeling secondary to aberrant luteinizing hormone
and cancer [87–90]. Shh acts by binding to its receptor (LH)-dependent proliferation and gonadal differentia-
Patched1 (Ptch1), and subsequently releasing the inhi- tion of subcapsular adrenocortical progenitor cells. Fur-
bition on the seven transmembrane domain protein ther studies demonstrated that LH signaling specifically
Smoothened (Smo). Activation of Smo changes the ratio upregulates expression of TGFβ2 in the subcapsular
of Gli repressor and activator forms, allowing for vari- region of the adrenal cortex, leading to aberrant Smad3
able downstream effects proportional to the magnitude activation in Inha–/– adrenal glands [91,92]. A switch
of the Hh signal. from predominant expression of Gata6 (endogenous to
Shh starts to express early in rodent adrenal glands. the adrenal cortex) to Gata4 (defines cellular identity in
In the adrenal cortex of embryonic mice, Shh mRNA the ovary) in the Inha–/– adrenal [93] drives both ovar-
expression can be detected as early as E12.5, in the ian theca and granulosa cell lineages in the adrenal, sug-
peripheral adrenocortical cells [87–90] and is main- gesting a role of inhibin α in the maintenance of adrenal
tained in the same pattern throughout embryogenesis versus gonadal fate of the adrenocortical progenitor
[32,33,87,89] and adulthood [32]. However, the major cells. Binding of the synergized β-catenin/Sf1 complex
ligand in the adrenal, Gli1, is expressed primarily in to the promoter region can stimulate inhibin α gene
the capsule. As mentioned above, lineage-tracing stud- expression in rat [94].
ies using an inducible cre system demonstrated that in Pod1 is a transcription factor with the basic helix–
adult mice, both Shh- and Gli-positive cells can give loop–helix (bHLH) motif, and has been shown to play
rise to differentiated adrenocortical cells and thus are crucial roles in cell fate determination and differentia-
considered potential candidates for stem/progeni- tion in a variety of tissues, including the gonads. Loss
tor cells in the adrenal gland. Research on Shh mutant of Pod1 results in increased expression of Sf1 and an
adrenals reported defects in both the capsule and cor- increased number of fetal Leydig cells [95–97]. Although
tex. The mutant capsule was thinner with less prolif- its function in the adrenal cortex remains elusive, the
eration, while the cortex has many fewer cortical cells finding that expression of Pod1 is restricted to the adre-
but normal zonation compared to normal controls. nal capsule [98] and is significantly decreased in adrenal
MOlECulAR MECHAnisMs THAT REgulATE ADREnAl DEvElOPMEnT 11
tumors (Lotfi and Hammer, unpublished data), suggests the cellular senescence of this population resulting
a regulatory role in the adrenal cortex. from the loss of function of the mouse tpp/acd gene
Telomerase is a reverse transcriptase that adds (adrenocortical dysplasia – a component of the telo-
DNA sequence repeats to the 3' end of DNA strands in mere capping complex, shelterin) is rescued in the
the telomere regions. It carries its own RNA molecule absence of P53, albeit at the expense of adrenocortical
and uses it as a template when elongating telomeres, carcinoma [101], indicating a critical role of telomere
which are shortened after each replication cycle. protection in the maintenance of adrenocortical stem/
Telomerase is expressed in embryonic stem cells, progenitor cells. Indeed, adrenocortical carcinomas
allowing cells to divide repeatedly during organogen- display significantly enhanced telomerase activity
esis. In adults, telomerase is only present in cells that compared to benign adrenocortical tumors [102], mak-
need to divide regularly (e.g. in the immune system), ing telomerase a potential marker for malignancy in
but not in normal resting cells. In accordance with pre- adrenal tumor diagnosis [103]. Further study indicates
vious findings on adrenal stem/progenitor cell loca- that hormone levels (estrogen) can inhibit telomerase
tion, the RNA component of telomerase is exclusively activity and thus reduce cell proliferation in the adre-
found under the capsular region of the adult adrenal, nal gland in mice [104].
suggesting the presence of active telomerase in pro- All the factors involved in adrenal gland development
liferating progenitor populations [99,100]. Moreover, have been summarized in Table 2.1.
TABLE 2.1 summary of Factors involved in Adrenal Development, their name, location and Proposed Function
Molecule Adrenal localization/source Proposed function
Nuclear receptor Pod1 Capsule Expressed in the adrenal capsule. Inhibits Sf1 in gonad and
in vitro
Gli1 Capsule Sf1-negative capsular (stem/progenitor) cell gives rise to
underlying cortex
β-Catenin Subcapsular region and fetal cortex Effector of Wnt signaling
Dax-1 Subcapsular region Activated by Wnt signaling and glucocorticoids. Mediator
of undifferentiated state (stem/progenitor cells). Inhibits Sf1
steroidogenesis
Gata6 Subcapsular region Adrenal versus gonadal cell specification. Co-regulator of Sf1-
mediated transcription
Sf1 Cortex Transcription of steroidogenic enzymes. Adrenal cell
specification, proliferation and differentiation (stem/progenitor
cells)
Pbx Cortex Induces Sf1 expression through the FAdE. Required for growth
maintenance of the adult adrenal cortex
Inhibin α Endocrine signal Prevents TGFβ2-dependent initiation of signaling. Gatekeeper
of adrenal cell fate of subcapsular (stem/progenitor) cells
Secreted factor IGF2 Capsule MAPK- and AKT-dependent growth and survival. Stabilization
of β-catenin
Shh Subcapsular region Expressed in subcapsular (stem/progenitor) cells to signal to
Gli1-positive capsular (stem/progenitor) cells. Gives rise to
differentiated cortex
Wnt ligands Ligand-dependent Many Wnt ligands activate subcapsular β-catenin. Contribute
to multipotency of undifferentiated (stem/progenitor) cells
ACTH Pituitary Steroidogenesis. Differentiation (stem/progenitor cells). Sf1
transactivation. Sf1 phosphorylation
Adrenal steroids Cortex Glucocorticoid receptor-dependent Dax-1 transcription
(glucocorticoid)
Enzyme Telomerase Subcapsular region Maintenance of telomeres in rapidly proliferating subcapsular
(stem/progenitor) cells
