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The adrenal cortex is an endocrine organ organized into concentric zones that are specialized to produce specific steroid hormones essential for life. The development and maintenance of the adrenal cortex are complex, as a fetal adrenal is first formed from a common primordium with the gonads, followed by its separation in a distinct primordium, the invasion of the adrenal primordium by neural crest-derived cells to form the medulla, and finally its encapsulation. The fetal cortex is then replaced by a definitive cortex, which will establish zonation and be maintained throughout life by regeneration relying on the proliferation, centripetal migration, and differentiation of several stem/progenitor cell populations whose activities are sex-specific. Here, we highlight the advances made, using transgenic mouse models, to delineate the molecular mechanisms regulating these processes., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Triadimefon is a broad-spectrum antifungal agent, which is widely used in agriculture to control mold and fungal infections. It is considered an endocrine disruptor. Whether triadimefon exposure can inhibit the development of fetal adrenal glands and the underlying mechanism remain unclear. Thirty-two pregnant female Sprague-Dawley rats were randomly divided into four groups. Dams were gavaged triadimefon (0, 25, 50, and 100 mg/kg/day) daily for 10 days from gestational day (GD) 12 to GD 21. Triadimefon significantly reduced the thickness of the zona fasciculata of male fetuses at 100 mg/kg, although it did not change the thickness of the zona glomerulosa. It significantly reduced the serum aldosterone levels of male fetuses at a dose of 100 mg/kg, and significantly reduced serum corticosterone and adrenocorticotropic hormone levels at doses of 50 and 100 mg/kg. Triadimefon significantly down-regulated the expression of Agtr1, Mc2r, Star, Cyp11b1, Cyp11b2, Igf1, Nr5a1, Sod2, Gpx1, and Cat, but did not affect the mRNA levels of Scarb1, Cyp11a1, Cyp21, Hsd3b1, and Hsd11b2. Triadimefon markedly reduced AT1R, CYP11B2, IGF1, NR5A1, and MC2R protein levels. Triadimefon significantly reduced the phosphorylation of AKT1 and ERK1/2 at 100 mg/kg without affecting the phosphorylation of AKT2. In contrast, it significantly increased AMPK phosphorylation at 100 mg/kg. In conclusion, exposure to triadimefon during gestation inhibits the development of fetal adrenal cortex in male fetuses. This inhibition is possibly due to the reduction of several proteins required for the synthesis of steroid hormones, and may be involved in changes in antioxidant contents and the phosphorylation of AKT1, ERK1/2, and AMPK., (Copyright © 2021. Published by Elsevier B.V.)

The adrenal cortex plays pivotal roles in the maintenance of blood volume, responsiveness to stress and the development of gender characteristics. Gender differences of human adrenal cortex have been recently reported and attracted increasing interests. Gender differences occur from the developing stage of the adrenal, in which female subjects had more activated stem cells with higher renewal capacity resulting in gender-associated divergent structures and functions of cortical zonations of human adrenal. Female subjects generally have the lower blood pressure with the lower renin levels and ACE activities than male subjects. In addition, HPA axis was more activated in female than male, which could possibly contribute to gender differences in coping with various stressful events in our life. Of particular interest, estrogens were reported to suppress RAAS but activate HPA axis, whereas androgens had opposite effects. In addition, adrenocortical disorders in general occur more frequently in female with more pronounced adrenocortical hormonal abnormalities possibly due to their more activated WNT and PRK signaling pathways with more abundant activated adrenocortical stem cells present in female adrenal glands. Therefore, it has become pivotal to clarify the gender influence on both clinical and biological features of adrenocortical disorders. We herein reviewed recent advances in these fields., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The adrenal cortex is the main steroid producing organ of the human body. Studies on adrenal tissue renewal have been neglected for many years, but recent intensified research has seen tremendous progress in our understanding of the formation and homeostasis of this organ. However, cell turnover of the adrenal cortex appears to be complex and several cell populations have been identified that can differentiate into steroidogenic cells and contribute to adrenal cortex renewal. The purpose of this review is to provide an overview of how the adrenal cortex develops and how stem cell populations relate to its developmental progenitors. Finally, we will summarize present and future approaches to harvest the potential of progenitor/stem cells for future cell replacement therapies., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The adrenal gland plays a pivotal role in an organism's health span by controlling the endocrine system. Decades of research on the adrenal gland have provided multiscale insights into the development and maintenance of this essential organ. A particularly interesting finding is that founder stem/progenitor cells participate in adrenocortical development and enable the adult adrenal cortex to regenerate itself in response to hormonal stress and injury. Since major advances have been made in understanding the dynamics of the developmental process and the remarkable regenerative capacity of the adrenal gland, understanding the mechanisms underlying adrenal development, maintenance, and regeneration will be of interest to basic and clinical researchers. Here, we introduce the developmental processes of the adrenal gland and discuss current knowledge regarding stem/progenitor cells that regulate adrenal cortex remodeling and regeneration. This review will provide insights into the fascinating ongoing research on the development and regeneration of the adrenal cortex.

It has recently been shown that the loss of the Hippo signaling effectors Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) in adrenocortical steroidogenic cells impairs the postnatal maintenance of the adrenal gland. To further explore the role of Hippo signaling in mouse adrenocortical cells, we conditionally deleted the key Hippo kinases large tumor suppressor homolog kinases 1 and -2 (Lats1 and Lats2, two kinases that antagonize YAP and TAZ transcriptional co-regulatory activity) in steroidogenic cells using an Nr5a1-cre strain (Lats1flox/flox;Lats2flox/flox;Nr5a1-cre). We report here that developing adrenocortical cells adopt characteristics of myofibroblasts in both male and female Lats1flox/flox;Lats2flox/flox;Nr5a1-cre mice, resulting in a loss of steroidogenic gene expression, adrenal failure and death by 2 to 3 weeks of age. A marked accumulation of YAP and TAZ in the nuclei of the myofibroblast-like cell population with an accompanying increase in the expression of their transcriptional target genes in the adrenal glands of Lats1flox/flox;Lats2flox/flox;Nr5a1-cre animals suggested that the myofibroblastic differentiation could be attributed in part to YAP and TAZ. Taken together, our results suggest that Hippo signaling is required to maintain proper adrenocortical cell differentiation and suppresses their differentiation into myofibroblast-like cells., (© Endocrine Society 2020. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The X-zone is a transient cortical region enriched in eosinophilic cells located in the cortical-medullary boundary of the mouse adrenal gland. Similar to the X-zone, the fetal zone in human adrenals is also a transient cortical compartment, comprising the majority of the human fetal adrenal gland. During adrenal development, fetal cortical cells are gradually replaced by newly formed adult cortical cells that develop into outer definitive zones. In mice, the regression of this fetal cell population is sexually dimorphic. Many mouse models with mutations associated with endocrine factors have been reported with X-zone phenotypes. Increasing findings indicate that the cell fate of this aged cell population of the adrenal cortex can be manipulated by many hormonal and nonhormonal factors. This review summarizes the current knowledge of this transient adrenocortical zone with an emphasis on genes and signaling pathways that affect X-zone cells.

The observation of two precursor groups of the early stem cells (Groups I and II) leads to the realization that a first amount of fetal stem cells (Group I) migrate from the AMG (Aortal-Mesonephric-Gonadal)-region into the aorta and its branching vessels. A second group (Group II) gains quite a new significance during human development. This group presents a specific developmental step which is found only in the human. This continuation of the early development along a different way indicates a general alteration of the stem cell biology. This changed process in the stem cell scene dominates the further development of the human stem cells. It remains unclear where this phylogenetic step first appears. By far not all advanced mammals show this second group of stem cells and their axonal migration. Essentially only primates seem to be involved in this special development.

In mammalian cells TPP1, encoded by the Acd gene, is a key component of the shelterin complex, which is required for telomere length maintenance and telomere protection. In mice, a hypomorphic mutation in Acd causes the adrenocortical dysplasia (acd) phenotype, which includes limb and body axis anomalies, and perinatal lethality. p53 deficiency partially rescues limb and body axis anomalies in acd mutant embryos, but not perinatal lethality, implicating p53-independent mechanisms in the acd phenotype. Loss of function of most shelterin proteins results in early embryonic lethality. Thus, study of the hypomorphic acd allele provides a unique opportunity to understand telomere dysfunction at an organismal level. The aim of this study was to identify transcriptome alterations in acd mutant and acd, p53 double mutant embryos to understand the p53-dependent and -independent factors that contribute to the mutant phenotypes in the context of the whole organism. Genes involved in developmental processes, cell cycle, metabolic pathways, tight junctions, axon guidance and signaling pathways were regulated by p53-driven mechanisms in acd mutant embryos, while genes functioning in immune response, and RNA processing were altered independently of p53 in acd, p53 double mutant embryos. To our best of knowledge, this is the first study revealing detailed transcriptomic alterations, reflecting novel p53-dependent and -independent pathways contributing to the acd phenotype. Our data confirm the importance of cell cycle and DNA repair pathways, and suggest novel links between telomere dysfunction and immune system regulation and the splicing machinery. Given the broad applicability of telomere maintenance in growth, development, and genome stability, our data will also provide a rich resource for others studying telomere maintenance and DNA damage responses in mammalian model systems., (Copyright © 2018 Elsevier B.V. All rights reserved.)

This review summarizes key knowledge regarding the development, growth, and growth disorders of the adrenal cortex from a molecular perspective. The adrenal gland consists of two distinct regions: the cortex and the medulla. During embryological development and transition to the adult adrenal gland, the adrenal cortex acquires three different structural and functional zones. Significant progress has been made in understanding the signaling and molecules involved during adrenal cortex zonation. Equally significant is the knowledge obtained regarding the action of peptide factors involved in the maintenance of zonation of the adrenal cortex, such as peptides derived from proopiomelanocortin processing, adrenocorticotropin and N-terminal proopiomelanocortin. Findings regarding the development, maintenance and growth of the adrenal cortex and the molecular factors involved has improved the scientific understanding of disorders that affect adrenal cortex growth. Hypoplasia, hyperplasia and adrenocortical tumors, including adult and pediatric adrenocortical adenomas and carcinomas, are described together with findings regarding molecular and pathway alterations. Comprehensive genomic analyses of adrenocortical tumors have shown gene expression profiles associated with malignancy as well as methylation alterations and the involvement of miRNAs. These findings provide a new perspective on the diagnosis, therapeutic possibilities and prognosis of adrenocortical disorders.

The adrenocortical gland undergoes structural and functional remodelling in the fetal and postnatal periods. After birth, the fetal zone of the gland undergoes rapid involution in favor of the definitive cortex, which reaches maturity with the emergence of the zona reticularis(zR) at the adrenarche. The mechanisms underlying the adrenarche, the process leading to pre-puberty elevation of plasma androgens in higher primates, remain unknown, largely due to lack of any experimental model. By following up fetal and definitive cortex cell lines in mice, we showed that activation of protein kinase A (PKA) signaling mainly impacts the adult cortex by stimulating centripetal regeneration, with differentiation and then conversion of the zona fasciculata into a functional zR. Animals developed Cushing syndrome associated with primary hyperaldosteronism, suggesting possible coexistence of these hypersecretions in certain patients. Remarkably, all of these traits were sex-dependent: testicular androgens promoted WNT signaling antagonism on PKA, slowing cortical renewal and delaying onset of Cushing syndrome and the establishment of the zR in male mice, this being corrected by orchidectomy. In conclusion, zR derives from centripetal conversion of the zona fasciculata under cellular renewal induced by PKA signaling, determining the size of the adult cortex. Finally, we demonstrated that this PKA-dependent mobilization of cortical progenitors is sexually dimorphic and could, if confirmed in humans, account for female preponderance in adrenocortical pathologies., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

The nuclear receptor steroidogenic factor 1 (Sf1, Nr5a1, Ad4bp) is crucial for formation, development and function of steroidogenic tissues. A fetal adrenal enhancer (FAdE) in the Sf1 gene was previously identified to direct Sf1 expression exclusively in the fetal adrenal cortex and is bound by both Sf1 and Dax1. Here, we have examined the function of Sf1 SUMOylation and its interaction with Dax1 on FAdE function . A diffused prolonged pattern of FAdE expression and delayed regression of the postnatal fetal cortex (X-zone) were detected in both the SUMOylation-deficient- Sf1 2KR/2KR and Dax1 knockout mouse lines, with FAdE expression/activity retained in the postnatal 20αHSD-positive postnatal X-zone cells. In vitro studies indicated that Sf1 SUMOylation, although not directly influencing DNA binding, actually increased binding of Dax1 to Sf1 to further enhance transcriptional repression of FAdE. Taken together, these studies define a crucial repressor function of Sf1 SUMOylation and Dax1 in the physiological cessation of FAdE-mediated Sf1 expression and the resultant regression of the postnatal fetal cortex (X-zone)., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)

Context: The human fetal adrenal (HFA) is an integral component of the fetoplacental unit and important for the maintenance of pregnancy. Low kisspeptin levels during pregnancy are associated with miscarriage, and kisspeptin and its receptor are expressed in the HFA. However, the role of kisspeptin in fetal adrenal function remains unknown., Objective: To determine the role of kisspeptin in the developing HFA., Design: Experiments using H295R and primary HFA cells as in vitro models of the fetal adrenal. Association of plasma kisspeptin levels with HFA size in a longitudinal clinical study., Setting: Academic research center and tertiary fetal medicine unit., Participants: Thirty-three healthy pregnant women were recruited at their 12-week routine antenatal ultrasound scan., Main Outcome Measures: The spatiotemporal expression of Kiss1R in the HFA. The production of dehydroepiandrosterone sulfate (DHEAS) from HFA cells after kisspeptin treatment, alone or in combination with adrenocorticotropic hormone or corticotropin-releasing hormone. Fetal adrenal volume (FAV) and kisspeptin levels at four antenatal visits (∼20, 28, 34, and 38 weeks' gestation)., Results: Expression of Kiss1R was present in the HFA from 8 weeks after conception to term and was shown in the inner fetal zone. Kisspeptin significantly increased DHEAS production in H295R and second-trimester HFA cells. Serial measurements of kisspeptin confirmed a correlation with FAV growth in the second trimester, independent of sex or estimated fetal weight., Conclusions: Kisspeptin plays a key role in the regulation of the HFA and thus the fetoplacental unit, particularly in the second trimester of pregnancy.

The adrenal gland is an endocrine organ that plays essential roles in stress responses. This organ consists of two types of tissues, adrenomedulla and adrenocortex, deriving from different embryonic origins. Whereas it is well accepted that the adrenomedulla derives from neural crest cells, the origin of the adrenocortex remains elusive. In addition, the adrenocortex and gonads, two major steroid hormone-producing tissues, have been thought to share the same origin, although the experimental evidence is lacking. In this study, to identify the origin of adrenocortex and to compare it to that of gonads, we scrutinized the medial portion of the coelomic epithelium (CE) after the lateral plate mesoderm has split into two CE components with a concomitant opening of the coelomic cavity in between them. We found that early medial CE consists of a two-cell layer-thick band of epithelial-like cells, the outer and inner CEs. The outer CE faces the coelomic cavity, whereas the inner CE is juxtaposed to nascent blood vessels. Combining direct cell labeling with early molecular markers, we found that outer CE was the origin of the gonad but not the adrenocortex. The adrenocortex, instead, appears to derive from inner CE. Thus, the adrenocortical and gonadal progenitors are already segregated from each other when the coelomic cavity has opened. This study provides a new basis for understanding how the adrenal gland forms and how steroid hormone-producing tissues arise during development., (© 2017 Japanese Society of Developmental Biologists.)

The adrenal cortex is a critical steroidogenic endocrine tissue, generated at least in part from intermediate mesoderm of the anterior urogenital ridge. Previous work has pinpointed a minor role of the FGFR2IIIb isoform in expansion and differentiation of the fetal adrenal cortex in mice but did not address the complete role of FGFR2 and FGFR1 signaling in adrenocortical development. Here, we show that a Tbx18(cre) line mediates specific recombination in the coelomic epithelium of the anterior urogenital ridge which gives rise by a delamination process to the adrenocortical primordium. Mice with conditional (Tbx18(cre)-mediated) deletion of all isoforms of Fgfr2 exhibited severely hypoplastic adrenal glands around birth. Cortical cells were dramatically reduced in number but showed steroidogenic differentiation and zonation. Neuroendocrine chromaffin cells were also reduced and formed a cell cluster adjacent to but not encapsulated by steroidogenic cells. Analysis of earlier time points revealed that the adrenocortical primordium was established in the intermediate mesoderm at E10.5 but that it failed to expand at subsequent stages. Our further experiments show that FGFR2 signaling acts as early as E11.5 to prevent apoptosis and enhance proliferation in adrenocortical progenitor cells. FGFR1 signaling does not contribute to early adrenocortical development. Our work suggests that FGFR2IIIb and IIIc isoforms largely act redundantly to promote expansion of the adrenocortical primordium., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

This study was designed to determine the role of the MEK/ERK1/2 and PI3K/Akt pathways in cortisol production and endothelial nitric oxide synthase (eNOS) phosphorylation (peNOS) in the ovine fetal adrenal in response to long-term hypoxia (LTH). Pregnant ewes were maintained at high altitude (3820 m) for the last 100 days of gestation (dGa). At 138 to 142 dGa, fetal adrenal cortical cells (FACs) were collected from LTH and age-matched normoxic fetuses. Cortisol production and peNOS were measured in response to pretreatment with the MEK/ERK1/2 pathway inhibitor UO126 (UO) and adrenocorticotropic hormone (ACTH) stimulation. UO126 reduced ACTH-stimulated cortisol in both normoxic and LTH FACs. UO126 alone or in combination with ACTH reduced peNOS in the normoxic group, while ACTH alone or ACTH + UO inhibited peNOS in LTH FACs. Additionally, cortisol was measured in response to pretreatment with UO and treatment with 22R-hydroxycholesterol (22R-OHC) or water-soluble cholesterol (WSC) with and without ACTH stimulation. UO126 had no effect on 22R-OHC-treated cells, but reduced cortisol in cells treated with WSC and/or ACTH. Cortisol and peNOS were also measured in response to pretreatment with PI3K/Akt pathway inhibitor Wortmannin (WT) and ACTH stimulation. Wortmannin further increased cortisol under ACTH-stimulated conditions and, like ACTH, reduced peNOS in LTH but not normoxic FACs. Together, these data suggest that in LTH FACs MEK/ERK1/2 does not regulate peNOS but that UO acts downstream from eNOS, possibly at cholesterol transport, to affect cortisol production in LTH FACs, while the PI3K/Akt pathway, along with ACTH, regulates peNOS and plays a role in the fetal adaptation to LTH in FACs., (© The Author(s) 2015.)

The adrenal glands consist of an outer cortex and an inner medulla, and their primary purposes include hormone synthesis and secretion. The adrenal cortex produces a complex array of steroid hormones, whereas the medulla is part of the sympathetic nervous system and produces the catecholamines epinephrine and norepinephrine. In the mouse, GATA binding protein (GATA) 4 and GATA6 transcription factors are coexpressed in several embryonic tissues, including the adrenal cortex. To explore the roles of GATA4 and GATA6 in mouse adrenal development, we conditionally deleted these genes in adrenocortical cells using the Sf1Cre strain of animals. We report here that mice with Sf1Cre-mediated double deletion of Gata4 and Gata6 genes lack identifiable adrenal glands, steroidogenic factor 1-positive cortical cells and steroidogenic gene expression in the adrenal location. The inactivation of the Gata6 gene alone (Sf1Cre;Gata6(flox/flox)) drastically reduced the adrenal size and corticosterone production in the adult animals. Adrenocortical aplasia is expected to result in the demise of the animal within 2 weeks after birth unless glucocorticoids are provided. In accordance, Sf1Cre;Gata4(flox/flox)Gata6(flox/flox) females depend on steroid supplementation to survive after weaning. Surprisingly, Sf1Cre;Gata4(flox/flox)Gata6(flox/flox) males appear to live normal lifespans as vital steroidogenic synthesis shifts to their testes. Our results reveal a requirement for GATA factors in adrenal development and provide a novel tool to characterize the transcriptional network controlling adrenocortical cell fates.

The human adult adrenal cortex is composed of the zona glomerulosa (zG), zona fasciculata (zF), and zona reticularis (zR), which are responsible for production of mineralocorticoids, glucocorticoids, and adrenal androgens, respectively. The final completion of cortical zonation in humans does not occur until puberty with the establishment of the zR and its production of adrenal androgens; a process called adrenarche. The maintenance of the adrenal cortex involves the centripetal displacement and differentiation of peripheral Sonic hedgehog-positive progenitors cells into zG cells that later transition to zF cells and subsequently zR cells., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The adrenal gland consists of two distinct parts, the cortex and the medulla. Molecular mechanisms controlling differentiation and growth of the adrenal gland have been studied in detail using mouse models. Knowledge also came from investigations of genetic disorders altering adrenal development and/or function. During embryonic development, the adrenal cortex acquires a structural and functional zonation in which the adrenal cortex is divided into three different steroidogenic zones. Significant progress has been made in understanding adrenal zonation. Recent lineage tracing experiments have accumulated evidence for a centripetal differentiation of adrenocortical cells from the subcapsular area to the inner part of the adrenal cortex. Understanding of the mechanism of adrenocortical cancer (ACC) development was stimulated by knowledge of adrenal gland development. ACC is a rare cancer with a very poor overall prognosis. Abnormal activation of the Wnt/β-catenin as well as the IGF2 signaling plays an important role in ACC development. Studies examining rare genetic syndromes responsible for familial ACT have played an important role in identifying genetic alterations in these tumors (like TP53 or CTNNB1 mutations as well as IGF2 overexpression). Recently, genomic analyses of ACT have shown gene expression profiles associated with malignancy as well as chromosomal and methylation alterations in ACT and exome sequencing allowed to describe the mutational landscape of these tumors. This progress leads to a new classification of these tumors, opening new perspectives for the diagnosis and prognostication of ACT. This review summarizes current knowledge of adrenocortical development, growth, and tumorigenesis., (© 2015 American Physiological Society.)

We have previously shown that estrogen selectively suppresses growth of the fetal zone of the baboon fetal adrenal cortex, which produces the C19-steroid precursors, eg, dehydroepiandrosterone sulfate, which are aromatized to estrogen within the placenta. In the present study, we determined whether fetal adrenal expression of cell cycle regulators are altered by estrogen and thus provide a mechanism by which estrogen regulates fetal adrenocortical development. Cyclin D1 mRNA levels in the whole fetal adrenal were increased 50% (P < .05), and the number of cells in the fetal adrenal definitive zone expressing cyclin D1 protein was increased 2.5-fold (P < .05), whereas the total number of cells in the fetal zone and fetal serum dehydroepiandrosterone sulfate levels were elevated 2-fold (P < .05) near term in baboons in which fetal serum estradiol levels were decreased by 95% (P < .05) after maternal administration of the aromatase inhibitor letrozole and restored to normal by concomitant administration of letrozole plus estradiol throughout second half of gestation. However, fetal adrenocortical expression of cyclin D2, the cyclin-dependent kinase (Cdk)-2, Cdk4, and Cdk6, and Cdk regulatory proteins p27(Kip1) and p57(Kip2) were not changed by letrozole or letrozole plus estradiol administration. We suggest that estrogen controls the growth of the fetal zone of the fetal adrenal by down-regulating cyclin D1 expression and thus proliferation of progenitor cells within the definitive zone that migrate to the fetal zone. We propose that estrogen restrains growth and function of the fetal zone via cyclin D1 to maintain estrogen levels in a physiological range during primate pregnancy.

An incidental, bilateral, retained fetal adrenal cortex was detected in a male cynomolgus macaque (age, approximately 2.4 y) used in a 4-week toxicology study. Microscopic examination of the adrenal gland cortex zone revealed the presence of additional solid sheets and columns of cells supported by vascular capillary bed and composed of large polyhedral cells with abundant eosinophilic, slightly finely vacuolated cytoplasm that surrounded the entire circumference of the medulla. Nuclei were vesicular, round to oval with prominent small nucleoli. There was no evidence for inflammation or cellular degeneration. Based on the microscopic examination, a diagnosis of retained fetal cortex of the adrenal gland was made. This morphologic change resembles fetal cortex in human infants. To our knowledge, this case description is the first report of a cynomolgus macaque with the rare entity of retained fetal cortex, which should not be misinterpreted as a test article-related change., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

Background: The sonic hedgehog (SHH) pathway plays a key role in rodent adrenal cortex development and is involved in tumorigenesis in several human tissues, but data in human adrenal glands are limited., Objectives: The objectives of the study were to analyze the involvement of the SHH pathway in human adrenal development and tumorigenesis and the effects of SHH inhibition on an adrenocortical tumor (ACT) cell line., Patients and Methods: Expression of SHH pathway components was evaluated by immunohistochemistry in 51 normal adrenals (33 fetal) and 34 ACTs (23 pediatric) and by quantitative PCR in 81 ACTs (61 pediatric) and 19 controls (10 pediatric). The effects of SHH pathway inhibition on gene expression and cell viability in the NCI-H295A adrenocortical tumor cell line after cyclopamine treatment were analyzed., Results: SHH pathway proteins were present in fetal and postnatal normal adrenals and showed distinct patterns of spatiotemporal expression throughout development. Adult adrenocortical carcinomas presented with higher expression of PTCH1, SMO, GLI3, and SUFU compared with normal adult adrenal cortices. Conversely, pediatric ACTs showed lower mRNA expression of SHH, PTCH1, SMO, GLI1, and GLI3 compared with normal pediatric adrenal cortices. In vitro treatment with cyclopamine resulted in decreased GLI3, SFRP1, and CTNNB1 mRNA expression and β-catenin staining as well as decreased cell viability., Conclusions: The SHH pathway is active in human fetal and postnatal adrenals, up-regulated in adult adrenocortical carcinomas, and down-regulated in pediatric ACTs. SHH pathway antagonism impaired cell viability. The SHH pathway is deregulated in ACTs and might provide a new target therapy to be explored.

The lineage relationships of fetal adrenal cells and adrenal capsular cells to the differentiated adrenal cortex are not fully understood. Existing data support a role for each cell type as a progenitor for cells of the adult cortex. This report reveals that subsets of capsular cells are descendants of fetal adrenocortical cells that once expressed Nr5a1. These fetal adrenocortical cell descendants within the adrenal capsule express Gli1, a known marker of progenitors of steroidogenic adrenal cells. The capsule is also populated by cells that express Tcf21, a known inhibitor of Nr5a1 gene expression. We demonstrate that Tcf21-expressing cells give rise to Nr5a1-expressing cells but only before capsular formation. After the capsule has formed, capsular Tcf21-expressing cells give rise only to non-steroidogenic stromal adrenocortical cells, which also express collagen 1a1, desmin and platelet-derived growth factor (alpha polypeptide) but not Nr5a1. These observations integrate prior observations that define two separate origins of adult adrenocortical steroidogenic cells (fetal adrenal cortex and/or the adrenal capsule). Thus, these observations predict a unique temporal and/or spatial role of adult cortical cells that arise directly from either fetal cortical cells or from fetal cortex-derived capsular cells. Last, the data uncover the mechanism by which two populations of fetal cells (fetal cortex derived Gli1-expressing cells and mesenchymal Tcf21-expressing mesenchymal cells) participate in the establishment of the homeostatic capsular progenitor cell niche of the adult cortex.

Developmental signalling pathways are implicated in the formation and maintenance of the adrenal gland, but their roles are currently not well defined. In recent years it has emerged that Sonic hedgehog (Shh) and Wnt/β catenin signalling are crucial for the growth and development of the adrenal cortex. Here we demonstrate that Fibroblast growth factor receptor (Fgfr) 2 isoforms IIIb and IIIc are expressed mainly in the adrenal subcapsule during embryogenesis and that specific deletion of the Fgfr2 IIIb isoform impairs adrenal development, causing reduced adrenal growth and impaired expression of SF1 and steroidogenic enzymes. The hypoplastic adrenals also have thicker, disorganised capsules which retain Gli1 expression but no longer express Dlk1. Fgfr2 ligands were detected in both the capsule and the cortex, suggesting the importance of signalling between the capsule and the cortex in adrenal development., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

MicroRNAs (miRNAs) are small, endogenous, non-protein-coding RNAs that are an important means of posttranscriptional gene regulation. Deletion of Dicer, a key miRNA processing enzyme, is embryonic lethal in mice, and tissue-specific Dicer deletion results in developmental defects. Using a conditional knockout model, we generated mice lacking Dicer in the adrenal cortex. These Dicer-knockout (KO) mice exhibited perinatal mortality and failure of the adrenal cortex during late gestation between embryonic day 16.5 (E16.5) and E18.5. Further study of Dicer-KO adrenals demonstrated a significant loss of steroidogenic factor 1-expressing cortical cells that was histologically evident as early as E16.5 coincident with an increase in p21 and cleaved-caspase 3 staining in the cortex. However, peripheral cortical proliferation persisted in KO adrenals as assessed by staining of proliferating cell nuclear antigen. To further characterize the embryonic adrenals from Dicer-KO mice, we performed microarray analyses for both gene and miRNA expression on purified RNA isolated from control and KO adrenals of E15.5 and E16.5 embryos. Consistent with the absence of Dicer and the associated loss of miRNA-mediated mRNA degradation, we observed an up-regulation of a small subset of adrenal transcripts in Dicer-KO mice, most notably the transcripts coded by the genes Nr6a1 and Acvr1c. Indeed, several miRNAs, including let-7, miR-34c, and miR-21, that are predicted to target these genes for degradation, were also markedly down-regulated in Dicer-KO adrenals. Together these data suggest a role for miRNA-mediated regulation of a subset of genes that are essential for normal adrenal growth and homeostasis.

This study assessed the role of the extracellular signal-regulated kinase (ERK) signaling pathway on the previously observed enhanced cortisol secretion in response to adrenocorticotropic hormone (ACTH) treatment in fetal adrenocortical cells (FACs) from long-term hypoxic (LTH) ovine fetuses. Ewes were maintained at high altitude (3,820 m) from ~40 to 138-141 days gestation when FACs were collected and challenged with either ACTH (10 nM) or 8-bromoadenosine 3',5'-cyclic monophosphate (8-bromo-cAMP, 10 mM) in the presence or absence of the mitogen-activated protein kinase/extracellular signal-regulated protein kinase (MEK)/ERK inhibitor UO126 (10 μM). FACs from age-matched normoxic fetuses served as controls. Media and FACs were collected at selected time intervals after ACTH or 8-bromo-cAMP stimulation for cortisol measurement and Western analysis of ERK1/2 and phospho-ERK1 and -2 (pERK1/2). After ACTH or 8-bromo-cAMP treatment, cortisol production was greater in the LTH group compared with control (P < 0.05). UO126 reduced ACTH and 8-bromo-cAMP-mediated cortisol output in both groups (P < 0.01 vs. ACTH or 8-bromo-cAMP alone). Under basal conditions, ERK1/2 and pERK1/2 were not different between LTH and normoxic fetuses. In response to ACTH or 8-bromo-cAMP treatment, ERK1/2 were not different between groups; however, pERK1/2 were elevated in the LTH FACs compared with normoxic control FACs. ERK1/2 phosphorylation declined following ACTH treatment in the control group, but UO126 had no effect on ERK1/2 compared with untreated levels. Both ACTH and 8-bromo-cAMP treatment resulted in a decline of protein levels. UO126 pretreatment virtually eliminated pERK1/2 expression. We conclude that basal ERK signaling in FACs is necessary for normal cortisol production and sustained pERK in LTH adrenals enhances cortisol production.

The adrenal gland controls a plethora of crucial physiological functions, and dysfunction is associated with severe morbidity. Because of the vital importance of appropriate adrenal function, the development and function of the gland have been intensively studied, and these investigations have revealed fascinating developmental origins and a remarkable remodeling and regenerative capacity in the adult. This chapter, focusing on the adrenal cortex, will describe our current understanding of the development and maintenance of the adrenal gland, which has been advanced over recent years by the use of sophisticated genetic models in the study of both normal function and disease. This work has shed light on the transcriptional networks and signaling pathways involved in development and maintenance of the gland and in its pathology; these are discussed in the light of the wealth of physiological information gathered in studies of human and rodent adrenal development and function., (© 2013 Elsevier Inc. All rights reserved.)

The autonomic nervous system, which includes the sympathetic neurons and adrenal medulla, originates from the neural crest. Combining avian blood vessel-specific gene manipulation and mouse genetics, we addressed a long-standing question of how neural crest cells (NCCs) generate sympathetic and medullary lineages during embryogenesis. We found that the dorsal aorta acts as a morphogenetic signaling center that coordinates NCC migration and cell lineage segregation. Bone morphogenetic proteins (BMPs) produced by the dorsal aorta are critical for the production of the chemokine stromal cell-derived factor-1 (SDF -1) and Neuregulin 1 in the para-aortic region, which act as chemoattractants for early migration. Later, BMP signaling is directly involved in the sympatho-medullary segregation. This study provides insights into the complex developmental signaling cascade that instructs one of the earliest events of neurovascular interactions guiding embryonic development.

Steroidogenic acute regulatory protein (StAR) facilitates cholesterol transfer into the inner mitochondrial membrane in the acute phase of steroidogenesis. Mice lacking StAR (Star(-/-)) share phenotypes with human individuals having congenital lipoid adrenal hyperplasia including compromised production of steroid hormones and florid accumulation of cholesterol esters in adrenal glands and gonads. To define a specific pattern of molecular changes with StAR deficiency, we performed transcriptome analysis of adrenal cells selectively isolated by fluorescent-activated cell sorting at embryonic d 17.5 or 18.5 in seven wild-type (Star(+/+)) or four Star(-/-) mice having the transgene targeting the enhanced green fluorescent protein to cell lineages that express StAR. A gene expression profile was obtained by whole-mouse genome microarray and confirmed by quantitative real-time PCR, identifying 1206 and 767 significantly up-regulated and down-regulated genes, respectively, in Star(-/-) mice compared with Star(+/+) mice (fold difference ≥ 2 and P value < 0.05 with false discovery rate < 0.2). In Star(-/-) mice, expression levels of genes involved in cholesterol efflux and the inflammatory response were significantly up-regulated, whereas those related to steroid hormone biosynthesis or cholesterol biosynthesis and influx were not significantly changed. Immunoreactive Iba1 or F4/80 (macrophage marker) in adrenal glands of Star(-/-) mice was detected not only in an increased number of resident macrophages but also in most adrenocortical cells. These findings expand our understanding of the pathophysiology of adrenal glands with the disruption of StAR and propose a reciprocal interaction between adrenocortical cells and resident macrophages inside adrenal glands of Star(-/-) mice.

Complications act as stress-inducers during pregnancy so the fetus can develop functional compensatory mechanisms or morphologic changes. The cases analyzed are with congenital malformations or acute stress; chronic included cases with ascending infection (AI) and perinatal hypoxia/anoxia (PHA). The hematoxylin-eosin (H&E) was done to analyze the vacuolization, and the immunohistochemistry to the phagocytosis. The discreet standard of vacuolization was observed in 52.6% of the cases, 22.1% moderate, and 25.3% severe. The number of macrophages was higher in PHA. Changes in these organs are closely related to the cause of death and to the period during which the harmful agent.

Unlabelled: The (As) arsenic exposure is a risk factor for causing disturbances in the endocrine organs., Objective: To evaluate if sub-chronic As exposure during the pre- and postnatal development causes disturbances in the puberty. Moreover, determine adverse effects of As on the ovarian follicle and adrenocortical cell maturation., Methods: Females adult Wistar rats were exposed to sodium arsenite at 3 ppm calculated as As in drinking water from mating, gestation. Following the birth, the female offspring continued exposured to As via lactation. Weaned pups received the same As treatment as mothers, until they were 1-4 months (mo) old. At these ages, blood sampling and tissue harvest were done. The tissues were fixed in situ with 4% paraformaldehyde in phosphate buffer. After the perfusion the ovaries, uterus, adrenal glands were harvested, dissected out, weighted. The ovaries and the adrenal glands were processed to paraffin and sectioned at 5 μM and stained with hematoxylin and eosin for light microscopy., Statistical Analysis: Comparisons between groups were made by unpaired t-test or nonparametric Mann-Whitney test as appropriate., Results: 100% As treated rats at 1 mo of age were at diestrous stage, with low estradiol E2. As treatment caused disturbances in the morphology of the ovarian cell consisting in DNA damage evidenced by picknotic chromatin, cariorexis, significant cytoplasmic vacuolization and also vasculature damaged. Arrest in follicle maturation was also present., Conclusions: We found that the onset of puberty in the As treated rats was 1 mo delayed since vagina was still closed, the vaginal smear showed that they were at diestrus stage with plasma low E2 levels., (Copyright © 2010 Elsevier GmbH. All rights reserved.)

Steroidogenic acute regulatory protein (StAR) mediates the rate-limiting step in the synthesis of steroid hormones, essential to fetal development. We have reported that the StAR expression in fetal adrenal is inhibited in a rat model of nicotine-induced intrauterine growth retardation (IUGR). Here using primary human fetal adrenal cortex (pHFAC) cells and a human fetal adrenal cell line NCI-H295A, we show that nicotine inhibits StAR expression and cortisol production in a dose- and time-dependent manner, and prolongs the inhibitory effect on cells proliferating over 5 passages after termination of nicotine treatment. Methylation detection within the StAR promoter region uncovers a single site CpG methylation at nt -377 that is sensitive to nicotine treatment. Nicotine-induced alterations in frequency of this point methylation correlates well with the levels of StAR expression, suggesting an important role of the single site in regulating StAR expression. Further studies using bioinformatics analysis and siRNA approach reveal that the single CpG site is part of the Pax6 binding motif (CGCCTGA) in the StAR promoter. The luciferase activity assays validate that Pax6 increases StAR gene expression by binding to the glucagon G3-like motif (CGCCTGA) and methylation of this site blocks Pax6 binding and thus suppresses StAR expression. These data identify a nicotine-sensitive CpG site at the Pax6 binding motif in the StAR promoter that may play a central role in regulating StAR expression. The results suggest an epigenetic mechanism that may explain how nicotine contributes to onset of adult diseases or disorders such as metabolic syndrome via fetal programming., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Mice showing mosaic expression of an appropriate marker gene that is activated during development provide simple tools for investigating cell lineages. We used the mosaic β-galactosidase staining patterns in adrenal cortices of 21OH/ LacZ transgenic mice to study both organogenesis and maintenance of the adult tissue. Randomly orientated mosaic patterns present in embryonic day 14.5 (E14.5) adrenals changed progressively during the perinatal period from discrete spots, via patches and radial arrays, to radial stripes, which first emerged between postnatal days 0 and 7 (P0 and P7). The mosaic radial stripe pattern was fully established by P21 and remained unchanged throughout the adult period (8-52 weeks). The mouse adrenal gland grew continuously between E14.5 and P21, including the period during which stripes emerge. Ki67-positive, proliferative cells in the adrenal cortex were mainly localized to the outer cell layers between E18.5 and P3. By P10, cell proliferation had increased, and the proliferative region had expanded but was still mainly confined to the outer cortex. Correlation of changes in mosaic patterns in 21OH/LacZ adrenal cortices with the locations of adrenocortical cell proliferation suggest that the radial stripes arise by edge-biased growth during the perinatal period, even if they are maintained by stem cells in adults. The stability of the adult stripe pattern suggests that stem cell function is unchanged between 8 and 52 weeks.

Continuous efforts have been devoted to unraveling the biophysiology and development of the human fetal adrenal cortex, which is structurally and functionally unique from other species. It plays a pivotal role, mainly through steroidogenesis, in the regulation of intrauterine homeostasis and in fetal development and maturation. The steroidogenic activity is characterized by early transient cortisol biosynthesis, followed by its suppressed synthesis until late gestation, and extensive production of dehydroepiandrosterone and its sulfate, precursors of placental estrogen, during most of gestation. The gland rapidly grows through processes including cell proliferation and angiogenesis at the gland periphery, cellular migration, hypertrophy, and apoptosis. Recent studies employing modern technologies such as gene expression profiling and laser capture microdissection have revealed that development and/or function of the fetal adrenal cortex may be regulated by a panoply of molecules, including transcription factors, extracellular matrix components, locally produced growth factors, and placenta-derived CRH, in addition to the primary regulator, fetal pituitary ACTH. The role of the fetal adrenal cortex in human pregnancy and parturition appears highly complex, probably due to redundant and compensatory mechanisms regulating these events. Mounting evidence indicates that actions of hormones operating in the human feto-placental unit are likely mediated by mechanisms including target tissue responsiveness, local metabolism, and bioavailability, rather than changes only in circulating levels. Comprehensive study of such molecular mechanisms and the newly identified factors implicated in adrenal development should help crystallize our understanding of the development and physiology of the human fetal adrenal cortex.

Unlabelled: With 14 figures and 3 tables, Summary: Each adrenal gland consisted of cortex and medulla that developed from different embryological origins and presented different cellular organization. One hundred male or female camel embryos or fetuses with crown vertebral rump lengths (CVRL) that ranged from 0.8 to 117 cm were examined. The adrenal cortex, which is derived from intermediate mesoderm, was first observed in the 0.8-cm CVRL camel embryo. The adrenal cortex initially was combined with the gonad as a thickened region of proliferating cells derived from splanchnic intermediate mesoderm. Adrenocortical tissue was first separated from the gonadal tissue in the 2-cm CVRL camel fetus and was observed as a separate dorso-medial mass of cells. At 2.5-cm CVRL, the adrenocortical tissue was surrounded by a capsule of undifferentiated mesenchymal cells, except at its proximal pole, where an invagination was located through which chromaffinoblast cells entered the cortex. The chromaffinoblast cells migrated from the neural crest to form the medulla of the developing adrenal gland. In the 3.5-cm CVRL camel fetus, the adrenocortical cells differentiated into two layers: the inner fetal cortex and the outer definitive cortex. As development proceeded, the fetal cortex degenerated and the definitive cortex formed the zona glomerulosa and zona fasciculata. The zona reticularis did not form until the end of gestation. During prenatal life, the adrenal medulla was much thicker than the cortex., (© 2010 Blackwell Verlag GmbH.)

The germline R337H mutation in the TP53 gene is considered to be responsible for the increased incidence of adrenocortical tumors (ACTs) in children from Brazil. High level production of hormones in ACTs (>95%) cause virilization alone (60%), Cushing syndrome (<5%), the mixed type (30%), or other rarer manifestations. ACT probably develops owing to events occurring during the final stages of intrauterine life based on the very common early onset of signs and symptoms shortly after birth. In this study, we determined by immunohistochemistry and enzyme assays whether placental alkaline phosphatase (PLAP) is expressed in pediatric ACTs. Immunohistochemical analysis revealed positive p53 expression in 88% of the tested ACTs (29 of 33). PLAP was detected at a slightly lower frequency based on immunohistochemical (17 of 33, 51%) and enzyme activity analyses (9 of 16, 56%). In conclusion, probably at a certain time point during adrenocortical development (end of gestation to early postnatal period), some fetal zone cells survive owing to defective apoptosis and develop into childhood ACT, maintaining some characteristics of the embryonal period, such as PLAP expression. Further studies of PLAP should investigate the functional role, if any, of PLAP in such tumors.

The origins of our understanding of the cellular and molecular mechanisms by which signaling pathways and downstream transcription factors coordinate the specification of adrenocortical cells within the adrenal gland have arisen from studies on the role of Sf1 in steroidogenesis and adrenal development initiated 20 years ago in the laboratory of Dr. Keith Parker. Adrenocortical stem/progenitor cells have been predicted to be undifferentiated and quiescent cells that remain at the periphery of the cortex until needed to replenish the organ, at which time they undergo proliferation and terminal differentiation. Identification of these stem/progenitor cells has only recently been explored. Recent efforts have examined signaling molecules, including Wnt, Shh, and Dax1, which may coordinate intricate lineage and signaling relationships between the adrenal capsule (stem cell niche) and underlying cortex (progenitor cell pool) to maintain organ homeostasis in the adrenal gland., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

The orphan nuclear receptor AD4BP/SF-1 (adrenal-4-binding protein/steroidogenic factor-1 (NR5A1)) is essential for the proper development and function of reproductive and steroidogenic tissues. Although the expression of Ad4BP/Sf-1 is specific for those tissues, the mechanisms underlying this tissue-specific expression remain unknown. Our transgenic studies have identified the tissue-specific enhancers for the fetal adrenal cortex, ventromedial hypothalamus, and pituitary in Ad4BP/Sf-1 gene. The adrenal cortex forms morphologically distinct compartments, the inner (fetal) and outer (definitive or adult) zones. Despite considerable effort, the mechanisms that mediate the differential development of the fetal and adult adrenal cortex remain incompletely understood. It remained controversial whether a true fetal type adrenal cortex is present in mice, and this argument was complicated by the postnatal development of the so-called X-zone. Using transgenic mice with lacZ driven by the fetal adrenal enhancer (FAdE), we clearly identified a fetal adrenal cortex in mice, and the X-zone is the fetal adrenal cells accumulated at the juxtamedullary region after birth. We combined the FAdE with the Cre/loxP system to trace cell lineages in which the FAdE was active at some stage in development. These lineage tracing studies establish definitively that the adult cortex derives from precursor cells in the fetal cortex in which the FAdE was activated before the organization into two distinct zones. The potential of these fetal adrenocortical cells to enter the pathway that eventuate in cells of the adult cortex disappeared by E14.5. Thus, these studies demonstrate a direct link between the fetal and adult cortex involving a transition that must occur before a specific stage of development., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

Sonic hedgehog signaling was recently demonstrated to play an important role in murine adrenal cortex development. The organization of the rat adrenal differs from that of the mouse, with the zona glomerulosa and zona fasciculata separated by an undifferentiated zone in the rat, but not in the mouse. In the present study we aimed to determine the mRNA expression patterns of Sonic hedgehog and the hedgehog signaling pathway components Patched-1 and Gli1 in the developing and adult rat adrenal. Sonic hedgehog expression was detected at the periphery of the cortex in cells lacking CYP11B1 and CYP11B2 expression, while signal-receiving cells were localized in the overlying capsule mesenchyme. Using combined in situ hybridization and immunohistochemistry we found that the cells expressing Sonic hedgehog lie between the CYP11B2 and CYP11B1 layers, and thus Sonic hedgehog expression defines one cell population of the undifferentiated zone., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

During the early phases of development, adrenal glands share a common origin with kidneys and gonads. The action of diverse transcription factors, signalling pathways and endocrine signals is required for the individualization of the adrenal primordium and its subsequent differentiation into an adult adrenal gland, with massive remodelling taking place around the time of birth in humans. Here I summarize the most important steps by which the adrenal cortex is shaped and present an overview of the current understanding of the genes and molecular pathways implicated in adrenal development and involved in the pathogenesis of its congenital diseases. Evidence is accumulating that some pivotal factors acting during adrenocortical development also play an important role to regulate the growth of adrenocortical tumors, representing promising therapeutical targets for a biology-oriented therapy., (2010 Elsevier Ltd. All rights reserved.)

Neural crest-derived cells are believed to migrate into the fetal adrenal cortex from the medially-located hilus. However, there appears to be a paucity of observations of the migration and distribution of medullary cells in humans. In sagittal as well as horizontal sections of human fetuses between 9 and 16 weeks of gestation, we identified chromaffin, ganglion and Schwann-like cells in the developing adrenal gland using immunohistochemistry. Cells showing tyrosine hydroxylase (TH) immunoreactivity (i.e., candidate ganglion cells) entered the fetal cortex mainly from the medial half of the adrenal, but the path of entry also included the ventral, dorsal and caudal aspects. These cells displayed linear arrangements, forming a connection between the peripheral and central areas of the gland. S100 protein-immunoreactive cells (i.e., Schwann-like cells) accompanied most (but not all) of the TH-positive cells. The distribution of chromogranin A-immunoreactive cells (i.e., chromaffin cells) was similar to and overlapped with that of TH-positive cells. Chromogranin A-positive cells were observed around the aorta as well as in the adrenal. The entry of neural crest-derived cells does not appear to be restricted to a hypothetical medial hilus, but occurs widely around the cortex, with or without the accompaniment of Schwann-like cells. These cells advance in lines through the fetal cortex in a cord-like arrangement without destruction of the cortical architecture. Some of the TH-positive cells very likely express chromogranin A before entry into the adrenal.

The adrenal capsule is postulated to harbor stem/progenitor cells, the progenies of which contribute to the growth of adrenocortex. We discovered that cells in the adrenal capsule are positive for Ptch1 and Gli1, genes indicative of responsiveness to the stimulation of Hedgehog (Hh) ligands. On the other hand, Sonic hedgehog (Shh), one of the mammalian Hh ligands, is expressed in the adrenocortex underneath the adrenal capsule, possibly acting upon the Hh-Responsive capsule. To investigate the functional significance of Shh in adrenal growth, we ablated Shh in an adrenocortex-specific manner using the Steroidogenic factor 1-Cre mouse. Loss of Shh in the adrenocortex led to reduced proliferation of capsular cells and a 50-75% reduction in adrenocortex thickness and adrenal size. The remaining adrenocortex underwent proper zonation and was able to synthesize steroids, indicating that Shh is dispensable for differentiation of adrenocortex. When these animals reached adulthood, their adrenocortex did not undergo compensatory growth in response to a high level of plasma ACTH, and the size of the adrenal remained significantly smaller than the control adrenal. Using a genetic lineage-tracing model, we further demonstrated that the Hh-responding cells in the adrenal capsule migrated centripetally into the adrenocortex. Our results not only provide the genetic evidence to support that the adrenal capsule contributes to the growth of adrenocortex in both fetal and adult life but also identify a novel role of Shh in this process.

Importance of the Field: DAX-1 (NR0B1) is an unusual orphan nuclear receptor whose function is essential for the development of the human adrenal cortex and onset of puberty. Recent data have implicated this transcription factor also in embryonic stem cell and cancer biology., Areas Covered in This Review: The role of DAX-1 in the regulation of development and function of the adrenal cortex, reproductive axis, embryonic stem cells and a few types of cancer., What the Reader Will Gain: Here we review the past and present milestones in DAX-1 research and try to provide hints about the development and fields of application of DAX-1-targeted drugs in the future., Take Home Message: The unusual structure and restricted expression pattern of DAX-1 may offer unique opportunities for drug discovery.

Adrenocortical dysplasia (acd) is a spontaneous autosomal recessive mouse mutation that exhibits a pleiotropic phenotype with perinatal lethality. Mutant acd embryos have caudal truncation, vertebral segmentation defects, hydronephrosis, and limb hypoplasia, resembling humans with Caudal Regression syndrome. Acd encodes Tpp1, a component of the shelterin complex that maintains telomere integrity, and consequently acd mutant mice have telomere dysfunction and genomic instability. While the association between genomic instability and cancer is well documented, the association between genomic instability and birth defects is unexplored. To determine the relationship between telomere dysfunction and embryonic malformations, we investigated mechanisms leading to the caudal dysgenesis phenotype of acd mutant embryos. We report that the caudal truncation is caused primarily by apoptosis, not altered cell proliferation. We show that the apoptosis and consequent skeletal malformations in acd mutants are dependent upon the p53 pathway by genetic rescue of the limb hypoplasia and vertebral anomalies with p53 null mice. Furthermore, rescue of the acd phenotype by p53 deficiency is a dosage-sensitive process, as acd/acd, p53(-/-) double mutants exhibit preaxial polydactyly. These findings demonstrate that caudal dysgenesis in acd embryos is secondary to p53-dependent apoptosis. Importantly, this study reinforces a significant link between genomic instability and birth defects.

Context: X-linked congenital adrenal hypoplasia with hypogonadotropic hypogonadism (AHCH) is known to be caused by coding mutations in the nuclear receptor subfamily 0, group B, member 1 (NR0B1) gene, encoding the transcriptional repressor dosage-sensitive sex-reversal adrenal hypoplasia critical region on the X chromosome protein 1 (DAX1)., Objective/patients: Four males in a family were affected by AHCH. Our aim was to locate the genetic cause of their disease, knowing that they had no mutation in the obvious candidate gene, NR0B1., Design: Linkage analysis of the X chromosome and mutational screening of conserved noncoding regions upstream of NR0B1 were performed. To functionally characterize the genetic defect, studies of transcription and expression of DAX1 and steroidogenic factor 1 (SF-1) were done., Results: A 60 Mb inversion on the X chromosome with one of the inversion breakpoints located in a conserved noncoding region 4 kb upstream of NR0B1 was detected. The inversion causes relocation of a putative SF-1 binding site implicated in murine gonadal development. A reporter construct lacking this enhancer element upstream of NR0B1 was unresponsive to SF-1 transcriptional activation. Immunohistochemistry suggested that the inversion leads to SF-1 silencing in the patients' testes both in childhood and in adult life., Conclusion: We report a noncoding mutation causing AHCH, an inversion resulting in a phenotype similar to what is caused by intragenic NR0B1 null mutations. The inversion seems to disrupt and/or relocate regulatory sites crucial in DAX1 expression.

Scientists have long hypothesized the existence of tissue-specific (somatic) stem cells and have searched for their location in different organs. The theory that adrenocortical organ homeostasis is maintained by undifferentiated stem or progenitor cells can be traced back nearly a century. Similar to other organ systems, it is widely believed that these rare cells of the adrenal cortex remain relatively undifferentiated and quiescent until needed to replenish the organ, at which time they undergo proliferation and terminal differentiation. Historical studies examining cell cycle activation by label retention assays and regenerative potential by organ transplantation experiments suggested that the adrenocortical progenitors reside in the outer periphery of the adrenal gland. Over the past decade, the Hammer laboratory, building on this hypothesis and these observations, has endeavored to understand the mechanisms of adrenocortical development and organ maintenance. In this review, we summarize the current knowledge of adrenal organogenesis. We present evidence for the existence and location of adrenocortical stem/progenitor cells and their potential contribution to adrenocortical carcinomas. Data described herein come primarily from studies conducted in the Hammer laboratory with incorporation of important related studies from other investigators. Together, the work provides a framework for the emerging somatic stem cell field as it relates to the adrenal gland.

The Cre-loxP system combined with gene targeting strategies has proven to be very useful for gene inactivation in specific tissues and/or cell types. To achieve adrenal cortex specific recombination in vivo, we used a 0.5-kb fragment of the 5'-flanking region of the akr1b7 gene to drive Cre expression in adrenocortical cells. The resulting 0.5 akr1b7-Cre mice express Cre in all steroidogenic zones of the adrenal cortex but not in the gonads. Although recombination of the ROSA26R reporter locus was not observed in all cortical cells, we provide evidence that Cre is expressed in all the cells of the cortex in adult mice. In addition, Cre activity was found in collecting ducts and maturing glomeruli of the kidney. This line is the first to show specific Cre expression in the adrenal cortex in the absence of Cre expression in the gonads. This transgene thus provides a valuable tool for specific gene recombination in the adrenal cortex and kidney.

Human sexual differentiation is a critical process whereby a strict dimorphism is established that enables future reproductive success as phenotypic males and females. Significant components of this differentiation pathway unfold during the first three months of gestation when they are sensitive to disruption by abnormal hormonal influences. Excessive exposure of female development to androgens in conditions such as congenital adrenal hyperplasia causes virilization. However, recently we have suggested that female development normally takes place in the presence of low, yet significant, levels of androgen, implying a need for strict regulation to avoid virilization and the potential for a biological role of androgens in females that has not been fully elucidated. Here, we review androgen-dependent male differentiation of the external genitalia in humans, and link this to current understanding of female development and steroidogenesis in the developing adrenal cortex.