Adrenal Zonation Research Articles

Loss of LGR4/GPR48 causes severe neonatal salt wasting due to disrupted WNT signaling altering adrenal zonation.

Disorders of isolated mineralocorticoid deficiency, which cause potentially life-threatening salt-wasting crisis early in life, have been associated with gene variants of aldosterone biosynthesis or resistance; however, in some patients no such variants are found. WNT/β-catenin signaling is crucial for differentiation and maintenance of the aldosterone-producing adrenal zona glomerulosa (zG). Herein, we describe a highly consanguineous family with multiple perinatal deaths and infants presenting at birth with failure to thrive, severe salt-wasting crises associated with isolated hypoaldosteronism, nail anomalies, short stature, and deafness. Whole exome sequencing revealed a homozygous splice variant in the R-SPONDIN receptor LGR4 gene (c.618-1G>C) regulating WNT signaling. The resulting transcripts affected protein function and stability and resulted in loss of Wnt/β-catenin signaling in vitro. The impact of LGR4 inactivation was analyzed by adrenal cortex-specific ablation of Lgr4, using Lgr4fl/fl mice mated with Sf1:Cre mice. Inactivation of Lgr4 within the adrenal cortex in the mouse model caused decreased WNT signaling, aberrant zonation with deficient zG, and reduced aldosterone production. Thus, human LGR4 mutations establish a direct link between LGR4 inactivation and decreased canonical WNT signaling, which results in abnormal zG differentiation and endocrine function. Therefore, variants in WNT signaling and its regulators should systematically be considered in familial hyperreninemic hypoaldosteronism.

Flow-dependent differentiation of cultured adrenal cells under different stimuli.

It still remains unclear how the functional organisation of the adrenal cortex arises. One aim of this study was to create a setup which allows for the establishment of a concentration gradient in vitro. This was achieved by a continuous flow of medium through the culture flask which caused differences in glucose and cortisol concentrations as well as in pH values between the sites of inflow and outflow of medium. Using real-time polymerase chain reaction, we found that a continuous supply of 1ml medium per hour significantly increased the expression of MC2R, CYP11B1 and CYP17A1 genes of NCI-H295R cells in the distal area of the flask as compared with the proximal part. The expression of the AT1R showed a reverse regulation. The addition of dexamethasone to the medium led to an increase in gene expression of MC2R while AT1R was downregulated. Moreover, we detected a higher expression of CYP11B2 and a decreased expression of CYP11B1 when endothelial cell-conditioned medium (ECCM) was added to the inflow. Our experiments show that a directed medium delivery system creates different gradients and affects the functional differentiation of the NCI-H295R cells. Also, our results emphasise that products of endothelial cells have additional effects on the differentiation of the cultured adrenal cortical cells. Our results are in support that the regulation of the adrenal zonation is possible through different concentration gradients.

β-catenin in adrenal zonation and disease

The Wnt signaling pathway is a critical mediator of the development and maintenance of several tissues. The adrenal cortex is highly dependent upon Wnt/β-catenin signaling for proper zonation and endocrine function. Adrenocortical cells emerge in the peripheral capsule and subcapsular cortex of the gland as progenitor cells that centripetally differentiate into steroid hormone-producing cells of three functionally distinct concentric zones that respond robustly to various endocrine stimuli. Wnt/β-catenin signaling mediates adrenocortical progenitor cell fate and tissue renewal to maintain the gland throughout life. Aberrant Wnt/β-catenin signaling contributes to various adrenal disorders of steroid production and growth that range from hypofunction and hypoplasia to hyperfunction, hyperplasia, benign adrenocortical adenomas, and malignant adrenocortical carcinomas. Great strides have been made in defining the molecular underpinnings of adrenocortical homeostasis and disease, including the interplay between the capsule and cortex, critical components involved in maintaining the adrenocortical Wnt/β-catenin signaling gradient, and new targets in adrenal cancer. This review seeks to examine these and other recent advancements in understanding adrenocortical Wnt/β-catenin signaling and how this knowledge can inform therapeutic options for adrenal disease.

SUN-LB39 ATAC-Seq Reveals Dynamic Changes in Chromatin Accessibility Following PKA Activation in NCI-H295R Adrenocortical Cells

The adrenal cortex is comprised of distinct concentric zones that produce hormones essential for life - the outermost zona glomerulosa (zG) produces aldosterone, and the innermost zona fasciculata (zF) produces cortisol. Adrenal zonation is maintained by a balance between paracrine (Wnt/β-catenin) and endocrine (ACTH/PKA) signaling. Wnt/β-catenin signaling is maintained in the outer cortex (zG, upper zF) by a gradient of Wnt ligands that diminish centripetally, and PKA signaling is maintained in the inner cortex (zF) by ACTH. Recent studies in vivo suggest that sustained PKA signaling promotes zF proliferation, enabling lineage conversion towards zF by inhibiting Wnt/β-catenin transcriptional programming. While these studies were crucial in elucidating mechanisms supporting adrenal zonation, it remains unclear if PKA-mediated repression of Wnt/β-catenin is carried out at the chromatin level or if it is secondary to ligand-dependent modulation of paracrine signaling as may happen in vivo. To address this question, we utilized the adrenocortical cell line (NCI-H295R), which harbors a mutation in CTNNB1 rendering Wnt/β-catenin signaling constitutively active. We stimulated PKA in NCI-H295R using forskolin, and assessed genome-wide chromatin accessibility by ATAC-seq and transcriptome changes by RNA-seq. Motif analysis of ATAC-seq from baseline NCI-H295R revealed that chromatin accessibility is dominated by transcription factors SF1 (master regulator of the adrenal cortex and steroidogenesis), AP1 (effector of PKA) and LEF1 (effector of Wnt/β-catenin). Following forskolin administration, we observed decreased accessibility in chromatin containing LEF1 binding motifs, and increased accessibility in chromatin bearing AP1 and SF1 motifs, suggesting that PKA activation drives AP1/SF1-dependent transcription and inhibits Wnt/β-catenin-dependent transcription at the chromatin level. Indeed, RNA-seq revealed that forskolin administration decreased the expression of zG and Wnt/β-catenin target genes, while simultaneously increasing expression of AP1/SF1 target genes. Collectively, these data demonstrate that PKA activation leads to profound chromatin remodeling that enables zF identity even in the setting of constitutive Wnt/β-catenin signaling. Ongoing studies are aimed at elucidating how chromatin modifiers and transcriptional machinery coordinate the dynamic regulation of differentiation programs required for adrenocortical homeostasis and zonation.

SUN-210 Differentially Expressed Mirnas in Zona Reticularis (ZR) Cells of the Human Adrenal Cortex

The human adrenal cortex, involved in adaptive responses to stress, fluid homeostasis, and secondary sexual characteristics, arises from a tightly regulated development of a zone and cell type-specific secretory pattern. However, the molecular mechanisms governing adrenal zonation, particularly postnatal ZR development, which produces adrenal androgens in a life-time-specific manner, remain poorly understood. The hallmark of ZR is the low expression of type 2 3β-hydroxysteroid dehydrogenase (HSD3B2). However, the mechanisms underlying HSD3B2 downregulation in the ZR remain unknown. MiRNAs are seen as regulators of cell phenotypes. The objective of the study was to compare miRNA expression profiles in human adrenal ZR and zona fasciculata (ZF). ZF and ZR were microdissected from human adrenals tissues (n=5, from boys aged 16, 17 and 19 yr and girls aged 12 and 16 yr) by laser capture microdissection (LCM). Total RNA was extracted from 5 ZF/ZR pairs and next-generation sequencing (NGS) was used to perform the microRNA expression profiling. 281 mature microRNAs were identified in human adrenal cortex. Among them, 7 microRNAs were significantly differentially expressed between ZF and ZR. The expression of miR-375-3p, miR-483-3p and miR-7-5p was higher in ZR compared with its paired ZF by 2-fold or greater. Multiple available bioinformatic algorithms (TargetScan, miRanda, DianaLab and PicTar) were employed to search for its target genes. Among predicted target genes, several genes (GATA-6, GATA-4, SF1, NR4A2, and IGF-1) are known to be involved in HSD3B2 regulation. In summary, LCM combined with NGS provided a robust approach to explore the adrenal zone-specific micro-RNA profiling. Our data gave first hints that miRNAs might be novel regulatory modules associated with human adrenal ZR cell-specific transcript regulation underlying developmental androgen production.

Hormonal and spatial control of SUMOylation in the human and mouse adrenal cortex.

SUMOylation is a highly conserved and dynamic post-translational mechanism primarily affecting nuclear programs for adapting organisms to stressful challenges. Alteration of SUMOylation cycles leads to severe developmental and homeostatic defects and malignancy, but signals coordinating SUMOylation are still unidentified. The adrenal cortex is a zonated endocrine gland that controls body homeostasis and stress response. Here, we show that in human and in mouse adrenals, SUMOylation follows a decreasing centripetal gradient that mirrors cortical differentiation flow and delimits highly and weakly SUMOylated steroidogenic compartments, overlapping glomerulosa, and fasciculata zones. Activation of PKA signaling by acute hormonal treatment, mouse genetic engineering, or in Carney complex results in repression of small ubiquitin-like modifier (SUMO) conjugation in the inner cortex by coordinating expression of SUMO pathway inducers and repressors. Conversely, genetic activation of canonical wingless-related integration site signaling maintains high SUMOylation potential in the outer neoplastic cortex. Thus, SUMOylation is tightly regulated by signaling pathways that orchestrate adrenal zonation and diseases.-Dumontet, T., Sahut-Barnola, I., Dufour, D., Lefrançois-Martinez, A.-M., Berthon, A., Montanier, N., Ragazzon, B., Djari, C., Pointud, J.-C., Roucher-Boulez, F., Batisse-Lignier, M., Tauveron, I., Bertherat, J., Val, P., Martinez, A. Hormonal and spatial control of SUMOylation in the human and mouse adrenal cortex.

Contribution of retinoic acid receptor signalling to adrenal cortex morphology and functional zonation through modulation of WNT/[beta]-catenin pathway

Contribution of retinoic acid receptor signalling to adrenal cortex morphology and functional zonation through modulation of WNT/[beta]-catenin pathway

EZH2: a master regulator of adrenal cortex homeostasis and zonation

EZH2: a master regulator of adrenal cortex homeostasis and zonation

Editorial: Adrenal Cortex: From Physiology to Disease

Editorial: Adrenal Cortex: From Physiology to Disease

CO-34: Retinoic acid receptor signaling contributes to adrenal morphology and functional zonation

Background Primary aldosteronism is the most common form of secondary hypertension. Aldosterone producing adenoma (APA) and bilateral adrenal hyperplasia together account for ~95% of cases. Recurrent somatic mutations in KCNJ5, CACNA1D, ATP1A1 and ATP2B3 have been identified in APA. This has allowed us to establish a pathophysiological model involving modifications in intracellular ionic homeostasis and membrane potential leading to the activation of calcium signaling, the major trigger for aldosterone production. In parallel, we have identified retinoic acid receptor (RAR) signaling as a central molecular network involved in APA formation independently of the mutation status. The aim of our study was to evaluate the role of RAR signaling in the development of APA. Methods To this purpose we have investigated the modulation of RAR signaling in two different cell lines derived from the human adrenal cortex as well as in different mouse models. Results Whereas treatment of H295R cells with 10–6M of all trans retinoic acid (ATRA, a RARα agonist) and 9-cis retinoic acid (9-cis RA, a RXRα agonist) did not affect cell viability, 10–5M of ATRA dramatically decreased cell number in a time-dependent manner. This effect was due to decreased cell proliferation and increased cell apoptosis. However, 9-cis RA did not modify tumor growth in a mouse xenograft model. RARα invalidation by shRNA transduction in H295R cells did not affect their proliferative properties, but induced a major change in cellular phenotype with cells forming spheroid structures able to grow in suspension. Investigation of the adrenal phenotype of rarα knock-out mice demonstrated that in young (12 weeks) and old (40 weeks) rarα-/- mice the characteristic cellular arrangement of the adrenal cortex was replaced by an enlarged zona glomerulosa and a disorganized zona fasciculata, this effect being more pronounced in old mice. Conclusions Our results suggest that RAR signaling contributes to normal adrenal morphology and functional zonation. Disruption of RAR signaling could trigger abnormal proliferation of cells in the adrenal cortex, creating a propitious environment for the emergence of specific mutations affecting ionic channels and ATPases leading to increased aldosterone production.

Adrenocortical growth and cancer.

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.

Alteration of Subcapsular Adrenocortical Zonation in Humans with Aging

Few studies have examined functional adrenal zonation throughout human life. Adrenals from 61 surgical/autopsy patients from 1 day old to 92 years old who had no clinical endocrinological/mineralocorticoid abnormalities were assessed for immunohistochemically defined adrenal zonation. The zona glomerulosa (zG) was well developed in all 11 patients ranging in age from newborn to the 30s. After 40 years of age, however, the zG occupied less than one-quarter of the adrenal circumference, suggestive of zG involution. The other subcapsular areas were occupied by the progenitor zone (zP), which expressed neither cytochrome P450(aldo) nor P450(11β) but 3β-hydroxysteroid dehydrogenase and P450scc, although some autopsy cases had adrenals with zG zonation because of secondary aldosteronism, and others who had experienced severe stresses showed subcapsular zona fasciculata (zF). In conclusion, the adrenal cortex consists of homogeneous zG-topped columns from birth to adolescence. Subsequently, in the fifth decade of life, the cortex is reconstituted by integration of three types of cortical columns: scattered zG-topped columns and zonal zP-topped columns, the latter having the ability for bidirectional differentiation into either zG-topped columns or zF-topped columns, according to secondary aldosteronism or the presence of severe stresses. Such adrenocortical remodeling is ascribed to high-sodium/low-potassium diets.

Invalidation of TASK1 potassium channels disrupts adrenal gland zonation and mineralocorticoid homeostasis

TASK1 (KCNK3) and TASK3 (KCNK9) are two-pore domain potassium channels highly expressed in adrenal glands. TASK1/TASK3 heterodimers are believed to contribute to the background conductance whose inhibition by angiotensin II stimulates aldosterone secretion. We used task1-/- mice to analyze the role of this channel in adrenal gland function. Task1-/- exhibited severe hyperaldosteronism independent of salt intake, hypokalemia, and arterial 'low-renin' hypertension. The hyperaldosteronism was fully remediable by glucocorticoids. The aldosterone phenotype was caused by an adrenocortical zonation defect. Aldosterone synthase was absent in the outer cortex normally corresponding to the zona glomerulosa, but abundant in the reticulo-fasciculata zone. The impaired mineralocorticoid homeostasis and zonation were independent of the sex in young mice, but were restricted to females in adults. Patch-clamp experiments on adrenal cells suggest that task3 and other K+ channels compensate for the task1 absence. Adrenal zonation appears as a dynamic process that even can take place in adulthood. The striking changes in the adrenocortical architecture in task1-/- mice are the first demonstration of the causative role of a potassium channel in development/differentiation.

Shift from Conn’s syndrome to Cushing’s syndrome in a recurrent adrenocortical carcinoma

Adrenocortical tumors may originate from the zona glomerulosa, zona fasciculata, or zona reticularis and be associated with syndromes due to overproduction of mineralocorticoids, glucocorticoids, or androgens respectively. We report an unusual case of recurrent adrenocortical carcinoma (ACC), which seems to contradict the paradigm of functional adrenal zonation. A male patient presented with severe primary aldosteronism due to an ACC, which relapsed after adrenalectomy and adjuvant mitotane therapy. After removal of the tumor recurrence and eight cycles of chemotherapy with etoposide, doxorubicin and cisplatin, the patient presented again with ACC masses, but in association with overt Cushing's syndrome and normal aldosterone levels. Extensive pathologic examination showed that this shift in steroid hormone production was paralleled by an attenuation of tumor cell atypia and polymorphism, whereas gene expression profile analysis demonstrated a change in expression of adrenal steroidogenic enzymes. Moreover, cancer progression was associated with overexpression of the inhibin-alpha subunit, which could have contributed to the phenotypic changes. This case of recurrent ACC demonstrates that adrenocortical cells can reverse their differentiation program during neoplastic progression and change their specific hormone synthesis, as a consequence of modifications in the expression profile of steroidogenic enzymes and cofactors. We hypothesize that this shift in steroid hormone secretion is a consequence of chromosome amplification induced by chemotherapy. These findings, besides opening new perspectives to study adrenocortical cell plasticity and potential, demonstrate how conventional clinical and pathologic evaluation can be combined with genomic analysis in order to dissect thoroughly the biology of cancer.

The Orphan Nuclear Receptor NGFIB Regulates Transcription of 3β-Hydroxysteroid Dehydrogenase: IMPLICATIONS FOR THE CONTROL OF ADRENAL FUNCTIONAL ZONATION

3beta-Hydroxysteroid dehydrogenase type 2 (HSD3B2) is a steroid-metabolizing enzyme that is essential for adrenal production of mineralocorticoids and glucocorticoids. Thus, HSD3B2 is expressed at high levels in the glomerulosa and fasciculata, where these steroids are produced. In contrast, the production of dehydroepiandrosterone (DHEA) and DHEA sulfate in the adrenal reticularis is inversely correlated with the expression of HSD3B2. The reasons for the zonal expression of HSD3B2 are not known but represent an important aspect in the biochemical zonation of the adrenal. Using microarray, real time reverse transcriptase-PCR, immunohistochemistry, and HSD3B2 promoter analysis, we demonstrate that the NGFIB family of nuclear hormone receptors plays a critical part in the regulation of HSD3B2 transcription and may play an important role in the functional zonation of the adrenal gland. Microarray analysis of cortisol- versus DHEA sulfate-producing adrenal tissue demonstrated that NGIFB paralleled expression of HSD3B2 with expression much higher in cortisol-producing adrenal tissue; this observation was also demonstrated using real time reverse transcriptase-PCR analysis. In addition, immunohistochemistry confirmed that within adult and fetal adrenal gland NGFIB expression paralleled expression of HSD3B2. Transient transfections into H295R adrenal cells demonstrated that NGFIB family members enhanced HSD3B2 reporter activity but had no effect on a 17alpha-hydroxylase (CYP17) promoter construct. Deletion and mutational analyses of the 5'-flanking region of the HSD3B2 gene identified a consensus NGFIB response element that bound NGFIB in mobility shift assays. Infection of cultured human adrenal cells with adenovirus-containing NGFIB increased cortisol production by 8-fold and increased expression of HSD3B2 mRNA 26-fold over that observed in mock-infected cells. In primary cultures of adrenal cells, ACTH, an activator of HSD3B2, rapidly induced expression of NGFIB. These results suggest that NGFIB plays a crucial role in adrenal zonation by regulating HSD3B2 gene transcription.

A Role for the NGFI‐B Family in Adrenal Zonation and Adrenocortical Disease

The three zones of the human adrenal cortex are functionally distinct with the glomerulosa producing aldosterone, the fasciculata producing cortisol, and the reticularis producing DHEA/DHEAS. This functional zonation is largely due to the zone‐specific expression of steroidogenic enzymes. Recent evidence suggests a role for the NGFI‐B family of orphan nuclear receptors (particularly NURR1 and NGFI‐B) in the zone‐specific expression of two key steroidogenic enzymes, aldosterone synthase (CYP11B2) and 3β‐hydroxysteroid dehydrogenase (HSD3B2). Herein we discuss the evidence that suggests a role for NURR1 (NR4A2) in the expression of CYP11B2 in the glomerulosa as well as in the dysregulation of CYP11B2 gene expression as is seen in aldosterone‐producing adenoma (APA), a major cause of endocrine hypertension. NURR1 appears to be important for CYP11B2 transcription and is found at higher levels in glomerulosa and in APA. Its expression in adrenal cells is also readily increased by angiotensin II treatment. HSD3B2 is a steroid‐metabolizing enzyme that is essential for adrenal production of mineralocorticoids and glucocorticoids. Thus, HSD3B2 is expressed at high levels in the glomerulosa and fasciculata where these steroids are produced but at low levels in the adrenal reticularis, which produces mainly DHEA. We recently demonstrated that NGFI‐B (nur77 or NR4A1) plays an important role in the regulation of HSD3B2 transcription and may play an important role in the functional zonation of the adrenal gland. Immunohistochemistry confirmed that, within adult and fetal adrenal gland, NGFI‐B expression paralleled expression of HSD3B2. Transient transfections demonstrated that NGFI‐B family members enhanced HSD3B2 reporter activity but had no effect on a 17α‐hydroxylase (CYP17) promoter construct. Taken together these results suggest that the NGFI‐B family of transcription factors plays a role in establishing the functional zonation of the human adrenal by regulating CYP11B2 and HSD3B2 gene transcription.

Ultrastructural and histochemical study on the interrenal cells of the male stickleback (Gasterosteus aculeatus, Teleostea), in relation to the reproductive annual cycle.

The steroidogenic interrenal cells in the adrenal homologue of the male stickleback (Gasterosteus aculeatus) were studied in relation to the reproductive cycle by means of histological and ultrastructural observations, and using histochemical methods for the localisation of 3beta-hydroxysteroid dehydrogenase (3betaHSD) and 17beta-hydroxysteroid dehydrogenase (17betaHSD). To determine the various stages of the reproductive cycle, the testes were also examined by histological and histochemical methods (3betaHSD). The results indicate that in this teleost the interrenal cells can undergo a cycle in which phases characterised by different cytological aspects are observed. During this cycle there is a renewal of organelles, in particular mitochondria and SER. Periodic degenerative processes are also found. Organelle cytology showed that the cell cycle has at least 3 different aspects during the year. An analogy with some cytological aspects of the adrenal zonation in mammals is possible. It is postulated that the interrenal gland activity could substitute or supplement androgen production by the testes.

Adrenal zonation: clues from 11β-hydroxylase and aldosterone synthase

Aldosterone and cortisol are the major mineralocorticoid and glucocorticoid produced by the human adrenal. Circulating levels of angiotensin II and potassium control the adrenal production of aldosterone, while the production of cortisol is controlled mainly by adrenocorticotropin. The capacity of the adrenal cortex to differentially produce aldosterone and cortisol relies to a large degree on the expression of aldosterone synthase ( CYP11B2) and 11β-hydroxylase ( CYP11B1). CYP11B2 catalyzes the final steps in the biosynthesis of aldosterone and is expressed solely in the glomerulosa of the adrenal cortex, while CYP11B1 catalyzes the final steps in the biosynthesis of cortisol and is expressed in the fasciculata/reticularis. The zonal expression of these two isozymes appears to result from transcriptional regulation of the two genes. Herein, the recent progress in defining the cellular mechanisms that regulate transcription of these two isozymes and thus the capacity of the adrenal gland to differentially produce aldosterone and cortisol is discussed.

Development of adrenal zonation in fetal rats defined by expression of aldosterone synthase and 11beta-hydroxylase.

The adult rat adrenal cortex is comprised of three concentric steroidogenic zones that are morphologically and functionally distinguishable: the zona glomerulosa, zona intermedia, and the zona fasciculata/reticularis. Expression of the zone-specific steroidogenic enzymes, cytochrome P450 aldosterone synthase (P450aldo), and P450 11beta hydroxylase (P45011beta), produced by the zona glomerulosa and zona fasciculata/reticularis, respectively, can be used to define the adrenal cortical cell phenotype of these two zones. In this study, immunohistochemistry and in situ hybridization were used to determine the ontogeny of expression of P450aldo and P45011beta to monitor the pattern of development of the rat adrenal cortex. RIA was used to measure adrenal content of aldosterone and corticosterone, the resulting products of the two enzymatic pathways. Double immunofluorescent staining for both enzymes at gestational day 16 (E16) showed P45011beta protein expressed in cells distributed throughout most of the adrenal intermixed with a separate, but smaller, population of cells expressing P450aldo protein. Whereas expression of P45011beta protein retained a similar pattern of distribution from E16 to adulthood (ignoring distribution of SA-1 positive, presumptive medullary cells), P450aldo protein changed its pattern of distribution by E19, becoming localized in a discontinuous ring of cells adjacent to the capsule. By postnatal day 1, P450aldo protein distribution was similar to that observed in adult glands; P450aldo-positive cells formed a continuous zone underlying the capsule. In situ hybridization showed that the pattern of P45011beta messenger RNA expression paralleled protein expression at all times, whereas P450aldo messenger RNA paralleled protein at E19 and after, but was undetectable before E19. However, adrenal aldosterone and corticosterone, as measured by RIA, were detected by E16, supporting the functional capacity of both phenotypes for all ages studied. These data suggest that the development of the adrenal zona glomerulosa occurs in two distinct phases; initial expression of the glomerulosa phenotype in scattered cells of the inner cortex before E17, followed by a change in distribution to the outer cortex between E17 and E19. It is hypothesized that this change in distribution occurs via cell differentiation, rather than cell migration, and that a possible regulator of these events is the fetal renin-angiotensin system.

Development of Adrenal Zonation in Fetal Rats Defined by Expression of Aldosterone Synthase and 11 -Hydroxylase

Development of Adrenal Zonation in Fetal Rats Defined by Expression of Aldosterone Synthase and 11 -Hydroxylase