Adrenal Development in Mice Requires GATA4 and GATA6 Transcription Factors.
ABSTRACT: 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.
Project description:Transcription factor GATA6 is expressed in the fetal and adult adrenal cortex and has been implicated in steroidogenesis. To characterize the role of transcription factor GATA6 in adrenocortical development and function, we generated mice in which Gata6 was conditionally deleted using Cre-LoxP recombination with Sf1-cre. The adrenal glands of adult Gata6 conditional knockout (cKO) mice were small and had a thin cortex. Cytomegalic changes were evident in fetal and adult cKO adrenal glands, and chromaffin cells were ectopically located at the periphery of the glands. Corticosterone secretion in response to exogenous ACTH was blunted in cKO mice. Spindle-shaped cells expressing Gata4, a marker of gonadal stroma, accumulated in the adrenal subcapsule of Gata6 cKO mice. RNA analysis demonstrated the concomitant upregulation of other gonadal-like markers, including Amhr2, in the cKO adrenal glands, suggesting that GATA6 inhibits the spontaneous differentiation of adrenocortical stem/progenitor cells into gonadal-like cells. Lhcgr and Cyp17 were overexpressed in the adrenal glands of gonadectomized cKO vs control mice, implying that GATA6 also limits sex steroidogenic cell differentiation in response to the hormonal changes that accompany gonadectomy. Nulliparous female and orchiectomized male Gata6 cKO mice lacked an adrenal X-zone. Microarray hybridization identified Pik3c2g as a novel X-zone marker that is downregulated in the adrenal glands of these mice. Our findings offer genetic proof that GATA6 regulates the differentiation of steroidogenic progenitors into adrenocortical cells.
Project description:Adrenal cortex autotransplantation with ACTH stimulation may be an alternative therapy for patients with bilateral adrenalectomy to avoid adrenal crisis, but its underlying mechanism has not been elucidated. Previously, we detected Dhh upregulation in rat adrenocortical autografts after transplantation. Here, we investigated potential regulators such as Gata4, Gata6, Sry and Sox9 which affect Dhh transcription in adrenocortical autografts with or without ACTH stimulation. In ACTH-stimulated autografts, Gata4 and Gata6 were downregulated compared to control autografts. This response was linked to rDhh repression. A reporter assay using the upstream region of rDhh and a GATA binding motif revealed that rDhh promoters were significantly upregulated by co-transfection with Gata4 or Gata6 or both. Sry and Sox9 expression in autografts with or without ACTH stimulation were verified by PCR and RNAscope analyses. The ovarian differentiation factors Foxl2 and Rspo1 were also upregulated in the autografts. Gata4 and Gata6 were found to be significant factors in the regulation of rDhh expression and could be associated with adrenocortical autograft maintenance. Gonadal primordia with bipotential testicular and ovarian functions may also be present in these autografts.
Project description:Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) are functionally redundant transcriptional regulators that are downstream effectors of the Hippo signaling pathway. They act as major regulators of stem cell maintenance, cell growth, and differentiation. To characterize their roles in the adrenal cortex, we generated a mouse model in which Yap and Taz were conditionally deleted in steroidogenic cells (Yapflox/flox;Tazflox/flox;Nr5a1cre/+). Male Yapflox/flox;Tazflox/flox;Nr5a1cre/+ mice were characterized by an age-dependent degeneration of the adrenal cortex associated with an increase in apoptosis and a progressive reduction in the expression levels of steroidogenic genes. Evaluation of the expression levels of stem and progenitor cell population markers in the adrenal glands of Yapflox/flox;Tazflox/flox;Nr5a1cre/+ mice also showed the downregulation of sonic hedgehog (Shh), a marker of the subcapsular progenitor cell population. Gross degenerative changes were not observed in the adrenal glands of Yapflox/flox;Tazflox/flox;Nr5a1cre/+ females, although steroidogenic capacity and Shh expression were reduced, suggesting that mechanisms of adrenocortical maintenance are sex specific. These results define a crucial role for YAP and TAZ in the maintenance of the postnatal adrenal cortex.
Project description:Testicular adrenal rest tumours (TARTs) are benign adrenal-like testicular tumours that frequently occur in male patients with congenital adrenal hyperplasia. Recently, GATA transcription factors have been linked to the development of TARTs in mice. The aim of our study was to determine GATA expression in human TARTs and other steroidogenic tissues. We determined GATA expression in TARTs (<i>n</i>?=?16), Leydig cell tumours (LCTs; <i>n</i>?=?7), adrenal (foetal (<i>n</i>?=?6)?+?adult (<i>n</i>?=?10)) and testis (foetal (<i>n</i>?=?13)?+?adult (<i>n</i>?=?8)). We found testis-like <i>GATA4</i>, and adrenal-like <i>GATA3</i> and <i>GATA6</i> gene expressions by qPCR in human TARTs, indicating mixed testicular and adrenal characteristics of TARTs. Currently, no marker is available to discriminate TARTs from LCTs, leading to misdiagnosis and incorrect treatment. <i>GATA3</i> and <i>GATA6</i> mRNAs exhibited excellent discriminative power (area under the curve of 0.908 and 0.816, respectively), while immunohistochemistry did not. GATA genes contain several CREB-binding sites and incubation with 0.1?mM dibutyryl cAMP for 4?h stimulated <i>GATA3</i>, <i>GATA4</i> and <i>GATA6</i> expressions in a human foetal testis cell line (hs181.tes). Incubation of adrenocortical cells (H295RA) with ACTH, however, did not induce <i>GATA</i> expression <i>in vitro</i> Although ACTH did not dysregulate <i>GATA</i> expression in the only human ACTH-sensitive <i>in vitro</i> model available, our results do suggest that aberrant expression of GATA transcription factors in human TARTs might be involved in TART formation.
Project description:Transcription factor GATA6 is expressed in the fetal and adult adrenal cortex and has been implicated in steroidogenesis. To characterize the role of GATA6 in adrenocortical development and function, we generated mice in which Gata6 was conditionally deleted using Cre-LoxP recombination with Sf1-cre. The adrenal glands of adult Gata6 conditional knockout (cKO) mice were small and had a thin cortex with thickened capsule. Cytomegalic changes were evident in the adrenal glands of fetal and adult cKO mice, and chromaffin cells were ectopically located at the periphery of the glands. The secretion of corticosterone in response to exogenous ACTH was blunted in cKO mice. Cells expressing gonadal-like markers, including Gata4, Amhr2, and Tcf21, accumulated in the adrenal capsule and subcapsule of cKO mice, suggesting aberrant adrenocortical progenitor/stem cell differentiation. Gonadectomy triggered the overexpression of sex steroidogenic differentiation markers, such as Lhcgr and Cyp17, in the adrenal glands of male and female cKO mice. Nulliparous female and orchiectomized male cKO mice lacked an adrenal X-zone. Microarray hybridization identified Pik3c2g as a novel X-zone marker that is downregulated in the adrenal glands of nulliparous female Gata6 cKO mice. Our findings offer genetic proof of the longstanding hypothesis that GATA6 regulates the differentiation of steroidogenic progenitors into corticoid-producing cells. 3 replicates from both conditional knockout of Gata6 in the adrenal gland and control adrenal glands from non-knockout mice were compared
Project description:In response to gonadectomy certain inbred mouse strains develop sex steroidogenic adrenocortical neoplasms. One of the hallmarks of neoplastic transformation is expression of GATA4, a transcription factor normally present in gonadal but not adrenal steroidogenic cells of the adult mouse. To show that GATA4 directly modulates adrenocortical tumorigenesis and is not merely a marker of gonadal-like differentiation in the neoplasms, we studied mice with germline or conditional loss-of-function mutations in the Gata4 gene. Germline Gata4 haploinsufficiency was associated with attenuated tumor growth and reduced expression of sex steroidogenic genes in the adrenal glands of ovariectomized B6D2F1 and B6AF1 mice. At 12 months after ovariectomy, wild-type B6D2F1 mice had biochemical and histological evidence of adrenocortical estrogen production, whereas Gata4(+/-) B6D2F1 mice did not. Germline Gata4 haploinsufficiency exacerbated the secondary phenotype of postovariectomy obesity in B6D2F1 mice, presumably by limiting ectopic estrogen production in the adrenal glands. Amhr2-cre-mediated deletion of floxed Gata4 (Gata4(F)) in nascent adrenocortical neoplasms of ovariectomized B6.129 mice reduced tumor growth and the expression of gonadal-like markers in a Gata4(F) dose-dependent manner. We conclude that GATA4 is a key modifier of gonadectomy-induced adrenocortical neoplasia, postovariectomy obesity, and sex steroidogenic cell differentiation.
Project description:A link between elevated luteinizing hormone (LH) levels, GATA-4 and LH receptor (LHCGR) expression and gonadotropin-dependent adrenocortical tumorigenesis in humans and mice has been shown. To assess the mechanistic tumorigenic interrelationships between these factors, we transgenically expressed Gata4 under the 21-hydroxylase promoter (Cyp21a1, 21-OH) in C57Bl/6N mice. There was a gradual age-dependent increase of GATA-4 expression only in 21-OH-GATA-4 (TG) female adrenals, in association with slowly progressing neoplasia of non-steroidogenic spindle-shaped A cells in the subcapsular cortex. Gonadectomy (GDX), apparently through direct action of elevated serum LH, markedly enhanced the adrenocortical neoplasia, which now also appeared in GDX TG males. The neoplastic areas of the post-GDX TG adrenals contained, besides A cells, larger lipid-laden, steroidogenically active and LHCGR-positive B cells. Prolonged (>10 months) exposure to elevated post-GDX LH levels resulted in formation of adrenocortical adenomas in the TG mice. Intact and GDX TG mouse adrenals displayed elevated FOG-2 and decreased GATA-6 expression. Additionally, increased expression/activation of components of the Inhbb-Acvr2a-Acvr1c-Smad2/3 signaling system was observed in 12-month-old GDX TG adrenals. Our findings show that two distinct GATA-4-dependent populations of neoplastic adrenocortical cells form: non-steroidogenic LH-independent A cells and steroidogenic LH-dependent B cells.
Project description:Adrenal glands and gonads share a common primordium (AGP), but the molecular events driving differentiation are poorly understood. Here we demonstrate that the Wilms tumor suppressor WT1 is a key factor defining AGP identity by inhibiting the steroidogenic differentiation process. Indeed, ectopic expression of WT1 precludes differentiation into adrenocortical steroidogenic cells by locking them into a progenitor state. Chromatin immunoprecipitation experiments identify Tcf21 and Gli1 as direct targets of WT1. Moreover, cell lineage tracing analyses identify a long-living progenitor population within the adrenal gland, characterized by the expression of WT1, GATA4, GLI1, and TCF21, that can generate steroidogenic cells in vivo. Strikingly, gonadectomy dramatically activates these WT1(+) cells and leads to their differentiation into gonadal steroidogenic tissue. Thus, our data describe a mechanism of response to organ loss by recreating hormone-producing cells at a heterotopic site.
Project description:GATA4 and GATA6 are zinc-finger transcription factors that regulate specific genes involved in steroidogenesis. Using RNA interference (RNAi)-mediated reduction of GATA4 and/or GATA6 with microarray analysis, we aimed to identify novel GATA target genes in luteinizing porcine granulosa cells under vehicle- and cAMP-treated conditions. Microarray analysis identified IGF1 mRNA to be cAMP- and GATA-responsive, and real-time PCR demonstrated that the cAMP-induced increase in IGF1 mRNA was reduced under conditions of GATA6 depletion and GATA4 plus GATA6 depletion, but not GATA4 depletion. Insulin-like growth factor 1 protein levels in media were also decreased by GATA6 or GATA4 plus GATA6 reduction. IGFBP2 and IGFBP4 mRNAs were increased and IGFBP5 mRNA decreased with vehicle and cAMP treatment under GATA4 plus GATA6 RNAi conditions. GATA6 reduction alone increased basal IGFBP4 and decreased IGFBP5 with both vehicle and cAMP, and GATA4 reduction alone lowered cAMP IGFBP5 levels with cAMP. No changes in IGFBP3 mRNA were observed with GATA reduction relative to the control RNAi condition. Levels of insulin-like growth factor binding proteins 2-5 in media as assessed by Western ligand blotting were not altered by GATA reduction. Electromobility gel shift assays with two GATA-containing oligonucleotides of the IGF1 5'-regulatory region showed GATA4 and GATA6 could bind the more proximal GATA-B site. These studies indicate that although GATA4 and GATA6 can bind the porcine IGF1 5'-region, GATA6 is functionally most important for cAMP-stimulated mRNA levels. Using microarray analysis, we identified other mRNAs that were altered by GATA-reduced conditions, including ALDH1, DIO2, and EDNRB. Our findings further support GATA as a coordinator of endocrine/paracrine/autocrine signals in the ovary.
Project description:The enzyme P450c17 is required for glucocorticoid, sex steroid, and some neurosteroid biosynthesis. Defective human P450c17 causes sexual infantilism and 46,XY sex reversal but is compatible with life, whereas ablation of the corresponding mouse gene causes embryonic lethality at around E7. Normal mouse embryos express P450c17 protein and activity in the embryonic endoderm at E7. Adult adrenal and gonadal steroidogenesis requires steroidogenic factor-1 (SF-1), but SF-1 is not expressed in the early mouse embryo. We show that P450c17 is expressed in differentiated mouse parietal and visceral endoderm lineages, in cultured mouse F9 embryonic carcinoma stem cells, in mouse embryonic stem cells, and in cultured mouse P19 stem cells. Bases -110 to -55 (which contain an SF-1 site and two potential GATA sites) of the rat cyp17 gene confer promoter activity in F9 cells. Overexpression of SF-1 has no effect, whereas overexpression of GATA4 in F9 cells increases transcription from -110/-55 fused to a reporter and increases endogenous P450c17 mRNA. Chromatin immunoprecipitation assays show that GATA4 binds to -215/+55 of mouse cyp17. Stimulating F9 cells with retinoic acid and cAMP differentiates them into visceral and parietal endoderm. Commensurate with cell differentiation, quantitative PCR showed increased GATA4 and GATA6 mRNAs, temporally followed by increased P450c17 mRNA. Small interfering RNA inhibition of GATA4 or GATA6 in undifferentiated or differentiated F9 cells diminished endogenous cyp17 expression. Thus, P450c17 is expressed in mouse embryonic stem cells, its expression increases upon differentiation to an early embryonic endoderm lineage, and GATA4/6 are responsible for activation of P450c17 gene expression at this early stage of embryonic development.