GATA4 is a critical regulator of gonadectomy-induced adrenocortical tumorigenesis in mice.

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.

GATA4 deficiency impairs ovarian function in adult mice.

Transcription factor GATA4 is expressed in granulosa cells and, to a lesser extent, in other ovarian cell types. Studies of mutant mice have shown that interactions between GATA4 and its cofactor, ZFPM2 (also termed FOG2), are required for proper development of the fetal ovary. The role of GATA4 in postnatal ovarian function, however, has remained unclear, in part because of prenatal lethality of homozygous mutations in the Gata4 gene in mice. To circumvent this limitation, we studied ovarian function in two genetically engineered mouse lines: C57BL/6 (B6) female mice heterozygous for a Gata4-null allele, and 129;B6 female mice in which Gata4 is deleted specifically in proliferating granulosa cells using the Cre-loxP recombination system and Amhr2-cre. Female B6 Gata4(+/-) mice had delayed puberty but normal estrous cycle lengths and litter size. Compared to wild-type mice, the ovaries of gonadotropin-stimulated B6 Gata4(+/-) mice were significantly smaller, released fewer oocytes, produced less estrogen, and expressed less mRNA for the putative GATA4 target genes Star, Cyp11a1, and Cyp19. Gata4 conditional knockout (cKO) mice had a more severe phenotype, including impaired fertility and cystic ovarian changes. Like Gata4(+/-) mice, the ovaries of gonadotropin-stimulated cKO mice released fewer oocytes and expressed less Cyp19 than those of control mice. Our findings, coupled with those of other investigators, support the premise that GATA4 is a key transcriptional regulator of ovarian somatic cell function in both fetal and adult mice.

GATA4 regulates Sertoli cell function and fertility in adult male mice.

Transcription factor GATA4 is expressed in Sertoli and Leydig cells and is required for proper development of the murine fetal testis. The role of GATA4 in adult testicular function, however, has remained unclear due to prenatal lethality of mice harboring homozygous mutations in Gata4. To characterize the function of GATA4 in the adult testis, we generated mice in which Gata4 was conditionally deleted in Sertoli cells using Cre-LoxP recombination with Amhr2-Cre. Conditional knockout (cKO) mice developed age-dependent testicular atrophy and loss of fertility, which coincided with decreases in the quantity and motility of sperm. Histological analysis demonstrated Sertoli cell vacuolation, impaired spermatogenesis, and increased permeability of the blood-testis barrier. RT-PCR analysis of cKO testes showed decreased expression of germ cell markers and increased expression of testicular injury markers. Our findings support the premise that GATA4 is a key transcriptional regulator of Sertoli cell function in adult mice.

Neural tube defects in mice with reduced levels of inositol 1,3,4-trisphosphate 5/6-kinase.

Inositol 1,3,4-trisphosphate 5/6-kinase (ITPK1) is a key regulatory enzyme at the branch point for the synthesis of inositol hexakisphosphate (IP(6)), an intracellular signaling molecule implicated in the regulation of ion channels, endocytosis, exocytosis, transcription, DNA repair, and RNA export from the nucleus. IP(6) also has been shown to be an integral structural component of several proteins. We have generated a mouse strain harboring a beta-galactosidase (betagal) gene trap cassette in the second intron of the Itpk1 gene. Animals homozygous for this gene trap are viable, fertile, and produce less ITPK1 protein than wild-type and heterozygous animals. Thus, the gene trap represents a hypomorphic rather than a null allele. Using a combination of immunohistochemistry, in situ hybridization, and betagal staining of mice heterozygous for the hypomorphic allele, we found high expression of Itpk1 in the developing central and peripheral nervous systems and in the paraxial mesoderm. Examination of embryos resulting from homozygous matings uncovered neural tube defects (NTDs) in some animals and axial skeletal defects or growth retardation in others. On a C57BL/6 x 129(P2)Ola background, 12% of mid-gestation embryos had spina bifida and/or exencephaly, whereas wild-type animals of the same genetic background had no NTDs. We conclude that ITPK1 is required for proper development of the neural tube and axial mesoderm.

Molecular genetics of congenital diaphragmatic defects.

Congenital diaphragmatic hernia (CDH) is a severe birth defect that is accompanied by malformations of the lung, heart, testis, and other organs. Patients with CDH may have any combination of these extradiaphragmatic defects, suggesting that CDH is often a manifestation of a global embryopathy. This review highlights recent advances in human and mouse genetics that have led to the identification of genes involved in CDH. These include genes for transcription factors, molecules involved in cell migration, and extracellular matrix components. The expression patterns of these genes in the developing embryo suggest that mesenchymal cell function is compromised in the diaphragm and other affected organs in patients with CDH. We discuss potential mechanisms underlying the seemingly random combination of diaphragmatic, pulmonary, cardiovascular, and gonadal defects in these patients.

Adrenocortical tumorigenesis, luteinizing hormone receptor and transcription factors GATA-4 and GATA-6.

Luteinizing hormone (LH/hCG) responsiveness of normal and pathological human adrenal glands as well as the possibility of constitutive expressions of luteinizing hormone receptor (LHR) in adrenal cortex has been reported. Some recent studies showed a correlation between the LHR and abundant GATA-4 expression in both metastasizing and non-metastasizing human adrenocortical tumors, but not in normal adrenals, implicating the putative relevance of LHR and GATA-4 for adrenocortical pathophysiology. However, the physio- and pathophysiological significance of LHR and GATA-4 in the mechanism of adrenocortical tumorigenesis remains unclear. The paucity of suitable models for adrenal tumorigenesis makes the establishment of proper animal models highly important. LHR expression in the murine adrenal gland is an exception and not found in wild-type (WT) animal. We have previously shown that ectopic LHR expression in the murine adrenal gland can be induced by chronically elevated LH levels. We have generated a gonadotropin-responsive adrenal tumor model in gonadectomized transgenic (TG) mice expressing the inhibin alpha promoter/Simian Virus 40 T antigen transgene (inhalpha/Tag). Given the induction of expression and regulation of GATA-4 and GATA-6 zinc finger transcription factors in the gonads by gonadotropins, this review will explore their relationship to LHR expression and their role in adrenocortical tumorigenesis. A functional link between LHR and GATA-4 actions in the adrenal pathophysiology is proposed.

GATA transcription factors in adrenal development and tumors.

Of the six GATA transcription factors, GATA-4 and GATA-6 are expressed in the mouse and human adrenal with distinct developmental profiles. GATA-4 is confined to the fetal cortex, i.e. to the less differentiated proliferating cells, while GATA-6 is expressed both in the fetal and adult adrenal. In vitro, GATA-4 regulates inhibin-alpha and steroidogenic factor-1 implicated in normal adrenal function. GATA-6 probably has roles in the development and differentiation of adrenocortical cells, and in the regulation of steroidogenesis. GATA-4 expression is dramatically upregulated and GATA-6 downregulated in gonadotropin dependent mouse adrenocortical tumors. This is accompanied by the appearance of luteinizing hormone receptor (LHR). In vitro, GATA-4 transactivates LHR promoter, and gonadotropins upregulate GATA-4 levels. Human adrenal tumors occasionally express GATA-4, whereas GATA-6 levels are usually lower than normal.

GATA-4 is required for sex steroidogenic cell development in the fetal mouse.

The transcription factor GATA-4 is expressed in Sertoli cells, steroidogenic Leydig cells, and other testicular somatic cells. Previous studies have established that interaction between GATA-4 and its cofactor FOG-2 is necessary for proper Sry expression and all subsequent steps in testicular organogenesis, including testis cord formation and differentiation of both Sertoli and fetal Leydig cells. Since fetal Leydig cell differentiation depends on Sertoli cell-derived factors, it has remained unclear whether GATA-4 has a cell autonomous role in Leydig cell development. We used two experimental systems to explore the role of GATA-4 in the ontogeny of testicular steroidogenic cells. First, chimeric mice were generated by injection of Gata4-/- ES cells into Rosa26 blastocysts. Analysis of the resultant chimeras showed that in developing testis Gata4-/- cells can contribute to fetal germ cells and interstitial fibroblasts but not fetal Leydig cells. Second, wild-type or Gata4-/- ES cells were injected into the flanks of intact or gonadectomized nude mice and the resultant teratomas examined for expression of steroidogenic markers. Wild-type but not Gata4-/- ES cells were capable of differentiating into gonadal-type steroidogenic lineages in teratomas grown in gonadectomized mice. In chimeric teratomas derived from mixtures of GFP-tagged Gata4+/+ ES cells and unlabeled Gata4-/- ES cells, sex steroidogenic cell differentiation was restricted to GFP-expressing cells. Collectively these data suggest that GATA-4 plays an integral role in the development of testicular steroidogenic cells.

Impaired mesenchymal cell function in Gata4 mutant mice leads to diaphragmatic hernias and primary lung defects.

Congenital diaphragmatic hernia (CDH) is an often fatal birth defect that is commonly associated with pulmonary hypoplasia and cardiac malformations. Some investigators hypothesize that this constellation of defects results from genetic or environmental triggers that disrupt mesenchymal cell function in not only the primordial diaphragm but also the thoracic organs. The alternative hypothesis is that the displacement of the abdominal viscera in the chest secondarily perturbs the development of the heart and lungs. Recently, loss-of-function mutations in the gene encoding FOG-2, a transcriptional co-regulator, have been linked to CDH and pulmonary hypoplasia in humans and mice. Here we show that mutagenesis of the gene for GATA-4, a transcription factor known to functionally interact with FOG-2, predisposes inbred mice to a similar set of birth defects. Analysis of wild-type mouse embryos demonstrated co-expression of Gata4 and Fog2 in mesenchymal cells of the developing diaphragm, lungs, and heart. A significant fraction of C57Bl/6 mice heterozygous for a Gata4 deletion mutation died within 1 day of birth. Developmental defects in the heterozygotes included midline diaphragmatic hernias, dilated distal airways, and cardiac malformations. Heterozygotes had any combination of these defects or none. In chimeric mice, Gata4(-/-) cells retained the capacity to contribute to cells in the diaphragmatic central tendon and lung mesenchyme, indicating that GATA-4 is not required for differentiation of these lineages. We conclude that GATA-4, like its co-regulator FOG-2, is required for proper mesenchymal cell function in the developing diaphragm, lungs, and heart.

Gonadotropin-induced adrenocortical neoplasia in NU/J nude mice.

In response to prepubertal gonadectomy certain inbred mouse strains, including DBA/2J, develop sex steroid-producing adrenocortical neoplasms. This phenomenon has been attributed to a lack of gonadal hormones or a compensatory increase in gonadotropins. To assess the relative importance of these mechanisms, we created a new inbred model of adrenocortical neoplasia using female NU/J nude mice. These mice developed adrenocortical neoplasms in response to either gonadectomy or gonadotropin elevation from xenografts of human chorionic gonadotropin (hCG)-secreting Chinese hamster ovary cells. In each instance the adrenal tumors resembled the neoplasms found in gonadectomized DBA/2J mice and were composed of spindle-shaped A cells and lipid-laden B cells. Both cell populations were defined by ectopic expression of GATA-4 and an absence of the adrenocortical markers melanocortin-2-receptor and steroid 21-hydroxylase, but only B cells expressed the gonadal steroidogenic markers inhibin-alpha, LH receptor, P450c17, and P450c19. Expression of sex steroidogenic markers was attenuated in the neoplastic adrenal cortex of hCG-treated vs. gonadectomized mice. Whereas neoplastic adrenals were an obvious source of estradiol in gonadectomized mice, ovaries appeared to be the major source of this hormone in hCG-treated mice. Gonadectomy and hCG treatment elicited comparable increases in serum estradiol, but testosterone levels increased significantly only in hCG-treated mice. We conclude that chronic gonadotropin elevation, caused by either gonadectomy or hCG administration, signals a population of cells in the adrenal subcapsular region of permissive mice to undergo differentiation along a gonadal rather than an adrenal lineage. Thus, NU/J nude mice can be used as a model to study both neoplasia and adrenogonadal lineage specification.

Mouse strain susceptibility to gonadectomy-induced adrenocortical tumor formation correlates with the expression of GATA-4 and luteinizing hormone receptor.

Certain inbred strains of mice, including DBA/2J, develop adrenocortical tumors in response to gonadectomy. Spindle-shaped cells with limited steroidogenic capacity, termed A cells, appear in the subcapsular region of the adrenal gland, followed by sex steroid-producing cells known as B cells. These changes result from unopposed gonadotropin production by the pituitary, but the adrenocortical factors involved in tumorigenesis have not been characterized. GATA-4, a transcription factor normally expressed in fetal, but not adult, adrenocortical cells, was found in neoplastic cells that proliferate in the adrenal cortex of gonadectomized DBA/2J mice. GATA-4 mRNA was detected in the adrenal glands of female mice 0.5 months after ovariectomy and reached a maximum by 4 months. Castrated male mice developed adrenocortical tumors more slowly than gonadectomized females, and the onset of GATA-4 expression in the adrenal was delayed. In situ hybridization and immunohistochemistry revealed GATA-4 mRNA and protein in A and B cells, but not in normal adrenocortical cells. mRNA encoding another factor associated with adrenocortical tumorigenesis, LH receptor (LHR), was detected in A and B cells. In addition, transcripts for P450 17 alpha-hydroxylase/C17-C20 lyase, an enzyme essential for the production of sex steroids, and inhibin-alpha were found in B cells. Unilateral ovarian regeneration, a phenomenon known to occur in gonadectomized mice, was observed in a subset of DBA/2J mice undergoing complete ovariectomy. In these animals, adrenocortical tumor progression was arrested; A cells and GATA-4 expression were evident, but there was no expression of LHR or P450 17 alpha-hydroxylase/C17-C20 lyase. Strain susceptibility to adrenocortical tumorigenesis (DBA/2J >> FVB/N) correlated with the expression of GATA-4 and LHR, implicating these factors in the process of adrenocortical neoplasia in response to continuous gonadotropin stimulation.

Transcription factor GATA-6 is expressed in malignant endoderm of pediatric yolk sac tumors and in teratomas.

Transcription factors GATA-4 and GATA-6 play critical roles in mammalian yolk sac differentiation and function. Previously, we showed that GATA-4 is a potential marker for malignant yolk sac endoderm in pediatric germ cell tumors. This highly malignant tissue can cause diagnostic problems because yolk sac components may be difficult to differentiate from other, especially immature, tissue types in teratomas. In the search for new molecular markers for germ cell tumors, we have surveyed GATA-6 expression in benign and malignant pediatric germ cell tumors using mRNA in situ hybridization and immunohistochemistry. GATA-6 was expressed in most yolk sac tumors examined and also in nonmalignant tissues including gut/respiratory epithelium, sebocytes, and neuroepithelium in mature and immature teratomas. Given that GATA-6 has not been discovered in sebocytes before, this finding was confirmed by immunohistochemistry of normal mouse samples, indicating a function for this transcription factor in the mammalian skin. Taken together, GATA-6 can be used to identify yolk sac components in pediatric germ cell tumors. Furthermore, it is also expressed in specific tissues in teratomas. GATA-6, together with GATA-4, can thus be used as a novel molecular marker in characterizing of pediatric germ cell tumors.

Genetic mosaic analysis reveals that GATA-4 is required for proper differentiation of mouse gastric epithelium.

During mouse embryogenesis GATA-4 is expressed first in primitive endoderm and then in definitive endoderm derivatives, including glandular stomach and intestine. To explore the role of GATA-4 in specification of definitive gastric endoderm, we generated chimeric mice by introducing Gata4(-/-) ES cells into ROSA26 morulae or blastocysts. In E14.5 chimeras, Gata4(-/-) cells were represented in endoderm lining the proximal and distal stomach. These cells expressed early cytodifferentiation markers, including GATA-6 and ApoJ. However, by E18.5, only rare patches of Gata4(-/-) epithelium were evident in the distal stomach. This heterotypic epithelium had a squamous morphology and did not express markers associated with differentiation of gastric epithelial cell lineages. Sonic Hedgehog, an endoderm-derived signaling molecule normally down-regulated in the distal stomach, was overexpressed in Gata4(-/-) cells. We conclude that GATA-4-deficient cells have an intrinsic defect in their ability to differentiate. Similarities in the phenotypes of Gata4(-/-) chimeras and mice with other genetically engineered mutations that affect gut development suggest that GATA-4 may be involved in the gastric epithelial response to members of the TGF-beta superfamily.