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Endocrinology Vol. 143, No. 8 3136-3143
Copyright © 2002 by The Endocrine Society

ARTICLE

Differential Expression of GATA-4 and GATA-6 in Fetal and Adult Mouse and Human Adrenal Tissue

Sanne Kiiveri, Jianqi Liu, Mia Westerholm-Ormio, Naoko Narita, David B. Wilson, Raimo Voutilainen and Markku Heikinheimo

Children’s Hospital (S.K., M.W.-O., M.H.), Program for Developmental and Reproductive Biology (S.K., M.H.), Biomedicum Helsinki, and Department of Pathology (J.L., R.V.), University of Helsinki, 00290 Helsinki, Finland; Departments of Pediatrics (N.N., D.B.W., M.H.) and Molecular Biology and Pharmacology (D.B.W.), Washington University, St. Louis, Missouri 63110; and Department of Pediatrics, Kuopio University Hospital (R.V.), 70210 Kuopio, Finland

Address all correspondence and requests for reprints to: Markku Heikinheimo, M.D., Ph.D., Program for Developmental and Reproductive Biology, Biomedicum Helsinki, P.O. Box 63 (Haartmaninkatu 8), 00014 University of Helsinki, Finland. E-mail: . markku.heikinheimo{at}helsinki.fi


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Earlier work implicates transcription factors GATA-4 and GATA-6 in murine adrenal function. We have now studied their expression during mouse and human adrenal development in detail. GATA-4 and GATA-6 mRNAs and protein are readily detectable from embryonic d 14 and gestational wk 19 onwards in the mouse and human adrenal cortex, respectively. In the postnatal adrenal, GATA-4 expression is down-regulated, whereas GATA-6 mRNA and protein continue to be expressed. To clarify the significance of GATA-4 for early adrenocortical development, Gata4-/- ES cells were injected into eight-cell-stage embryos derived from ROSA26 mice, a transgenic line expressing ß-galactosidase in all cell types, including the adrenocortical cells. The resultant chimeric embryos were stained with X-gal to discriminate ES cell- and host-derived tissue. Gata4-/- cells contributed to adrenocortical cells in these chimeras, and these cells also expressed GATA-6. Taken together, our findings suggest that GATA-6 expression is needed throughout adrenal development from fetal to adult age. GATA-4, on the other hand, may serve a role in the fetal adrenal gene regulation, although it is not essential for early adrenocortical differentiation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE ADRENAL GLANDS have essential roles during fetal life in regulating intrauterine homeostasis and in preparing fetal organ systems, such as the lungs, for extrauterine life (reviewed in Ref. 1). The adrenal cortex undergoes rapid growth during fetal period and has high steroidogenic activity. In the human fetus, the adrenal cortex has two morphologically recognizable zones: an outer, narrow definitive zone and an inner fetal zone, which occupies about 80% of the cortical volume. Between these regions is a functionally distinct transitional zone. The fetal zone produces dehydroepiandrosterone sulfate (DHEA-S) for placental estrogen synthesis. Due to a lack of 3-ß-hydroxysteroid dehydrogenase expression (2), the fetal zone cannot synthesize cortisol from cholesterol or pregnenolone, but it is able to convert placental progesterone to cortisol (3). The fetal zone atrophies during the first months of postnatal life. The three adult cortical zones glomerulosa, fasciculata and reticularis, from capsule toward medulla, are established by 10–12 yr of age.

The mouse adrenal cortex differs structurally from that of humans. In mice, the adrenal gland is recognizable by embryonic day (E) 13, and it consists principally of cortical tissue (4). Medullary cells become distinguishable as late as E17. The adult mouse adrenal has cortical layers resembling those of humans: zona glomerulosa, intermedia, fasciculata, and reticularis (5). Mouse adrenal cortex also contains a so-called X-zone, which develops after birth adjacent to the inner aspect of the zona reticularis. It is well differentiated between d 13 and 20 of postnatal life. The function of the X-zone is uncertain, but it may have steroidogenic activity (6). The X-zone disappears after puberty in males and during the first pregnancy in females.

Transcription factors required for normal adrenocortical development and function include steroidogenic factor 1 (SF-1) and dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X-chromosome, gene 1 (DAX-1). Mice homozygous for the disruption of the Ftz-F1 gene, which encodes SF-1, completely lack adrenal glands (7). In normal adrenals SF-1 is thought to have a key role in regulating the steroid hydroxylases (reviewed in Ref. 8). Mutations in DAX-1 gene give rise to X-linked congenital adrenal hypoplasia (9, 10). DAX-1 has been implicated as one of the target genes for SF-1 (11, 12), and on the other hand DAX-1 can inhibit the transcriptional activity of SF-1 (13).

Another class of transcription factors implicated in adrenal function is GATA proteins. These molecules belong to a family of structurally related zinc finger proteins that regulate gene expression, development, and proliferation in a variety of tissues. Six vertebrate GATA-proteins have been identified and they bind to the consensus sequence (A/T)GATA(A/G) in the promoter and enhancer regions of their target genes (14, 15). GATA-4 and GATA-6 are highly conserved across species. Mouse GATA-4 has 90% homology with the amino acid sequence of human GATA-4 (16). Similarly, GATA-6 protein is highly homologous in different mammalian species. GATA-4 is known to be essential for ventral morphogenesis (17, 18) and GATA-6 has an important role in the development of the extraembryonic endoderm (19, 20). In the endocrine system, GATA-4 and GATA-6 are both expressed in testis and ovary, where they are under hormonal regulation (21, 22, 23, 24). We have previously shown that GATA-6 is expressed in the adrenal cortex of adult mice (25). There is no GATA-4 expression in normal postnatal mouse adrenals, but adrenocortical carcinomas arising in inhibin-{alpha} promoter/SV40 T-antigen transgenic mice abundantly express GATA-4 (25).

Of interest, recent reports have linked GATA transcription factors to SF-1. GATA-4 and SF-1 interact at the protein level and synergistically regulate the Müllerian-inhibiting substance (MIS) gene in vitro (26). Both SF-1 and GATA-4 binding sites in the MIS promoter have been shown to be essential for the activity of this promoter. The inhibin-{alpha} promoter has also been synergistically activated by GATA-4 and SF-1 (27). In addition, GATA-4 and other members of the GATA family interact with SF-1 promoter as studied by in vitro transactivation experiments (26, 27). In vitro studies suggest that GATA-4 may regulate expression of mouse fetoprotein transcription factor, a gene related to SF-1 (28). Thus, GATA factors have been functionally linked to SF-1, an essential regulator for early adrenal development.

To gain a better understanding of the role of GATA proteins in the adrenal function, we have now characterized the expression of GATA-4 and GATA-6 in the developing adrenal of normal and chimeric mice as well as of humans. We find GATA-4 mRNA and protein expression in the fetal adrenal cortex followed by a dramatic down-regulation soon after birth. In contrast, GATA-6 is expressed within the mouse and human adrenal cortex throughout development.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mouse tissues
Mouse embryos and young neonatal mice were obtained by mating male and female CBA or NMRI mice. The day of the appearance of the vaginal plug was designated as E0. Fetal mouse adrenal glands were dissected during E14, E15, and E17, and postnatal samples were collected at 1, 2, 7, 14, and 60 d after birth. Tissues were dissected in DMEM supplemented with GlutaMAX (Life Technologies, Inc., Paisley, Scotland, UK) and then snap frozen plain or in O.C.T. (optimal cutting temperature) cryopreservation solution (Tissue tek, Miles, Inc., Elkhart, IN).

Human tissues
Human fetal adrenals and other tissues were obtained from autopsy specimen of fetuses at 15 and 19 wk of gestation. The study protocol was approved by the Ethics Committee of the Helsinki City Maternity Hospital. Normal adult human adrenal samples were obtained from patients who underwent nephrectomy for kidney tumors. Tissue specimens were snap frozen in liquid nitrogen and kept at -70 C for RNA extraction, or fixed in formalin and embedded in paraffin for immunohistochemical studies and in situ hybridization.

Preparation of chimeric embryos
Chimeric mice were prepared and analyzed as described previously (29). In brief, Gata4-/- ES cells were injected into eight-cell-stage embryos or blastocysts derived from ROSA26 mice, a transgenic line that expresses ß-galactosidase in all cell types. Chimeric embryos were stained with X-gal to discriminate ES cell- and host-derived tissue, using previously described methods (29).

Cloning of the human GATA-4 and GATA-6 cDNAs
A 575-bp human GATA-4 cDNA was synthesized by PCR from human granulosa cell total cDNA by using oligonucleotides 5'-CTC CTT CAG GCA GTG AGA GC and 5'-GAG ATG CAG TGT GCT CGT GC, designed according to a human GATA-4 cDNA (GenBank accession no. NM-002052). The cycling conditions for PCR were as follows: 94 C for 15 sec, 58 C for 30 sec, 72 C for 1 min with 30 cycles, followed by a final extension 20 min at 72 C. Similarly a 712-bp human GATA-6 cDNA was synthesized from the same source by using oligonucleotides 5'-ATG ACT CCA ACT TCC ACC TCT and 5'-CAG CCT CCA GAG ATG TGT AC, designed according to a human GATA-6 cDNA (GenBank accession no. NM-005257). The cycling conditions for PCR were the same as above except the annealing temperature at 56 C. The oligonucleotides were prepared at the Haartman Institute, University of Helsinki (Helsinki, Finland). The human GATA-4 and GATA-6 PCR products were ligated into the pCR 2.1-TOPO vector and subcloned into the EcoRI site of the pGEM-7Zf(+/-) vector (Promega Corp., Madison, WI). The sequences of the cloned PCR products were verified with an ABI PRISM 377 DNA sequencer (PE Applied Biosystems, Foster City, CA).

In situ hybridization
Paraffin-embedded and frozen tissue samples were cut into 8- or 10-µm sections. Frozen sections were fixed in 4% paraformaldehyde in PBS and all sections were subjected to in situ hybridization as previously described (30). A total of seven E15, eight E17, and four 2-d-old mice were analyzed. Human samples included three fetal and two adult samples. Tissue sections were incubated with 1.2 x 106 cpm 33P-labeled (1000–3000 Ci/mmol; Amersham Pharmacia Biotech, Arlington Heights, IL) antisense or sense riboprobe in a total volume of 80 µl. The riboprobes for mouse GATA-4 and GATA-6 were prepared as described previously (21, 31, 32) and the preparation of the human GATA-4 and GATA-6 cDNAs is described in detail above.

RNA isolation and Northern blot analysis
Total RNA was isolated from the frozen tissues by ultracentrifugation through a cesium chloride cushion (33). The concentration of RNA was determined by spectrophotometric analysis at 260 nm (Ultrospec 2000, Amersham Pharmacia Biotech, Cambridge, UK). Fourteen micrograms of denatured RNA was subjected to electrophoresis on a 1% denaturing agarose gel and then transferred onto nylon membranes. The membranes with mouse RNA were hybridized with 32P-labeled (>6000 Ci/mmol, Amersham Pharmacia Biotech) GATA-4 cDNA probe or GATA-6 riboprobe, respectively. GATA-4 mouse cDNA probe was prepared from a fragment of mouse GATA-4 cDNA (31). The riboprobe for mouse GATA-6 was the same as used for in situ hybridization. Ribosomal RNA (18S) (Ambion, Inc., Austin, TX; catalog no. 7328) was used as a loading control. Hybridizations were performed at 60 C overnight (HB-1 D hybridization oven, Techne, Cambridge, UK) and washed three times 20 min at 60 C with 1x saline sodium citrate/0.1% sodium dodecyl sulfate. Hybridization signals were detected by autoradiography using Agfa Curix Ortho ST-L film (Agfa-Gavaert N. V., Mortsel, Belgium).

RT-PCR analysis
Total RNA was isolated and measured the same way as for Northern analysis. One microgram of RNA was reverse transcribed using Random primer (Roche Molecular Biochemicals, Basel, Switzerland, catalog no. 1034731). Two microliters of the reverse transcriptase (RT) reaction was used for each PCR and the primers used for amplifying a 414-bp mouse GATA-4 cDNA were 5'-CTC TGG AGG CGA GAT GGG AC corresponding to nucleotides 1254–1273 and 5'-CGT CGT CAC TTC TCT ACG CG corresponding to nucleotides 1648–1667, designed according to the mouse GATA-4 mRNA (GenBank accession no. NM-008092). The PCR amplification was performed in the DNA Thermal Cycler (Perkin Elmer Cetus, Norwalk, CT) with the following cycle profile: 95 C for 1 min, 60 C for 1 min and 72 C for 1 min for 35 cycles, followed by a final extension for 10 min at 72 C. The quality of RNA extracted was tested by PCR amplification of a 213-bp sequence of human ß-actin mRNA (GenBank accession no. XM-037235) from the same RT-reaction as for GATA-4. The primers used were 5'-CGG GAA ATC GTG CGT GAC ATT AAG and 5'-TTC GTG GAT GCC ACA GGA CTC C corresponding to nucleotides 689–712 and 880–901, respectively. The cycle profile for ß-actin was: 95 C for 15 sec, 56 C for 20 sec, and 72 C for 30 sec for 25 cycles, followed by a final extension for 20 min at 72 C. Twenty microliters of GATA-4 and 10 µl of ß-actin PCR products were simultaneously electrophoresed on a 2% agarose gel stained with 0.5 µg/ml ethidium bromide and visualized under UV light, followed by scanning of the gel (Quantity One 4.2, Bio-Rad Laboratories, Inc., Hercules, CA). A 50-bp DNA marker (MBI Fermentas, Amherst, NY, catalog no. SMO373S,) was used to showing the fragment sizes for comparison.

Immunohistochemistry
Frozen mouse tissue samples were fixed in 4% paraformaldehyde. The mouse and human tissue sections were then subjected to immunohistochemistry using commercial polyclonal antimouse GATA-4 IgG (1:200) or antimouse GATA-6 IgG (1:200) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, catalog nos. sc-1237 and sc-9055), or nonimmune IgG (1:1000) as the primary antibody. The avidin-biotin immunoperoxidase system was used to visualize bound antibody (Vectastain Elite ABC Kit, Vector Laboratories, Inc., Burlingame, CA). We used 3-amino-9-ethylcarbazole or 3,3'-diaminobenzedine (Sigma, St. Louis, MO) as the chromogen, and the development reaction occurred in the presence of 0.03% H2O2. Hematoxylin was used as counterstain.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
GATA-4 and GATA-6 are expressed in the fetal adrenal cortex of mice and humans
We initially surveyed the expression of GATA-4 and GATA-6 mRNA in the mouse adrenal samples by RT-PCR or Northern blot analysis. Using Northern analysis, we could not detect significant GATA-4 transcripts (data not shown), but RT-PCR revealed GATA-4 mRNA in the fetal adrenal tissue from E15 onwards (Fig. 1AGo). GATA-6 mRNA was found as early as at E15 and E17 by Northern analysis (Fig. 1BGo). Next we used in situ hybridization to study the temporal and spatial expression of GATA-4 and GATA-6 mRNA in the mouse adrenal samples. GATA-4 message was scattered over the various cell types and layers of mouse fetal adrenal cortex in E15 and E17 (Fig. 2Go, A and B), but this transcript was not expressed in adrenal medulla. Intense GATA-6 mRNA signal was uniformly distributed over the fetal adrenal cortex (Fig. 2Go, D and E). Using immunohistochemistry, GATA-4 protein was detected in the nuclei of separate adrenal cells spread over the E14–17 adrenals (Fig. 3AGo). Embryonal adrenocortical cells also expressed GATA-6 protein (Fig. 3BGo).



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  		Figure 1. Expression of GATA-4 and GATA-6 mRNA during mouse adrenal development. A, RT-PCR analysis of GATA-4 (upper lanes) in mouse adrenals during E15 and E17, and P1, P7, P14, and P60. ß-actin (lower lanes) was used as a RNA quality control. Testis (Te) is shown as positive and spleen (Sp) and water (Aq) as negative controls The PCR products were of the expected sizes 414 bp (GATA-4) and 213 bp (ß-actin). B, Northern blot analysis of E15 and E17 and P1, P7, P14, and P60 mouse adrenals demonstrates intense GATA-6 expression in all fetal and postnatal samples. RNA from mouse testis (Te) was used as a positive control. 18S transcripts are shown as a control for loading. Exposure time overnight.

 


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  		Figure 2. RNA in situ hybridization of mouse adrenal at E15. The expression patterns of GATA-4 (A and B) and GATA-6 (D and E) mRNA in the adrenal cortex are demonstrated. Similar expression pattern for GATA-4 and GATA-6 was seen at E17 (data not shown). Sense probes were used as negative controls for GATA-4 (C) and GATA-6 (F). H&E (A and D) and darkfield views (B, C, E, and F). Original magnification, x100.

 


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  		Figure 3. Immunohistochemistry for GATA-4 and GATA-6 of mouse E17 adrenal. GATA-4 positive nuclei are in small clusters next to the adrenal capsule (A). Faint GATA-4 reactivity is also seen in some of the inner cells of the developing adrenal gland. Most of the cortical cells express GATA-6 protein (B). Original magnification, x200.

 
We used in situ hybridization also in human fetal adrenals to study the GATA-4 and GATA-6 expression. GATA-4 mRNA was detected in the fetal adrenal cortex at gestational week 19 (Fig. 4Go, A and B). The expression pattern was patchy, not following specifically any of the adrenocortical layers, although GATA-4 mRNA localized mainly to the fetal zone. GATA-6 mRNA expression was abundant in the human fetal adrenal (Fig. 4Go, D and E) and localized in both fetal and definitive adrenocortical zones, being, however, more prominent in the definitive zone (Fig. 4Go, D and E).



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  		Figure 4. RNA in situ hybridization of 19-wk-old human fetal adrenal. GATA-4 mRNA transcripts are seen scattered in the fetal zone (A, B), whereas GATA-6 mRNA signal is uniformly distributed throughout the adrenal cortex (D, E). Only background signal is seen with the sense probes for GATA-4 (C) and GATA-6 (F). H&E (A, D) and darkfield views (B, C, E, F). Original magnification, x100.

 
Expression of GATA-4, but not GATA-6, is down-regulated in the adrenal after birth
Postnatal mouse adrenal samples also showed GATA-4 mRNA by RT-PCR (Fig. 1AGo), whereas only a little mRNA was detected in the outermost adrenocortical layers during postnatal d (P) 1 by in situ hybridization (Fig. 5Go, A and B). Consistent with the in situ hybridization results, immunohistochemistry on newborn mouse adrenals revealed GATA-4 protein only in rare isolated cells in the subcapsular region (data not shown). GATA-6 was highly expressed postnatally (Figs. 1BGo and 5Go, C and D).



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  		Figure 5. RNA in situ hybridization of mouse adrenal on P2. Low expression of GATA-4 (A and B), but abundant GATA-6 (C and D) mRNA in the adrenal cortex is detected. H&E (A and C) and darkfield views (B and D). Original magnification, x100.

 
We did not have access to normal adrenal tissue from children, but we were able to examine normal adult human samples. GATA-4 mRNA was undetectable in in situ hybridization of human adult adrenal samples (Fig. 6Go, A and B). In contrast, GATA-6 mRNA was abundantly expressed in cortical zones fasciculata and reticularis, with a clear predominance in the zona reticularis (Fig. 6Go, C and D). Immunohistochemistry confirmed the findings of in situ hybridization; we could not detect any GATA-4 protein in the adult samples (Fig. 7AGo), whereas GATA-6 protein was present in a large proportion of adrenocortical cells (Fig. 7BGo).



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  		Figure 6. RNA in situ hybridization of human adult adrenal for GATA-4 and GATA-6. GATA-6 mRNA is expressed in the cortical zones fasciculata (z.f.) and reticularis (z.r.) (C and D), whereas no GATA-4 (A and B) can be detected in the same sample. z. g., Zona glomerulosa; m, medulla. H&E (A and C) and darkfield views (B, D). Original magnification, x100.

 


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  		Figure 7. Immunohistochemistry for GATA-4 and GATA-6 of adult human adrenal. No GATA-4 protein (A) is present, whereas GATA-6 protein is localized into the nuclei of adrenocortical cells (B). Original magnification, x100.

 
GATA-4 is not essential for fetal adrenal development
That GATA-4 is expressed in fetal adrenal gland and then down-regulated after birth suggests a role for this factor in early adrenal development. To test whether GATA-4 is required for adrenocortical development, we performed genetic mosaic analysis. GATA-4 null mice die before E9 (17, 18); thus, insights into the role of GATA-4 in adrenal gland development cannot be ascertained from crosses of heterozygotes. To circumvent this problem, we used chimeric mice to study the role of GATA-4 in adrenocortical differentiation and gene expression. Gata4-/- ES cells (or control ES cells) were injected into eight-cell-stage embryos or blastocysts derived from ROSA26 mice, a transgenic line that expresses ß-galactosidase in most cell types, including adrenal cells (34, 35, 36). The resultant chimeric embryos (E15–E18) were stained with X-gal to discriminate ES cell- and host-derived tissue.

Gata4-/- ES cells contributed to adrenocortical cells in these Gata4-/- {leftrightarrow} ROSA26 chimeras (Fig. 8Go, A and D, arrows). When stained with hematoxylin and eosin (H&E), the morphology was similar in the host-derived adrenocortex compared with the cortex derived from Gata4 deficient ES cells (Fig. 8Go, B and E). To further assess the functional integrity of the cortical areas arising from Gata4-/- cells, in situ hybridization was performed for GATA-6; the results demonstrated that GATA-6 mRNA is evenly distributed throughout the cortex (Fig. 8Go, C and F), i.e. in both wild-type (Gata4+/+) and Gata4-deficient areas. We conclude that GATA-4 is not essential for adrenocortical cell differentiation in fetal mice.



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  		Figure 8. GATA-4 deficient ES cells can contribute to the adrenal cortex. Cryosections from a Gata4-/- ES cell x Rosa26 chimeric embryo were stained with X-gal; the Gata4 deficient tissue is indicated by an arrow (A and D; the arrow indicates the very same Gata4-/- area in all the panels, A–F). Note that both wild-type (ß-galactosidase positive) and Gata4-/- cells contribute to the adrenocortical cells (arrow in B and E). In situ hybridization demonstrates even distribution of GATA-6 (brightfield in B and E, darkfield in C and F) mRNA throughout the adrenal cortex including the Gata4 deficient cells (arrow). Boxes in panels A–C indicate the area magnified in panels D–F. k indicates kidney. Original magnifications, x100 (A–C) and x200 (D–F).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study demonstrates that GATA-4 is expressed during fetal adrenocortical development in mice and humans. In our previous study, we could not detect GATA-4 in the adult mouse adrenals by immunohistochemistry or in situ hybridization (25). By using the highly sensitive RT-PCR, we could now find GATA-4 also in the adult mouse adrenals. On the contrary, GATA-6 is abundantly expressed throughout development in the mouse and human adrenal. Thus, the developmental expression patterns of GATA-4 and GATA-6 differ significantly, implying distinct roles for these factors during adrenal organogenesis and function. The expression pattern of GATA-4 suggests a specific role for this factor in the fetal adrenal.

GATA-4 has been functionally linked to SF-1 in regulating the MIS and StAR (steroidogenic acute regulatory protein) promoters (26, 27, 28, 37, 38). In rat ovarian cells GATA-4 is required for the transcription of StAR (39), involved in the biosynthesis of steroid hormones in both the ovary and adrenal gland (40, 41). Therefore it is possible that GATA-4 or GATA-6 regulate StAR in the adrenal. Inhibin-{alpha} has also been indicated (22) as a possible target gene for GATA-4 in gonads. In fetal adrenals, inhibin subunit genes are expressed during the second trimester of gestation, whereas adult adrenals express them to a lesser extent (42). This expression pattern thus closely resembles that of GATA-4 in the present study and implicates inhibin-{alpha} as a potential target of GATA-4 also in the adrenal.

During the fetal period, the adrenal gland undergoes rapid growth and is highly active in steroid hormone production. GATA-4 might be associated with the proliferative signals in the adrenal cortex, given that its expression has been connected to cell proliferation in other tissues. Accordingly, GATA-4 expression in ovary has been shown to be most abundant in granulosa cells during their active proliferative phase (21, 23). In addition, mouse testicular Sertoli cells have high GATA-4 expression at the time of their proliferation prepubertally (22, 43). On the other hand, GATA-4 mRNA is down-regulated in mouse granulosa cells through apoptosis before follicular atresia (21).

Our earlier study revealed that GATA-4 protein is present in human adrenocortical carcinomas and in a transgenic mouse model developing adrenocortical tumors (25). In light of this study, adrenocortical carcinoma expression pattern resembles the fetal type of gene expression. Previous studies have shown that GATA-4 expression is up-regulated by FSH and LH in granulosa, Sertoli, and Leydig cells (21, 22). Human chorionic gonadotropin has been suggested to participate in the regulation of the fetal adrenal function (reviewed in Ref. 1). Recent evidence also suggest links between gonadotropin receptors and GATA-4 because functional LH-receptors appear along with GATA-4 expression in adrenocortical carcinomas of inhibin-{alpha} SV40 T-antigen-transgenic mice (our unpublished results). Moreover, GATA-4 is up-regulated in a dose-dependent manner by LH in an immortalized cell line arising from these tumors. These data suggest a link between GATA-4 and gonadotropins during endocrine adrenal development and an interactive role for them in the adrenocortical tumors.

While various lines of evidence suggest that GATA-4 can regulate gene expression in the adrenal gland and other steroidogenic organs, our studies with Gata4-/- chimeric mice indicate that this factor is not required for adrenocortical cell differentiation in the fetal mouse. In addition, GATA-4 deficient adrenocortical cells expressed GATA-6 in the developing adrenal cortex. On the other hand, there may be redundancy between GATA-4 and GATA-6, and GATA-6 could have overtaken the lost functions of Gata4 in this experimental model. There is also evidence of elevated GATA-6 levels in Gata4 deficient embryoid bodies (44). We could not, however, demonstrate that GATA-6 was elevated in the Gata4-/- derived adrenocortical regions of the chimeric mice (this study).

In contrast to GATA-4, GATA-6 is expressed throughout fetal and postnatal development. In addition, StAR mRNA is expressed in embryonic and adult mouse adrenal similarly to GATA-6 (45). In fetal adrenals, GATA-6 is evenly distributed over various cortical zones, but in human adult adrenal the strongest expression is found in zona reticularis. DHEA-S is the principal steroid product of the primate fetal adrenal, and zona reticularis is the main source of adrenal sex steroids in adult adrenals. This similarity in the temporospatial expression pattern of DHEA-S and GATA-6 suggests a possible connection between them. Of note, GATA-6 is also expressed in ovarian and testicular cells involved in steroidogenesis (21, 22). The role of GATA-6 in the gene regulation in these tissues remains, however, unclear.

Taken together, our findings suggest a role for GATA-4 in the fetal adrenal gene regulation and proliferation. Conversely, GATA-6 expression is needed throughout the adrenal development and function from fetal to adult age.


   Acknowledgments
 
We thank Dr. Ilkka Ketola, Dr. Mika Laitinen, Dr. Mikko Anttonen, Ms. Merja Haukka, Ms. Ulla Kiiski, Ms. Ritva Löfman (University of Helsinki, Finland), and Dr. Malgorzata Bielinska (Washington University, St. Louis, MO) for their expert assistance.


   Footnotes
 
This work was supported by the Finnish Pediatric Research Foundation (to S.K.), the University of Helsinki (to M.H.), the University Central Hospital in Helsinki (to M.H. and S.K.), the Academy of Finland (to M.H. and R.V.), Juselius Foundation (to M.H. and D.B.W.), the Finnish Medical Society (to M.W.-O.), and Kuopio University Hospital (to R.V.) and NIH Grant HL-61006 (to D.B.W.). Chimeric mice were generated in the NIH-supported Washington University Department of Pediatrics CHRC Genetically Altered Mouse Core.

Abbreviations: DAX-1, Dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X-chromosome, gene-1; DHEA-D, dehydroepiandrosterone sulfate; E, embryonic day; H&E, hematoxylin and eosin; MIS, Müllerian-inhibiting substance; RT, reverse transcriptase; SF-1, steroidogenic factor 1; StaR, steroidogenic acute regulatory protein.

Received November 16, 2001.

Accepted for publication April 9, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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