Regulation of Adrenal Development

The fetal adrenal cortex in humans consists of a large inner zone, known as the fetal zone, and a thin outer rim of immature cells termed the definitive zone.  The fetal zone produces adrenal androgens, which the placenta converts to estrogens.  After birth the fetal zone atrophies, and the definitive zone partitions into functionally distinct layers: the zona glomerulosa (zG), zona fasciculata (zF), and zona reticularis (zR), which produce mineralocorticoids, glucocorticoids, and adrenal androgens, respectively.  In the mouse adrenal gland the zG and zF are well defined, but the zR is difficult to discern. The postnatal mouse adrenal cortex contains an additional layer, the X-zone, which develops adjacent to the adrenal medulla. The X-zone is derived from the fetal zone and disappears at puberty in males and during the first pregnancy in females.

In collaboration with Dr. Markku Heikinheimo of the University of Helsinki, we have shown that that targeted mutagenesis of the gene for transcription factor GATA6 in the adrenal cortex of mice results in a complex phenotype that includes: 1) aberrant differentiation of adrenocortical stem/progenitor cells into gonadal-like cells, 2) ectopic chromaffin cells, and 3) abnormal zonation.

The accompanying figure shows that the adrenal glands of Gata6Flox/Flox; Sf1-cre mice lack an X-zone.  The upper panels show trichrome-stained adrenals from 1-mo-old control (left) and mutant (right) nulliparous female mice. Note the absence of an X-zone (double-headed arrow) in the mutant adrenal.  The middle panels show electron micrographs of juxtamedullary cells in adrenals from 2-mo-old control (left) and mutant (right) nulliparous female mice.  Cells with the ultrastructural hallmarks of X-zone, including peculiar mitochondrial complexes consisting of flattened mitochondria alternating with ER cisternae, are present in the control but not the mutant adrenal.  The bottom panels show H&E stained adrenals from 2-mo-old control (left) and mutant (right) mice that were orchiectomized at 3 weeks of age.  The mutant adrenal lacks a secondary X-zone (double-headed arrow) and has pronounced subcapsular cell hyperplasia.

Regulation of Steroidogenic Cell Differentiation

GATA4 is expressed in Sertoli cells, steroidogenic Leydig cells, and other testicular somatic cells.  We have used two experimental systems to explore the role of GATA4 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.

Panels A & B in the accompanying figure show multilabel immunostaining of a teratoma derived from wild-type ES cells.  The white arrowhead designates a presumptive Leydig cell (lc) that co-expresses GATA4 and the steroidogenic enzyme P450scc.  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 (panels C-E).  Collectively these data suggest that GATA4 plays an integral role in the development of testicular steroidogenic cells.

Molecular Basis of Adrenocortical Neoplasia

Ferrets and certain inbred strains of mice develop sex steroid-producing adrenocortical tumors in response to gonadectomy.  This process has been attributed to continuous LH production by the pituitary, but the adrenocortical factors involved in tumorigenesis are not understood well.  Our studies implicate GATA4 in postgonadectomy tumor formation in ferrets and DBA/2J mice.  We postulate that ectopic expression of LH receptor in adrenocortical cells confers sensitivity to LH, which in conjunction with GATA4, leads to changes in cell proliferation, differentiation, and sex steroid production.

The adjoining figure shows GATA4 immunoperoxidase staining (panels 1, 2, 3b) or H&E staining (panel 3a) of adrenocortical carcinomas from neutered ferrets. 

Collaborators on these studies include Dr. Matti Kiupel of Michigan State University, Dr. Markku Heikinheimo of the University of Helsinki, and Dr. Robi Mitra of the Department of Genetics.

Metabolic Basis of Neural Tube Defects

Neural tube defects (NTDs) are serious birth defects that affect approximately 0.1 percent of newborns.  Women with low blood levels of the vitamin folic acid are at increased risk of giving birth to a child with a NTD.  Folic acid supplementation of pregnant women prevents 70 percent of NTDs; the remaining 30 percent of cases are considered folic acid resistant.

Animal studies suggest that administration of another vitamin, inositol, can further reduce the incidence of NTDs.  Clinical trials aimed at preventing human NTDs through inositol supplementation are underway, and preliminary results are encouraging.  The mechanism for the protective effect of inositol, however, remains an enigma. 

In collaboration with Dr. Monita Wilson and Dr. Philip Majerus of the Department of Internal Medicine, we have shown that mice homozygous for a hypomorphic allele of Itpk1, a gene invovled in inositol metabolism, are predisposed to NTDs (including both anencephaly and spina bifida).  We hypothesize that the subset of human NTDs that are folic acid resistant, but that respond to inositol treatment, can be mimicked by the Itpk1 mutant mouse model.  These studies should shed light on the role of inositol in normal and abnormal embryonic development and on gene-nutrient interactions that underlie NTDs in humans.

The accompanying figure shows a normal embryo (A) and Itpk1 mutant embryos (B-F).  Note the presence of anencephaly (B), spina bifida (C-E), and axial skeletal malformations (kyphoscoliosis and abnormal ribs) (F) in the Itpk1 hypomorphic embryos.  Abbreviations: drg, dorsal root ganglia; hl, hindlimb; mc, myelocele; ntc, notochord.

Molecular Genetics of Congenital Diaphragmatic Hernia

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.  Loss-of-function mutations in the gene encoding FOG2, a transcriptional co-regulator, have been linked to CDH and pulmonary hypoplasia in humans and mice. 

In collaboration with Dr. Patrick Jay, we found that mutagenesis of the gene for GATA4, a transcription factor known to functionally interact with FOG2, predisposes inbred mice to a similar set of birth defects.  A significant fraction of C57Bl/6 mice heterozygous for a Gata4 deletion mutation died within one day of birth. Developmental defects in the heterozygotes included midline diaphragmatic hernias, dilated distal airways (see accompanying figure), and cardiac malformations.  We propose that GATA4, like its co-regulator FOG2, is required for proper mesenchymal cell function in the developing diaphragm, lungs, and heart.

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