Projects
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 protecitive 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 Basis of Adrenocortical Neoplasia
Domestic ferrets and certain inbred strains of mice (e.g. DBA/2J, CE) 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 recent studies implicate GATA factors in postgonadectomy tumor formation in ferrets and DBA/2J mice. We postulate that ectopic expression of LH receptor in adrenocortical cells confers inappropriate 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. Note the nuclear immunoreactivity, which correlates with the degree of cellular and nuclear atypia.
Collaborators on these studies include Dr. Matti Kiupel of Michigan State University and Dr. Markku Heikinheimo of the University of Helskini.
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 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.