Exogenous corticosterone administration during pregnancy in mice alters placental and fetal thyroid hormone availability in females.

INTRODUCTION: Maternal prenatal psychological stress is associated with adverse pregnancy outcomes and increased risk of adverse health outcomes in children. While the molecular mechanisms that govern these associations has not been fully teased apart, stress-induced changes in placental function can drive sex-specific phenotypes in offspring. We sought to identify and examine molecular pathways in the placenta that are altered in response to maternal prenatal stress. METHODS: We previously employed a mouse model of maternal prenatal stress where pregnant dams were treated with stress hormone (CORT) beginning in mid-gestation. Using this model, we conducted RNAseq analysis of whole placenta at E18.5. We used qRT-PCR to validate gene expression changes in the placenta and in a trophoblast cell line. ELISAs were used to measure the abundance of thyroid hormones in maternal and fetal serum and in the placenta. RESULTS: Dio2 was amongst the top differentially expressed genes in response to exogenous stress hormone. Dio2 expression was more downregulated in placenta of female fetuses from CORT-treated dams than both control placenta from females and placenta from male fetuses. Consistent with Dio2's role in production of bioactive thyroid hormone (T3), we found that there was a reduction of T3 in placenta and serum of female embryos from CORT-treated dams at E18.5. Both T3 and T4 were reduced in the fetal compartment of the placenta of female fetuses from CORT-treated dams at E16.5. Exogenous stress hormone induced reduction in thyroid hormone in females was independent of circulating levels of TH in the dams. DISCUSSION: The placental thyroid hormone synthesis pathway may be a target of elevated maternal stress hormone and modulate fetal programming of health and disease of offspring in a sex-specific fashion.

The TFAP2A-IRF6-GRHL3 genetic pathway is conserved in neurulation.

Mutations in IRF6, TFAP2A and GRHL3 cause orofacial clefting syndromes in humans. However, Tfap2a and Grhl3 are also required for neurulation in mice. Here, we found that homeostasis of Irf6 is also required for development of the neural tube and associated structures. Over-expression of Irf6 caused exencephaly, a rostral neural tube defect, through suppression of Tfap2a and Grhl3 expression. Conversely, loss of Irf6 function caused a curly tail and coincided with a reduction of Tfap2a and Grhl3 expression in tail tissues. To test whether Irf6 function in neurulation was conserved, we sequenced samples obtained from human cases of spina bifida and anencephaly. We found two likely disease-causing variants in two samples from patients with spina bifida. Overall, these data suggest that the Tfap2a-Irf6-Grhl3 genetic pathway is shared by two embryologically distinct morphogenetic events that previously were considered independent during mammalian development. In addition, these data suggest new candidates to delineate the genetic architecture of neural tube defects and new therapeutic targets to prevent this common birth defect.

Chronic, Elevated Maternal Corticosterone During Pregnancy in the Mouse Increases Allergic Airway Inflammation in Offspring.

Allergic asthma is a chronic pulmonary disorder fundamentally linked to immune dysfunction. Since the immune system begins developing in utero, prenatal exposures can affect immune programming and increase risk for diseases such as allergic asthma. Chronic psychosocial stress during pregnancy is one such risk factor, having been associated with increased risk for atopic diseases including allergic asthma in children. To begin to define the underlying causes of the association between maternal stress and allergic airway inflammation in offspring, we developed a mouse model of chronic heightened stress hormone during pregnancy. Continuous oral administration of corticosterone (CORT) to pregnant mice throughout the second half of pregnancy resulted in an ~2-fold increase in circulating hormone in dams with no concomitant increase in fetal circulation, similar to the human condition. To determine how prolonged heightened stress hormone affected allergic immunity in offspring, we induced allergic asthma with house dust mite (HDM) and examined the airway immune response to allergen. Female mice responded to HDM more frequently and had a more robust immune cell response compared to their male counterparts, irrespective of maternal treatment. Male offspring from CORT-treated dams had a greater number of inflammatory cells in the lung in response to HDM compared to males from control dams, while maternal treatment did not affect immune cell numbers in females. Alternatively, maternal CORT caused enhanced goblet cell hyperplasia in female offspring following HDM, an effect that was not observed in male offspring. In summary, prenatal exposure to mild, prolonged heightened stress hormone had sexually dimorphic effects on allergic inflammation in airways of adult offspring.

Generation and characterization of a conditional allele of Interferon Regulatory Factor 6.

Interferon Regulatory Factor 6 (IRF6) is a critical regulator of differentiation, proliferation, and migration of keratinocytes. Mutations in IRF6 cause two autosomal dominant disorders characterized by cleft lip with or without cleft palate. In addition, DNA variation in IRF6 confers significant risk for non-syndromic cleft lip and palate. IRF6 is also implicated in adult onset development and disease processes, including mammary gland development and squamous cell carcinoma. Mice homozygous for a null allele of Irf6 die shortly after birth due to severe skin, limb, and craniofacial defects, thus impeding the study of gene function after birth. To circumvent this, a conditional allele of Irf6 was generated. To validate the functionality of the conditional allele, we used three "deleter" Cre strains: Gdf9-Cre, CAG-Cre, and Ella-Cre. When Cre expression was driven by the Gdf9-Cre or CAG-Cre transgenes, 100% recombination was observed as indicated by DNA genotyping and phenotyping. In contrast, use of the Ella-Cre transgenic line resulted in incomplete recombination, despite expression at the one-cell stage. In sum, we generated a novel tool to delete Irf6 in a tissue specific fashion, allowing for study of gene function past perinatal stages. However, recombination efficiency of this allele was dictated by the Cre-driver used.

Cell-autonomous and non-cell-autonomous roles for IRF6 during development of the tongue.

Interferon regulatory factor 6 (IRF6) encodes a highly conserved helix-turn-helix DNA binding protein and is a member of the interferon regulatory family of DNA transcription factors. Mutations in IRF6 lead to isolated and syndromic forms of cleft lip and palate, most notably Van der Woude syndrome (VWS) and Popliteal Ptyerigium Syndrome (PPS). Mice lacking both copies of Irf6 have severe limb, skin, palatal and esophageal abnormalities, due to significantly altered and delayed epithelial development. However, a recent report showed that MCS9.7, an enhancer near Irf6, is active in the tongue, suggesting that Irf6 may also be expressed in the tongue. Indeed, we detected Irf6 staining in the mesoderm-derived muscle during development of the tongue. Dual labeling experiments demonstrated that Irf6 was expressed only in the Myf5+ cell lineage, which originates from the segmental paraxial mesoderm and gives rise to the muscles of the tongue. Fate mapping of the segmental paraxial mesoderm cells revealed a cell-autonomous Irf6 function with reduced and poorly organized Myf5+ cell lineage in the tongue. Molecular analyses showed that the Irf6-/- embryos had aberrant cytoskeletal formation of the segmental paraxial mesoderm in the tongue. Fate mapping of the cranial neural crest cells revealed non-cell-autonomous Irf6 function with the loss of the inter-molar eminence. Loss of Irf6 function altered Bmp2, Bmp4, Shh, and Fgf10 signaling suggesting that these genes are involved in Irf6 signaling. Based on these data, Irf6 plays important cell-autonomous and non-cell-autonomous roles in muscular differentiation and cytoskeletal formation in the tongue.