Thyroid Hormone Action Controls Cell Signaling in the Developing Ventricular Epithelium

Developmental thyroid hormone (TH) insufficiency is associated with an array of neurodevelopmental disorders in children, although the precise mechanisms of TH action are often unclear. Previously, we characterized that transient developmental hypothyroidism alters cell adhesion, migration, and apoptosis in the neonatal rat brain. These cellular abnormalities were largely localized to the ventricular epithelium, a progenitor cell niche, and later resulted in periventricular heterotopia formation. Here we employed laser capture microdissection and RNA-Sequencing (RNA-Seq) to further evaluate how TH insufficiency may affect this cell population. Pregnant rats were treated with a low dose of propylthiouracil (PTU, 0.0003%) through the drinking water to induce maternal TH insufficiency from gestational day 6 until postnatal day 14 (PN14); controls received deionized water only. This goitrogen treatment significantly reduced total thyroxine (T4) and triiodothyronine (T3) in the sera of dams and pups during the postnatal period. Both T4 and T3 were also significantly reduced in the telencephalon of exposed neonates on PN2 and PN8 relative to controls. Next, frozen sections were collected from pup brains on PN2, the posterior ventricular epithelium microdissected, and total RNA sequenced using Illumina HiSeq. We identified 271 genes that were differentially expressed in the ventricular epithelium of PTU-exposed animals as compared to controls (adj. p-values <0.05). This included downregulation of Hairless (Hr) and Calcium Calmodulin Kinase IV (Camk4), consistent with our previous work. Several genes associated with pathways that control cell adhesion and apoptosis were also differentially expressed. Intriguingly, we identified a 245-fold upregulation of an unannotated gene (adj. p<0.001); bioinformatic analyses suggest that this is a paralog within the Alpha Mannosidase Class 1 family. This work supports the hypothesis that TH signaling controls vital processes in the ventricular epithelium and has identified novel pathways that hormone action may control. This knowledge could lead to the identification of other phenotypes resultant from thyroid disruption. This work does not reflect US EPA policy.

Impact/Purpose

Environmental contaminants like perchlorate, pesticides, and perfluorinated compounds can result in abnormal thyroid function in animal models and have been attributed to thyroid abnormalities in some human populations. As thyroid hormones are required for normal brain development, pregnant women and children may be especially sensitive to these chemical exposures. While severe cases of maternal hypothyroidism (low thyroid hormones in the blood) cause disorders like cretinism, it is now recognized that mild thyroid dysfunction can also lead to more mild effects like learning disabilities in children. However, the precise effects that hormones have on the brain is not clear in these more subtle cases. We previously have shown that a stem cell niche in the developing rat brain is especially sensitive to thyroid hormones, although we do not fully understand why. To understand the connection between thyroid hormones and stem cells, we exposed pregnant rats to a thyroid disrupting chemical; this treatment induced hypothyroidism during pregnancy. A subset of animals did not receive this chemical and were normal (controls). After the animals gave birth, we isolated the stem cell population in the newborn rat brain by laser capture microdissection, and then performed RNA sequencing. This permitted the identification of genes and developmental pathways that thyroid hormones can affect, which could have permanent consequences in the brain. In total we identified 271 genes that were differentially expressed in pups born to hypothyroid mothers as compared to normal individuals. We also discovered several molecular pathways that appear to be preferentially affected by thyroid disruption, including cell adhesion. This work reveals how thyroid signaling influences a vital stem cell niche in the mammalian brain and has implications for our understanding of neurodevelopmental disorders. These data will also facilitate the development of molecular biomarkers to identify abnormal brain development following exposure to environmental thyroid disrupting chemicals at US EPA.

Citation

OShaughnessy, Katherine, K. Bell, C. Riutta, J. Ford, A. Pandiri, T. Stoker, AND Mary E. Gilbert. Thyroid Hormone Action Controls Cell Signaling in the Developing Ventricular Epithelium. Society of Toxicology Annual Meeting (SOT)-Virtual, Virtual, NC, March 12 - 26, 2021.