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Thyroid Hormone Action Controls Cell Signaling in the Developing Ventricular Epithelium: Implications for a Mechanistic Adverse Outcome Pathway
Citation:
OShaughnessy, K., A. Sasser, T. Stoker, A. Pandiri, Mary E. Gilbert, J. Ford, B. McMichael, K. Bell, AND C. Riutta. Thyroid Hormone Action Controls Cell Signaling in the Developing Ventricular Epithelium: Implications for a Mechanistic Adverse Outcome Pathway. Developmental Neurotoxicology Society, Virtual, CA, June 22 - 25, 2022.
Impact/Purpose:
Insufficient levels of thyroid hormones (THs) during pregnancy can result in abnormal brain development in children. Severe TH deficiency has been associated with neurodevelopmental impairments, but there is little published literature investigating mild TH disruption. This work examines how maternal thyroid disruption, induced by the pharmaceutical goitrogen propylthiouracil (PTU), affects thyroid function in rat mothers and offspring, and specifically a stem cell niche in the developing brain. Pregnant rats were administered propylthiouracil (PTU, 0 or 3 ppm) via the drinking water from gestational day 6 (GD6) to postnatal day 14 (PN14), permitted to give birth, and the offspring analyzed. This concentration of PTU was chosen as it was expected to decrease the thyroid hormone T4, but not triiodothyronine (T3), in dams (previously published work). Dam and pup serum T4/T3 were assessed from birth until PN8; brain T4/T3 concentrations were evaluated at PN2 and PN8. The ventricular epithelium (stem cell niche) of the pup brain was isolated using laser microdissection and sequenced (RNA-Seq).We identified 268 genes that were differentially expressed in the ventricular epithelium of PTU-exposed animals compared to controls (adj. p-values <0.05). Using Ingenuity Pathway Analysis, we show that many of these genes are associated with cell junction maintenance, congruent with our previous studies. We then showed alterations in the morphology of cell junction proteins N-Cadherin and Claudin-5 shown by immunohistochemistry. These results support the hypothesis that differential expression of genes involved in cell junction structure and function are acutely affected in the hypothyroid brain, including at a crucial stem cell niche.
Description:
Developmental thyroid hormone (TH) insufficiency is associated with various neurodevelopmental disorders in children. Previously, we demonstrated that developmental hypothyroidism alters cell adhesion, migration, and apoptosis in the neonatal rat brain. These abnormalities were largely localized to the ventricular epithelium, a progenitor cell niche, and later resulted in a periventricular heterotopia. This permanent morphological abnormality now serves as an anchor in a putative adverse outcome pathway (AOP). To further elucidate key events in this AOP, we employed laser capture microdissection and RNA-Sequencing (RNA-Seq) of the ventricular epithelium to evaluate how reduced thyroid action may affect this cell population. Pregnant rats were treated with propylthiouracil (PTU, 0.0003%) through the drinking water to induce maternal TH insufficiency from gestational day 6 until postnatal day 14 (PN14). This goitrogen treatment significantly reduced total thyroxine (T4) but not triiodothyronine (T3) in the sera of dams, and both T4 and T3 were significantly reduced in the pups during the postnatal period. Both T4 and T3 were also significantly reduced in the telencephalon of exposed neonates on PN2 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 268 genes that were differentially expressed in the ventricular epithelium of PTU-exposed animals compared to controls (adj. p-values <0.05). Using Ingenuity Pathway Analysis, we show that many of these genes are associated with cell junction maintenance, congruent with our previous studies. These results support the hypothesis that differential expression of genes involved in cell junction structure and function may serve as crucial key event(s) of heterotopia formation, and these data increase the biological plausibility of our putative AOP. This work does not reflect US EPA policy.