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A Targeted Thyroid Hormone Receptor Gene Dominant-Negative Mutation (P398H) Selectively Impairs Gene Expression in Differentiated Embryonic Stem Cells

Molecular Endocrinology Laboratory and Research Service, Veterans Affairs Greater Los Angeles Healthcare System, Departments of Medicine and Physiology, University of California Los Angeles School of Medicine, Los Angeles, California 90073

Address all correspondence and requests for reprints to: Gregory A. Brent, Molecular Endocrinology Laboratory, Veterans Affairs Greater Los Angeles Healthcare System, Building 114, Room 230, 11301 Wilshire Boulevard, Los Angeles, California 90073. E-mail: . gbrent{at}ucla.edu

Thyroid hormone and retinoic acid (RA) are essential for normal neural development in vivo, yet all in vitro differentiation strategies of embryonic stem (ES) cells use only RA. We developed a novel differentiation strategy of mouse ES cells using T₃. A dominant-negative knock-in point mutation (P398H) was introduced into the thyroid hormone receptor gene to determine the influence of T₃ on ES cell differentiation. Differentiation promoted by T₃ (1 nM), RA (1 µM), or combined T₃/RA was assessed in wild-type (wt) and mutant (m) ES cells on the basis of neuronal-specific gene expression and cell cycle. T₃ alone stimulated neural differentiation in a similar fashion as that seen with RA in both wtES and mES cells. Expression of neurogranin and Ca²⁺/calmodulin-dependent kinase IV mRNA (identified in vivo as T₃-regulated genes), however, was markedly reduced in mES, compared with wtES cells. RA treatment enhanced apoptosis, significantly greater than that seen with T₃ stimulation. T₃ treatment given with RA significantly reduced the apoptotic effects of RA, an effect not seen in mES cells. T₃-induced ES cell neural differentiation of thyroid hormone mutant and wtES cells provides an in vitro model to study T₃-dependent gene regulation in neural development. This system could also be used to identify novel T₃-regulated genes. The modulation of the apoptotic effects of RA by T₃ may have implications for stem cell therapy.

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