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 T(3). A dominant-negative knock-in point mutation (P398H) 
was introduced into the thyroid hormone receptor alpha gene to determine the 
influence of T(3) on ES cell differentiation. Differentiation promoted by T(3) 
(1 nM), RA (1 microM), or combined T(3)/RA was assessed in wild-type (wt) and 
mutant (m) ES cells on the basis of neuronal-specific gene expression and cell 
cycle. T(3) alone stimulated neural differentiation in a similar fashion as that 
seen with RA in both wtES and mES cells. Expression of neurogranin and 
Ca(2+)/calmodulin-dependent kinase IV mRNA (identified in vivo as T(3)-regulated 
genes), however, was markedly reduced in mES, compared with wtES cells. RA 
treatment enhanced apoptosis, significantly greater than that seen with T(3) 
stimulation. T(3) treatment given with RA significantly reduced the apoptotic 
effects of RA, an effect not seen in mES cells. T(3)-induced ES cell neural 
differentiation of thyroid hormone alpha mutant and wtES cells provides an in 
vitro model to study T(3)-dependent gene regulation in neural development. This 
system could also be used to identify novel T(3)-regulated genes. The modulation 
of the apoptotic effects of RA by T(3) may have implications for stem cell 
therapy.