Session: OR34-Molecular Mechanisms in Thyroid Hormone Action & Cancer
Room 103 (Moscone Center)
Cortical neuronal differentiation of mouse ESC occurs in two phases, using specific conditioned medium in each phase. Utilizing this method, we were able to obtain a high percentage of nestin-positive progenitor neurons (50%) in the first phase, and cortical neurons (60%) in the second phase of differentiation. When cells were treated with T3 (1 nM), the number of nestin-positive progenitors was 15% (p<0.05) more than control (without T3) and b-tubulin III-positive neurons was 18% (p<0.05) more, as determined by FACS analysis. The mRNA level of cortical neuron markers (TBR1, EMX1 and RC3) was significantly enhanced, compared to without T3 treatment, suggesting that T3 has a stimulatory role in corticogenesis. We utilized lentiviral shRNA COUP-TF1 transduction of ESC cells, to analyze the influences of COUP-TF1 on cortical neural differentiation and compared with or without T3 treatment. We found that after knockdown of COUP-TF1, the number of nestin-positive progenitors was reduced to 24% compared to shRNA control (36%). With T3 treatment, the number of progenitors in COUP-TF1 knockdown cells was increased to 43%, similar to the control level. A similar pattern was observed in b-tubulin III-positive neuron cells. We measured cortical neuron markers after shRNA COUP-TF1 knockdown, using q-PCR analysis. We found that in the presence of T3, the peak expression of EMX1, TBR1 and RC3 was earlier, at day 18 of differentiation, compared to control cells at day 22. Furthermore, after COUP-TF1 knockdown, RC3 and CamKIV, well-characterized T3-regulated genes, were significantly upregulated with T3 treatment. These results suggest that COUP-TF1 plays an important role in modulate the timing of T3-stimulated gene expression required for corticogenesis.
Nothing to Disclose: XT, KYYL, WT, GAB
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