Figure 4. Estradiol increase TCF/LEF-dependent transcription in cortical neurons.

(A)-Cortical neurons from E18 embryos were nucleofected with TOPFlash or FOPFlash reporter plasmids and luciferase activity was analyzed after 2DIV. Estradiol treatment (60 min, 100 nM) selectively increased transcription from the TOPFlash reporter plasmid compared to FOPFlash, which shows no activity. Insulin treatment (5 µg/ml) was used as a control of induction. (B–C)- Expression of the LacZ gene in response to estradiol in transgenic mice. The scheme represents the lacZ transgene under the control of three consensus TCF/LEF-binding motifs upstream of the c-fos promoter, as described in Materials and Methods . (B)-Upper panel. Total extracts of cortical neurons (2DIV) were obtained from a TCF/LEF-lacZ transgenic mouse (see Materials and Methods) and the β-galactosidase (β-gal) expression was assessed in western blots after estradiol treatment (100–200 nM) for 3 h. Wnt3a (20 ng/ml) was used as a control of TCF-mediated induction. A slight increase in β-gal protein was observed after exposure to estradiol, as with recombinant Wnt3a protein. (B), Lower panel. Basal expression of β-galactosidase in neurons from transgenic mice was assessed by immunocytochemistry using specific antibodies against β galactosidase (green) and Phalloidin-labelled with Alexas 549. (C), Alternatively, after treatment with estradiol or Wnt3a, total Lac Z expression was quantified by RT-PCR using specific β-gal oligonucleotides and using actin (a housekeeping gene) as an internal standard (see Methods ). The amplification of both genes was analyzed on agarose gels and the graph represents the normalized data obtained from the Lightcycler analysis. Both treatments clearly increase transcriptional activity when compared to controls.