Fig. 8. ß-catenin overexpression results in a less restricted population of neurons compared with RA-derived neurons. The phenotype of neurons generated by ß-catenin N and C overexpression in high-density EB cultures was compared with neurons generated by RA treatment of low-density EB cultures. (A-D) Hoxc4, red; ß-tubulin 3, green; (E-H) NeuN, green; GABA, red; (I-L) tyrosine hydroxylase, red; ß-tubulin 3, green; Hoechst stain blue (white arrows indicate TH-positive neurons); (M-P) Map2, green; ß-tubulin 3, red; (Q-T) synaptophysin, green; ß-tubulin 3, red. (A-D) All neurons generated by either ß-catenin overexpression or RA treatment were positive for Hoxc4, a homeobox gene specific for caudal neurons. (E-H) Many neurons were positive for the neurotransmitter GABA and all neurons were positive for NeuN. (I-L) Some neurons induced by overexpression of ß-catenin were immunoreactive for tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis. By contrast, TH immunoreactivity was not observed in RA-treated conditions. (M-T) All ß-tubulin 3-immunoreactive cells also expressed Map2 and synaptophysin. (U) Quantification of the percentage of gabaergic neurons found in the cultures. There was no statistically significant difference between the RA-treated and untreated cultures (by ANOVA). (V) Neurons generated from untransfected ES cells differentiated at low density also express Hoxc4, suggesting that these are caudal neurons (green, Hoxc4; red, ß-tubulin 3).