Figure 4

GRG5 regulates ESC competence for neuroectodermal commitment by suppressing Wnt and Bmp signaling. (a,b) Luciferase assays using the SuperTOP-LUC (a) and BRE-LUC (b) reporter plasmids to monitor Wnt and Bmp signaling respectively in CONTROL, OE GRG5 and KD GRG5 ESCs. Mean + SD of n = 3 independent experiments. *P < 0.05 (c) CONTROL and KD GRG5 ESCs were cultured in neural differentiation conditions for 6 days. Immunofluorescence photos and quantification of cells expressing neural progenitor (SOX1, NESTIN) and neuronal (TUJ1, MAP2) markers at Day6. (Scale bar, 100 μm) Mean + SD of n = 4 independent experiments. *P < 0.05 (d) CONTROL and OE GRG5 ESCs were differentiated towards neural lineage for 6 days. Photos and quantification of cell immunofluorescence for neural progenitor (SOX1) and neuronal (TUJ1, MAP2) markers at days 4 and 6. (Scale bar, 100 μm) Mean + SD of n = 4 independent experiments. *P < 0.05 (e) Wnt and Bmp signaling pathways were stimulated using CHIR and BMP4 treatment in CONTROL and OE GRG5 ESCs, 24 h before induction of neural differentiation. Immunofluorescence analysis and quantification of TUJ1 positive neurons at Day6 of the neural specification procedure. (Scale bar, 100 μm) Mean + SD of n = 3 independent experiments. *P < 0.05.