Fig. 5
Wnt signaling components wnt1/4/8b, sfrp1, dkk1 and axin2 are spatially distributed along the R-C polarized neuroectoderm. a Montage of images taken from Supplementary Movie 5, showing upper panel; merged image of DIC (differential interference contrast), Rax::GFP and 7TCF::Cherry, lower panel; merged image of Rax::GFP and 7TCF::Cherry. b Kymograph analysis of time-lapse images by culture day 7, showing Rax::GFP and 7TCF::Cherry. White solid line indicates the time when Rax::GFP+ signals started to polarize. c Four way fractionation of day-6 cells by FACS, depending on Rax::GFP expression levels. d Confirmation of four fractions via FACS. e Quantification of Wnt signaling-related gene expression via RT-qPCR: wnt1, wnt4, wnt8b, axin2, dkk1, and sfrp1. Error bars indicate s.e.m of each FACS sorting experiment. f, g, h Immunohistochemistry was performed on cryosections of day-6 aggregates, showing Wnt1, 7TCF::H2B-Tomato, Sfrp1, Dkk1, Six3::Venus and Irx3::Tomato expressions. Dkk1 signals in Fig. 5h were also used to show in green in Supplementary Fig. 5J. i Schematic diagram of Wnt component distribution. R, rostral; C, caudal. Scale bars, 100 µm (a, f, g). These images were one of n = 3 experiments (a, f, g, h)
