Fig. 4

Genome-wide analysis of the polarized tissue reveals differential expression of Wnt signaling components in the caudal region. a Fluorescent images of a day-5 aggregate using a Fgf5::Turq//Six3::Venus//Irx3::Tomato ESC line. Fgf5::Turq signals were enhanced by the auto contrast function of ImageJ software due to its dim expression at culture day 5. b Schematic diagram of microarray sample collection. c A validation of microarray samples via RT-qPCR, showing rax and irx3 expression in FACS-sorted GFP⁺ and GFP⁻ cells. d Volcano plot drawn by plotting points of all probe sets with log2 fold-changes of GFP⁻ expression values to GFP⁺ (X-axis) and p-values of significant differences (Y-axis). Blue dots indicate log2 fold-change of more than 1 (i.e., more than two-fold). e, f Quantification of Wnt and Fgf signaling-related-gene expressions via RT-qPCR, following FACS sorting of GFP⁺ and GFP^- cells. g Immunohistochemistry was performed on cryosections of day-5 Rax::GFP aggregates with antibodies recognizing Wnt1, Irx3 and GFP, showing Wnt1, Irx3 and Rax::GFP signals. h, i Quantification of gene expressions via RT-qPCR, following time-specific treatment of bFGF and PD173074. j Schematic of Fgf/FGFR and Wnt signaling relationship in ESC-derived caudal differentiation in vitro. Error bars indicate s.e.m of each experiment (c, e, f, h, i). Significance was determined using unpaired t-test. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant (e, f, h, i). Scale bars, 100 µm (a, g). These images were one of n = 3 experiments (a, g)