Fig. 1

Identification of SP5 as a highly responsive WNT/β-catenin target gene in hPSCs. a Cell morphology changes in response to Wnt3a treatment. HESCs (H1/WA01) were treated with 1 nM Wnt3a for the indicated times and imaged using phase contrast microscopy. Scale bar = 100 μm. b Immunofluorescence (IF) for a differentiation marker. hESCs were treated with 1 nM Wnt3a for 2 days and analyzed by IF for SOX17 expression. Scale bar = 100 μm. c Heatmap of genes with significant changes in expression upon Wnt3a treatment. HESCs were treated with 1 nM Wnt3a for the indicated times and RNA was isolated and analyzed by RNA-Seq. About 511 genes with significant differential expression in response to Wnt3a fell into four main clusters i–iv. Refer to Supplementary Data 1 for complete gene set. d Transcription factors with greatest differential expression in response to Wnt3a. The top 10 genes encoding transcription factors were sorted by percent maximum reads per kilobase per million mapped reads (RPKM). ChIP-Seq analysis indicated that the SP5 promoter region shows a significant change in the histone mark H3K4me3 upon Wnt3a signaling. Supplementary Fig. 1c, d provides additional information about these transcription factors and display the genome browser tracks of all 10 genes for this histone mark. e SP5 protein accumulation in response to Wnt3a treatment. HESCs were treated with 1 nM Wnt3a for the indicated times and nuclear extracts were immunoblotted for SP5, SP1, or β-actin (a loading control). kD kilo Daltons. f Immunofluorescence of SP5. HESCs were treated with Wnt3a (1 nM) for 24 h and cells were fixed and stained for SP5 (red, rabbit anti-SP5) and DNA (blue, Hoechst). Untreated cells were incubated with an equivalent volume of WNT storage buffer. Scale bar = 100 μm. g Expression of SP/KLF family members in hESCs treated with Wnt3a. RPKM values for all SP/KLF genes were extracted from the RNA-Seq data described in c and displayed as a heatmap