Fig. 3

Bmp and Wnt activities regulate heart field specification in cardiac spheroids. a Ingenuity pathway analysis of genes differentially expressed in Hcn4-GFP⁺ and Tbx1-Cre, RFP⁺ CPCs in vivo. We focused our analysis on pathways involved with “organism growth and development”. Data are shown as logarithm of Benjamini–Hochberg adjusted p-value, with threshold for significance of p-value < 0.1. b RNA-seq heatmaps of selected differentially regulated genes (Benjamini–Hochberg adjusted p-value < 0.1) from Bmp signaling pathway and Wnt/β-catenin pathways. Data are shown as row-scaled regularized logarithmic transformation of counts as produced by the DESeq2 package. c Vertical scatter plot showing the effect of increasing Bmp4 on formation of Hcn4-GFP⁺ CPCs (green trend line). d Vertical scatter plot showing the effect of increasing Bmp4 on formation of Tbx1, RFP + CPCs (red trend line). Both analyses (c, d) were performed on GFP⁺/RFP⁺ percentages from flow cytometric analyses in Fig. 1d. e Number of Hcn4-GFP⁺ CPCs and Tbx1-Cre, RFP + CPCs after induction with increasing concentrations of Wnt3A. f Number of Hcn4-GFP⁺ CPCs and Tbx1-cre, RFP + CPCs after induction with Bmp4 (1.25 ng/mL) in combination with Wnt3A. g qPCR analyses of Tbx5, Hcn4, Tbx1, and Fgf10 after induction with Bmp4 (1.25 ng/mL) alone or in combination with Wnt3a (100 ng/mL), Wnt5A (100 ng/mL), Wnt11 (100 ng/mL), and IWP-2 (0.5 μM). h Representative FACS plots of Hcn4-GFP⁺ and Tbx1-cre, RFP⁺ CPCs at day 5.5. i qPCR analyses of Tbx5, Hcn4, Tbx1, and Fgf10 after induction with Bmp4 (1.25 ng/mL) alone or in combination with Noggin (100 ng/mL) or dorsomorphin (100 nM), K2288 (100 nM), and DMH1 (100 nM). j Topflash assay after induction (88 h of differentiation) with Bmp4 (1.25 ng/mL) or Wnt3A (100 ng/mL) alone or in combination with Wnt3a, IWP-2, Noggin or Dorsomorphin. All data are mean ± SEM; n = 3; *p < 0.05; **p < 0.01. p values were determined using a paired Student’s t test