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Fig. 3 - Supplemental 1 Blood flow and bone morphogenetic protein (BMP) signaling independently activates id2b expression. (A) Representative confocal maximal intensity projection of control (non-injected), bmp2b, bmp4, and bmp7a morpholino-injected id2b:eGFP; Tg(myl7:mCherry) hearts at 24 hr post-fertilization (hpf). White circles outline eGFP signal. (B) Quantification of mean fluorescence intensity of id2b:eGFP in (A). Data normalized to the mean fluorescence intensity of control hearts. n=(7, 9, 8, 10). (C) Representative confocal maximal intensity projection of DMSO and Dorsomorphin (DM)-treated id2b:eGFP hearts at 24, 48, and 60 hpf. Embryos were treated from 10 to 24 hpf, from 24 to 48 hpf, or from 36 to 60 hpf. White circles outline eGFP signal. (D) Quantification of mean fluorescence intensity of id2b:eGFP in (C). Data normalized to the mean fluorescence intensity of DMSO-treated hearts. n=(6, 10) (24 hpf); n=(14, 16) (48 hpf); n=(9, 9) (60 hpf). (E) Confocal optical sections of control, tricaine-treated, and tnnt2a morpholino-injected 72 hpf Tg(BRE:d2GFP); Tg(kdrl:mCherry) hearts. Yellow asterisks, endocardial cells. Yellow arrowheads, BMP signal. Numbers at the top of each panel indicate the ratio of representative images. (F) Schematic diagram of blood flow and BMP signaling-mediated id2b expression. Data are presented as mean ± s.e.m. Unpaired two-tailed Student’s t-tests were used to determine statistical significance.**p<0.01, ***p<0.001. ns, not significant. Scale bars, 50 μm.