Fig. S4

zeb1a is required to develop iSMCs in zebrafish embryos. a) zeb1a knockdown impairs iSMC development. Bright-field and fluorescent images of Tg(acta2:mCherry)^uto5 embryos injected with two different morpholinos (KD^ATG and KD^spl). Zeb1a KD in zebrafish did not alter embryonic development, although small moderate pericardial edema and craniofacial abnormalities were evident at 72 hpf. By contrast, zeb1a-deficient embryos displayed no iSMC development, as shown in Tg(acta2:mCherry)^uto5-injected embryos (arrows). The number of embryos showing the phenotype is indicated. Scale bar, 200 μm. Notochord: n; heart: h. b) Schematic diagram of the first 4 zeb1a exons (not to scale) showing the locations of binding sites of two different zeb1a morpholinos, one targeting the initiation codon (KD^ATG) and the other targeting the spice donor site for intron 2 (KD^spl). Black arrows indicate the primer used. As shown in the RT-PCR experiments, zeb1a KD^spl-injected embryos displayed altered zeb1a wild-type mRNA expression. c) zeb1a knockdown reduces iSMC marker expression. Histograms show acta2, tagln, myh11, and foxa3 mRNA levels in the trunk of 72 hpf embryos measured by qRT-PCR after zeb1a KD. The iSMC markers acta2, tagln and myh11 were significantly downregulated, whereas the endodermal compartment (foxa3) was normal. Stars represent the results of unpaired t-tests of mean difference = 0 (*p<0.05, **p<0.01, ***p<0.001). d) zeb1a knockdown does not alter LPM and endodermal differentiation. Histograms show that the hand2 and foxa3 mRNA levels measured in the trunks of 48 hpf embryos by qRT-PCR after zeb1a KD were equal to control levels. Stars represent the results of unpaired t-tests of mean difference = 0 (*p<0.05, **p<0.01, ***p<0.001) e) zeb1a knockdown does not alter LPM/hand2+ cell number. Box and whisker plots show the percentage of LPM cells isolated by fluorescent-activated cell sorting (FACS) experiments from the trunks of Tg(hand2:EGFP)^pd24 embryos at 48 hpf after zeb1a KD. Zeb1a KD did not alter LPM/hand2+ cell number. The boxplots show the maximum, minimum, upper and lower quartiles, and the sample median. Stars represent the results of unpaired t-tests of mean difference = 0 (*p<0.05, **p<0.01, ***p<0.001). (Left to right) n = 5 and n = 10 groups of 10-20 embryos.