TGFβ-driven LPM morphogenesis requires zeb1a. (A) Knockdown of the transcription factor zeb1a impairs iSMC differentiation. Fluorescent images of Tg(acta2:mCherry)uto5 embryos at 72 hpf after zeb1a morpholino injections. zeb1a knockdown embryos exhibit decreased mCherry and Tagln expression in the gut region (g) compared with controls (arrow). Scale bars: 200 μm. Insets show confocal transverse sections of the posterior gut region (dashed line) in embryos stained for Tagln (green). The number of embryos exhibiting the phenotype is indicated. Nuclei are in blue. Scale bars in insets: 10 μm. n, notochord; h, heart. (B) Knockdown of zeb1a does not alter endoderm morphology and differentiation. Box and whisker plots show the percentage of iSMCs or endodermal cells isolated by fluorescent-activated cell sorting (FACS) experiments from the trunk of double Tg(acta2:mCherry)uto5 (Xia.Eef1a1:GFP)s854 embryos at 72 hpf after zeb1a downregulation. Although the number of iSMCs is severely reduced by zeb1a knockdown, endodermal cells are normal. The boxplots show the maximum, minimum, upper and lower quartiles, and the sample median. Asterisks represent the results of unpaired t-tests of mean difference=0 (***P<0.001; n=6 groups of 10-20 embryos). (C) Knockdown of zeb1a impairs LPM ventral migration. Upper panel: confocal transverse sections of Tg(hand2:EGFP)pd24 stained with phalloidin (red). The number of embryos exhibiting this phenotype is indicated. Scale bars: 15 μm. Lower panel: schematic representation of impaired migration in zeb1a knockdown embryos. (D) zeb1a differentially regulates expression of genes associated with migrating phenotypes in LPM. Histograms show qPCR analyses of defined genes in LPM cells sorted from Tg(hand2:EGFP)pd24 after zeb1a knockdown and the relative controls at 48 hpf. Compared with controls, the knockdown of zeb1a upregulates genes (such as E-cadherin and occludin A) associated with non-migrating epithelial structures (*P<0.05).
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