Eomesa activation of vgll4l is through feedforward loops via sox32 and its upstream activators. (A) qRT-PCR analysis of vgll4l expression in early gastrulas (6 h.p.f.) in control embryos and those injected with sox32 mRNA at the 1 cell stage. Expression is represented as fold change relative to control normalised to 18S rRNA. (B) WISH analysis of the ability of vgll4l expression in early gastrulas (6 h.p.f.) in embryos injected with mRNAs at the one-cell stage as indicated, with and without Sox32 morpholino knockdown. N = 2. Total numbers of embryos scored per condition are indicated. Representative images of expression patterns per gene per category are shown. Animal views; dorsal to the right. Fisher’s Exact two-tailed probability test p values: *P ≤ 5 × 10−2; **P ≤ 5 × 10−4; ***P ≤ 5 × 10−8; ****P ≤ 5 × 10−12; N.S. = not significant. Orange asterisks indicate significant differences in fractions of embryos exhibiting ectopic expression vs. other categories. Grey asterisks indicate significant differences in fractions of embryos exhibiting loss of expression vs. other categories. (C) Model for Eomesa regulation of vgll4l expression in dorsal forerunner cells indicating a type 3 incoherent feedforward loop on the left as Eomesa represses vgll4l directly while activating via Sox32, and potential type 1 coherent feedforward loops on the right as Eomesa activates positive regulators of sox32 and potentially also vgll4l.
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