Figure 3.

Snail-positive regulation of Mef2 I-D[L] requires the Snail motif. (A) Luciferase assay in Kc cells. The X-axis indicates the amount of DNA transfected (ng), and the Y-axis is the average fold of luciferase across replicates (n = 3), normalized to Renilla. Snail significantly potentiates Twist-mediated Mef2 I-D[L] enhancer activation (dark-gray bars) (Students two-tailed t-test, P < 0.01). Disruption of two putative Snail motifs, shown in B, abrogates Snail's effect (light-gray bars). (B) Two mutated Snail motifs, Sna1 and Sna3, are highlighted in the sequence of the Mef2 I-D[L] enhancer (asterisks and red letters indicate the base exchanges). The Sna2 motif overlaps an essential Twist motif and was therefore not mutated. (C) In vivo activity of the Mef2 I-D[L] enhancer. (Top panel) In wild-type embryos, the Mef2 I-D[L] enhancer initiates reporter gene expression (lacZ; green) in mesoderm at stage 5, similar to the endogenous Mef2 gene (red). Enhancer activity and Mef2 expression are ablated in sna¹⁸ mutant embryos but maintained in sna^V2 mutant embryos. (Bottom panels) Mutation of the Snail motifs 1 and 3 (Mef2 I-D[L] ΔSna1,3) drastically reduces lacZ expression. Expression of sna (blue) marks the mesoderm, while derepression of sim (blue) in the mesoderm was used to distinguish sna mutant embryos from wild-type embryos. LacZ expression in the head fold is caused by the eve minimal promoter in the reporter vector.