Fig. S2

Fig. S2

(Related to Figure 3): MEF2C and RBPJk bind to the F2 enhancer, and overexpression of NICD in the endothelium can drive F2 expression in the vein, while MEF2C is dispensable for early arterial Dll4 expression. (A) Recombinant MEF2C protein was used in an electrophoretic mobility shift assay (EMSA) with a radiolabelled double-stranded oligonucleotide representing the F2-6 MEF2 binding site. Lane 1 contains reticulocyte lysate without recombinant MEF2C (represented by a minus sign). MEF2C efficiently bound to the F2-6 MEF2 site (lane 2) and this binding was efficiently competed by an excess of the unlabeled wild type F2-6 MEF2 element (lane 3), but not by an excess of a mutant form of this site (lane 4). (B) The F2-lacZ enhancer was crossed into the Mef2c^+/- background, and wild-type and homozygous mutant embryos were stained for X-gal at E8.5, 9.0, and E9.5. No difference in enhancer activity was observable in mutant embryos compared to wild-type (Mef2c^+/+) littermates at early stages of vascular development (i.e. E8.5-E9.0) (n e 5 for each genotype). The dorsal aorta is indicated by arrows. Scale bars = 500 μm (C) Expression of Dll4 mRNA was examined by in situ hybridization in Mef2c^+/+ and Mef2c^-/- embryos. No difference in expression was noted at E8.5 (n=3 for each genotype). Scale bars = 500 μm (D) NICD was over-expressed in the endothelium via Tie2-Cre-mediated activation of a R26^N1ICD allele. Whole-mount images (left; scale bar = 500 μm) and sections (right; scale bar = 100 μm) are shown. The presence of an arteriovenous malformation (AVM) is indicated by an asterisk and the dorsal aorta (arrow) and cardinal vein (caret) are also indicated. Expression of F2 was expanded into the cardinal vein in Notch over-expression embryos. (E) EMSA showing RBPJk binding to a probe corresponding to the F2-6 RBPJk site. Binding was competed by an excess of unlabeled wild-type but not RBPJk mutant probe.