Figure 2.

Effect of different mutations on expression of the etv2-2A-GFP reporter. The reporter was designed to mimic the insertion of 2A-Gal4 into the genomic etv2 locus. Wild-type (wt) construct contains 5’UTR and the N-terminal coding sequence of the etv2 gene, and 2A-GFP sequence inserted out-of-frame. Four potential translation-initiating ATG sequences are shown in Frame 1. Frame 2 contains a single initiating ATG which is in-frame with 2A-GFP translation. Mutant 1 (Mut1) has mutations in all four ATG sites present in frame 1. Mutant 2 (Mut2) has mutation in the ATG site within the frame 2. Mutant 3 (Mut3) has a mutation within the ATG sequence that initiates GFP translation. The scatter plot shows relative GFP fluorescence intensity values (plotted in log₂ scale) of embryos injected with different etv2–2A-GFP reporter constructs. Note increased GFP fluorescence in embryos injected with Mut1 construct and reduced expression in Mut2 and Mut3-injected embryos compared to embryos injected with the wt construct. ** p<0.01, *** p<0.001, ****p<0.0001, t-Student’s test (two-tailed).