Fig. 4

Fig. 4 Site-specific integration of targeting vectors into vegfaa.

a Schematic of vegfaa gene structure, location of the intron 1 guideRNA target site. b The targeting vectors pSA2-Gal4vp16/4xnrUAS-mTagBFP2 and pSA2-Gal4vp16_synCoTC/4xnrUAS-mTagBFP2, FRT and FRT3 sites are indicated by triangles. c Successful integration of the targeting plasmid to generate the Ti(vegfaa^int1-Gal4vp16/4xnrUAS-mTagBFP2) hereafter referred to as vegfaa^afpUTR and Ti(vegfaa^int1-Gal4vp16_synCoTC/4xnrUAS-mTagBFP2) hereafter referred to as vegfaa^synCoTC targeted insertion lines. d, evegfaa targeted insertion lines demonstrating loss of vegfaa phenotypes in homozygote mutants at 48 hpf. dvegfaa^afpUTR transgenic insertion line. Scale bar 250 μm. evegfaa^synCoTC transgenic insertion line. mTagBFP2 fluorescence (magenta) in d and e was captured with the same imaging settings, demonstrating more robust expression levels in the synCoTC-containing line. Scale bar 250 μm. f mRNA levels at 24 hpf demonstrate the fold increase of mTagBFP2 levels compared to native vegfaa from Gal4/UAS amplification. Error bars represent SEM for n = 3 (vegfaa^afpUTR) or n = 4 (vegfaa^synCoTC) independent biological replicates, consisting of 17 or 18 (vegfaa^afpUTR) or 14 (vegfaa^synCoTC) pooled embryos. g Vascular labelling by the Tg(kdrl:EGFP) reporter demonstrates absence of the dorsal aorta (yellow arrow in wildtype) in mutants from both CRIMP lines, and branching defects of the trunk intersegmental vessels at 72 hpf. Scale bar 250 μm. h, i Both Ti(vegfaa^int1-Gal4vp16/4xnrUAS-mTagBFP2) (vegfaa^afpUTR) and Ti(vegfaa^int1-Gal4vp16_synCoTC/4xnrUAS-mTagBFP2) (vegfaa^synCoTC) CRIMP vegfaa mutants do not undergo genetic compensation. vegfaa and vegfab mRNA levels in vegfaa CRIMP mutants demonstrate complete loss of native vegfaa transcript, without a change in vegfab (h), demonstrating genetic compensation has not been induced. Error bars represent SEM for n = 3 (vegfaa^afpUTR) or n = 4 (vegfaa^synCoTC) independent biological replicates, consisting of 17 or 18 (vegfaa^afpUTR) or 14 (vegfaa^synCoTC) pooled embryos. rpl13 was used as the reference gene. ivegfaa^afpUTR and vegfaa^synCoTC CRIMP mutants have a reduction in the number of branching intracerebral central arteries (CtAs) similar to the reported non-compensating vegfaa promoterless mutant. vegfaa^{afpUTR−/−}n = 17, vegfaa^{synCoTC−/−}n = 19, wildtype n = 6. Error bars represent SEM. vegfaa^afpUTRp value = 0.0000002, vegfaa^synCoTCp value = 0.0000000003. Source data are provided as a Source Data file. Statistical differences were determined using a two-tailed unpaired t-test.