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[Preprint]. 2023 Jul 5:2023.07.03.547455. [Version 2] doi: 10.1101/2023.07.03.547455

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Figure 3. — (A) Injection of fli1:she-2A-mCherry DNA plasmid together with Tol2 mRNA results in mosaic expression of she-2A-mCherry in endothelial cells. Note that mCherry positive segments of the DA (arrows, A) show reduced diameter compared to adjacent mCherry-negative segments. (B) Quantification of the DA diameter in mCherry+ and mCherry– DA segments in embryos from two independent experiments. n denotes the number of embryos analyzed. *** p<0.001 (Student’s t-test).

(D-F) Trunk vasculature of wild-type, fli1:she-2A-mCherry, she−/− and she−/−; fli1:she-2A-mCherry embryos in kdrl:GFP background at 28 hpf. Confocal imaging of GFP expression in live embryos obtained from stable transgenic lines; the diameter of the DA is marked with red line.

(G,H) Pericardial edema in she−/− mutants (E) is rescued in she−/−; fli1:she-2A-mCherry embryos at 4 dpf.

(I) DA diameter is reduced in fli1:she-2A-mCherry embryos compared to wild-type non-transgenic (no Tg) siblings. Similarly, DA diameter is reduced in she−/−; fli1:she-2A-mCherry embryos compared to she siblings without the transgene. Embryos were analyzed at 28 hpf in kdrl:GFP background. Embryos were obtained by incross of either she+/+ (referred to as wild-type); fli1:she-2A-mCherry+/− or she−/−; fli1:she-2A-mCherry+/− parents and separated based on mCherry expression. Note that wild-type and she−/− embryos cannot be directly compared in this experiment because they came from different parents and are not siblings; embryos from different pairs can show significant variability in the DA diameter. Error bars show SEM. **p<0.01, Student’s t-test.

(J) Percentage of she mutant embryos showing pericardial edema at 4 dpf. Note that vascular endothelial expression of full length She (fli1:she-2A-mCherry) and the construct carrying a deletion of the SH2 domain in She protein (fli1:sheDSH2–2A-mCherry) rescues the mutant phenotype, while overexpression of She construct with SH2 domain alone (fli1:sheSH2–2A-mCherry) fails to rescue the phenotype.