Fig. 5

Fig. 5

efnb2a;efnb2b double mutants develop a circulatory short-circuit phenotype and sox7 genetically interacts with efnb2a. (A) Schematic diagram of efnb2a^hu3393 and efnb2b^hu2971alleles with premature stop codons (red asterisks) at amino acids 86 and 78, respectively. Eph, ephrin domain (green), Cd, cupredoxin domain (blue). (B) Quantification of efnb2a^hu3393 and efnb2b^hu2971 single and double mutant phenotypes revealed circulatory short-looping in 11% of efnb2a^hu3393/;efnb2b^hu2971+/ embryos and a significant increase of short-looping in 42% of double homozygous mutants (Student′s t-test, P=0.003 compared with efnb2a^hu3393/;efnb2b^hu2971+/). The genotype combinations not shown all displayed 100% wild-type phenotype. Bars represent combined results of five independent experiments (total of 128 embryos). (C) Blood circulation in gata1:dsRed-positive efnb2a^hu3393;efnb2b^hu2971 single and double mutant embryos at 2.5dpf. Note normal circulation in single mutants (upper panel) and short-loop phenotype in efnb2a^hu3393;efnb2^hu2971 double mutants (lower panel). Schematic representation of blood flow in double mutants (lower right panel). Lines depict blood flow in arteries (red) and veins (blue). VA, ventral aorta; PHS, primary head sinus; CCV, common cardinal vein. (D) Combinations of sox7^hu5626, efnb2a^hu3393 and efnb2b^hu2971 loss-of-function alleles result in increasingly severe effects on the circulatory system. All sox7^hu5626;efnb2a^hu3393 double homozygous mutant embryos exhibit a significant (Student′s t-test, P=0.002) increase in the penetrance of the short-circuit phenotype compared with the only partially penetrant sox7^hu5626 single mutants or the efnb2a^hu3393 single mutants lacking the short-loop defect completely. The genotype combinations not shown all displayed 100% wild-type phenotype. Bars represent combined results of five independent experiments (total of 507 embryos). (B,D) Embryos were analyzed at 2.5dpf. Phenotypes classified as ‘others’ are edema, total lack of circulation and/or shunts in trunk region.