hu10125 is a cad (perplexed) mutant. A–E: Overview of the genetic mapping of cadhu10125 mutants. Recombinant embryos at multiple simple sequence length polymorphism markers identified the critical genetic interval indicated (A). Sequencing of the cad gene identified the presence of a mutation (P1850T) in hu10125 cDNA (B,C), affecting a conserved residue in the linker region of Cad (D,E). F,G: Overall morphology of wild-type siblings (77.5 % of the progeny) and cadhu10125; cadhi2694 embryos (22.5% of the progeny) at 4 dpf in a classical complementation assay (n = 165 embryos scored) (F). Quantification of the number of intersegmental arteries per embryo (out of 10 segments) presenting with hyperbranching in WT siblings (n = 21 scored) and cadhu10125;cadhi2694 (n = 19 scored) embryos at 4 dpf (G). H–J: In situ hybridization analysis of cad expression shows ubiquitous expression at the 12-somite stage (H), enrichment in the trunk and neural tissues 30 hpf (I) and enrichment within kidney and putative vascular tissues at 48 hpf (J). Arrows: putative vascular endothelial cells. K: Quantitative RT-PCR for markers of the blood vasculature in arterial endothelial cells (AECs) sorted based on TG(flt1:YFP) expression at 36 and 60 hpf: kdrl (expressed in all blood vascular endothelial cells), flt1 and dll4 (restricted to arterial endothelial cells), ccbe1 (not expressed in the vascular endothelium) and cad. Gene expression were normalised to rpl13.
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