R-spondin 1 and its receptor kremen 1 are required together for angiogenesis. (A) dtty135 genetic interval on linkage group 16, with the number of recombinants per meiosis, position of BAC clones and position of the rspo1 locus indicated. (B) Rspo1 protein domain structure, with two furin domains (FU) and a thrombospondin domain (TSP). The dtty135 mutation changes serine 193 to leucine in the TSP domain. (C) A zebrafish embryo with the red box highlighting the region imaged in D-K. (D,E) In situ hybridization of the trunk of 24 hpf wild-type zebrafish embryos probed for rspo1 (D) or krm1 (E), with expression observed in the axial vasculature (arrowheads). (F-K) Confocal images of trunk vessels in 26 hpf Tg(fli-EGFP)y1 wild-type zebrafish injected with either 5 ng rspo1 MO (F), 0.5 ng krm1 MO (G), 2.5 ng rspo1 MO (H), 0.25 ng krm1 MO (I), 5 ng control MO (J) or 2.5 ng rspo1 MO + 0.25 ng krm1 MO (K). (L) Quantitation of the intersegmental vessel (ISV) phenotypes of 26 hpf Tg(fli-EGFP)y1 dtty135 mutant or morpholino-injected embryos. (M,N) Confocal images of ISV in 26 hpf Tg(fli-EGFP)y1 dtty135 mutant embryos that were either not injected (M) or were injected intramuscularly in the trunk with murine R-spondin (N). (O) Quantitation of the intersegmental vessel (ISV) phenotypes of uninjected or mouse R-spondin-injected dtty135 zebrafish at 28 hpf. In L and O the bars show the percentages of ISV that have failed to sprout (blue), ISV that have grown only up to the horizontal myoseptum half way up the trunk (red) and ISV that have grown all the way to the dorsal trunk to form the DLAV (yellow). The number of segments counted is shown above each bar on the graphs. Scale bars: 100 μm in D; 50 μm in E; 50 μm in F-K,M,N.