y25/y54 are mutations in cds2. (A) Genetic map of the cds2 interval on linkage group LG5, showing the number of recombinants (of a total of 745 mutant embryos) for tested markers in the interval (numbers above line), BAC clones (gray boxes), the critical interval defined by 1 recombinant on each side (red bar), and the position of the cds2 gene. Missense mutations leading to premature stop codons in the 2 separate cds2 mutations are also noted. (B) In situ hybridization of 24-hours postfertilization (hpf) wild-type zebrafish embryo probed for cds2, showing vascular-enriched expression (arrows). (C) Quantitation of the intersegmental vessel phenotypes of wild-type–, CDS1-, or CDS2-morpholino–injected wild-type and y54 mutant 30-hpf Tg(fli-EGFP)^y1 zebrafish. (D) Quantitation of the intersegmental vessel phenotypes of 30-hpf Tg(fli-EGFP)^y1 y54 mutant zebrafish injected with either lacZ mRNA (left column) or wild-type cds2 mRNA (cds2wt; right column). The bars in panels C and D show the percentages of intersegmental vessels (ISVs) that have failed to sprout (blue), ISVs that have grown only up to the horizontal myoseptum half way up the trunk (red), and ISVs that have grown all the way to the dorsal trunk to form the dorsal longitudinal anastomotic vessel (DLAV; yellow). The number of embryos counted for each injection condition in panels C and D is shown at the top of each column, with 10 trunk segments counted per embryo. (E) Fluorescence microscopy images of the trunk of a 48-hpf Tg(fli-EGFP)^y1 y54 mutant injected at the 1 cell-stage with a Tol2(flk1:mCherry-2a-cds2) transgene driving simultaneous expression of mCherry (top panel, red) and wild-type CDS2 in endothelial cells (marked by EGFP expression in bottom panel). Growth of ISVs is rescued. Scale bars = 800 μm (B) and 200 μm (E). CDS indicates CDP-diacylglycerol synthetase; EGFP, enhanced green fluorescent protein; MO, morpholino oligonucleotide; and WT, wild-type.