Fig. 3

Morpholinos rescue the phenotype of cal^vu69 embryos. (A) Schematic and sequence information for the design of morpholinos directed toward the rescue of the mutant splicing in cal^vu69 . The WT exon is uppercase. The mutation is highlighted in red, and the WT splice acceptor is in green. Each morpholino-binding site is indicated by a line located 52 to the polypyrimidine tract. Morpholinos that cause phenotypic rescue are highlighted in pink. Morpholino i1 served as a control to demonstrate that blocking of proper splicing at this exon–exon boundary would result in the calamity phenotype (data not shown). (B and C) Side-by-side comparisons of zebrafish 48 h after fertilization. (Top) WT sibling from cal^vu69 intercross clutch. (Bottom) cal^vu69/vu69 -mutant embryo. (Middle) cal^vu69/vu69 embryo rescued by injection of morpholino i3. (D and E) Close-up view of cal^vu69/vu69 -uninjected embryo showing lack of melanin pigmentation over head region and notochord abnormalities (arrowhead). (F and G) Close-up view of morpholino i3-rescued cal^vu69/vu69 embryo showing restoration of melanin pigmentation and a notochord absent of defects. (H) Quantification of the extent of rescue. A single group of embryos derived from several clutches, half injected with rescuing morpholino (red bars; uninjected, blue bars), was scored for the presence of any or a significant number of melanocytes (>20) and the presence of notochord defects. The scale of the graph reflects the expected 25% ratio of homozygous mutants derived from the heterozygous intercross. (I and J) Six days after fertilization, embryos were either uninjected (I) or injected (J) with rescuing morpholino i3.5. Rescued embryos maintain near-normal body morphology, whereas unrescued embryos swell, possibly because of decreased extracellular matrix integrity due to the loss of lysyl oxidase activity.