Fig. 5

CASH screening methodology and mutant candidate identification.
(A) Schematic representation of mutagenized wild-type male zebrafish bred with wild-type females. Males from the resultant F₁ generation are raised and outcrossed with wild-type females. The resultant embryo clutches will be 50% wild type and 50% heterozygous with respect to a mutation generated in the parental (P) gametes. These F₂ embryos are treated with moderate levels of BHP and stained for SA-β-gal activity (C). Quantitation of the staining levels is then performed. Embryo counts are grouped into 500-pixel intensity cohorts and plotted. (B) Wild-type embryos show a tight Gaussian distribution of staining intensity. Significantly right-shifted distributions (dotted red line in D) identify Class 1 candidates in terms of their oxidative stress sensitivity (D). Class 2 mutant candidates are identified if direct phenotypic abnormalities (red arrows) exist in approximately 50% of the embryos of clutches only in the presence of exogenous oxidative stress (an example is shown in E and F), with significantly right-shifted distributions (dotted red line in F).