Phenotypes of f5 homozygous mutants reveal adult lethality and spontaneous hemorrhage in homozygous mutant adult fish. (A) Survival curves of zebrafish offspring from f5^+/− incrosses, starting at 2 months of age, demonstrate progressive loss of 90% of homozygotes by 7 mpf. Heterozygotes also demonstrate statistically significant decreased survival compared with wild-type fish, with 50% mortality by 1.5 years of age. (B) Genotype distributions of offspring from f5^+/− incrosses evaluated at various stages demonstrate loss of homozygotes beginning at 28 dpf. (C) Spontaneous hemorrhage distal to the dorsal fin (arrow) in a 4-week-old homozygous mutant fish. (D) Severe spontaneous periorbital hemorrhage (arrow) grossly visible in a 5-week-old homozygous mutant fish just before death. Similar bleeding was not observed by blinded observers in heterozygous or wild-type clutchmates. Whole mount in situ hybridization with an antisense probe demonstrates localization of f5 expression to the liver in f5^+/+ zebrafish at 5 dpf (E), but not in f5^−/− mutants (F). (G) Total mRNA was prepared from pooled larvae (4 dpf, n = 21 for each genotype) followed by quantitative real-time PCR. Error bars represent standard deviation and statistical significance was determined by a Student t test.