Notch pathway inhibition of wild-type embryos by the γ-secretase inhibitor CpdE similarly displays decreased cpa5 expression. WISH using a digoxigenin-labeled RNA antisense probe to zebrafish cpa5 was performed at 30 and 48 hpf. Bar graphs represent the percent of embryos categorized by WISH expression levels. (A) Treatment with 50μM CpdE results in a decrease in cpa5 expression at 30 hpf (n = 35), whereas treatment with 75μM CpdE shows absent cpa5 expression (n = 10) compared with untreated controls (n = 44). Similarly, at 48 hpf, treatment with CpdE shows a dose-dependent decrease in cpa5 expression (n = 25 for 50μM CpdE, n = 30 for 75μM CpdE, n = 24 for wild-type embryos). DMSO 0.5% was used as a control, and from this group 24 and 23 embryos were analyzed at 30 and 48 hpf, respectively. (B) Treatment with 25 or 50μM CpdE results in a dose-dependent decrease of cpa5 expression in mib^ta52b mutants at 48 hpf, both anteriorly and posteriorly (n = 12 for 25μM, n = 11 for 50μM CpdE). Insets: Magnified view of the area of interest (5× objective, MZ6 microscope; Leica).