FIGURE SUMMARY
Title

Biological effects of 6-formylindolo[3,2-b]carbazole (FICZ) in vivo are enhanced by loss of CYP1A function in an Ahr2-dependent manner

Authors
Wincent, E., Kubota, A., Timme-Laragy, A., Jönsson, M.E., Hahn, M.E., Stegeman, J.J.
Source
Full text @ Biochem. Pharmacol.

Impact of CYP1A knockdown on FICZ-mediated effects on mortality and phenotype. Groups of embryos, non-injected (NI) or injected with Ctrl-MO (Ctrl) or CYP1A-MO (CYP), were exposed to vehicle control (DMSO; D), or 10 or 100 nM FICZ (F10, F100). All endpoints were determined at 3 dpt (4 dpf); (A) cumulative mortality, (B) hatching rates, (C) swim bladder inflation and (E) rates of pericardial edema and circulatory failure. Severity of pericardial edema and circulatory failure was graded 0-3, “0” representing no effect and “1-3” representing effects of increasing severity. The severity scoring index for pericardial edema is shown in (D). In (B), (C) and (E) the hatched part of a bar indicates the mortality rate. Results are shown as% of total number of embryos at exposure start based on three separate experiments (n = 69-99). Statistically significant differences in numbers of affected and unaffected embryos between the MO injected groups (Ctrl or CYP) and the corresponding NI group were determined by Fisher’s exact test with Bonferroni’s correction and are shown by asterisks (**p < 0.01 and ***p < 0.001). For the statistical analysis of data in (E) “affected embryos” are represented by the pooled number of all living embryos with severity score 1-3 in a group.

Phenotypic and quantitated effects of FICZ and αNF alone and in combination. Groups of 1 dpf embryos were exposed to different doses of FICZ or vehicle control (DMSO) in combination with αNF starting at 1 dpf. (A) and(B) Illustrative pictures of phenotypic effects; (A) control embryo (top) and embryo treated with FICZ (1.0 nM) + αNF (0.5 µM) (bottom). (B) Embryo treated with FICZ (0.005 nM) + αNF (1.0 µM). Abbreviations: reduced growth (RG), body curvature (BC), craniofacial deformities (CD), reduced eye size (RE), pericardial- and yolk sac edema (PE, YSE), and tube-shaped heart (TSH). All pictures were taken at 3 dpt (4 dpf). (C)-(F) quantitated data on lethality and phenotypic effects after exposure to (C) FICZ (0.005-10 nM; F0.005 etc), or (D) αNF (0.1 to 1 µM; A0.1 etc), or (E) combinations of αNF and FICZ. (F) Embryos injected with Ahr2-MO (Ahr) were exposed to FICZ (10 nM), αNF (1 µM), or a combination of FICZ and αNF. DMSO (D) was used as a vehicle control for all exposures. At 3 dpt (4 dpf) frequencies of cumulative mortality (Dead), pericardial- or yolk sac edema (Edema), and failure of swim-bladder inflation (-SB, no edema) were determined as% of total number of embryos at exposure start based on one representative experiment (n = 25-30). Embryos with none of the effects listed were scored as normal. Statistically significant differences among groups were determined by Fisher’s exact test with Bonferroni’s correction. A difference in mortality rate is indicated by asterisks (**p < 0.01; ***p < 0.001) between a treatment group and the reference group (D, A1, A0.5, or Ahr + D) and by daggers (†††p < 0.001) between an Ahr2-MO group and its corresponding non-injected group. For embryo groups where no significant mortality was observed, the difference in rates of any effect between an exposed group and the reference group is indicated by double daggers (p < 0.05; ‡‡‡p < 0.001).

Acknowledgments
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