The tryptophan photoproduct 6-formylindolo[3,2-b]carbazole (FICZ) binds multiple AHRs and induces multiple CYP1 genes via AHR2 in zebrafish

The tryptophan photooxidation product 6-formylindolo[3,2-b]carbazole (FICZ) has been proposed as a physiological ligand for the mammalian aryl hydrocarbon receptor (AHR), which it binds with high-affinity, inducing expression of cytochrome P450 1A1 (CYP1A1). We investigated whether the response to FICZ is evolutionarily conserved in vertebrates by measuring FICZ binding to two zebrafish AHRs (AHR1B and AHR2) and its ability to induce zebrafish CYP1 genes (CYP1A, CYP1B1, CYP1C1, CYP1C2, and CYP1D1) in vivo. Exposure of zebrafish embryos (48 h-post-fertilization; hpf) to 10 nM FICZ for 6 h caused strong induction of CYP1A mRNA and a statistically significant but modest induction of CYP1B1 and CYP1C1. Neither CYP1C2 nor CYP1D1 expression was induced by FICZ under the conditions of dose, time or developmental stage examined here. CYP1A induction was significantly greater after 6 h than after 12 h of exposure to FICZ, suggesting a rapid degradation of inducer. The 6-h EC₅₀ values for induction of CYP1A and CYP1B1 by FICZ were 0.6 and 0.5 nM compared to 72-h EC₅₀ values of 2.3 and 2.7 nM for PCB126, indicating that in zebrafish embryos FICZ is a more potent inducer than PCB126. FICZ at 10 nM was able to completely displace binding of 2,3,7,8-tetrachloro-1,6[³H]-dibenzo-p-dioxin to in vitro-expressed zebrafish AHR2 and AHR1B. Inhibition of AHR2 translation in zebrafish embryos by an AHR2-specific morpholino antisense oligonucleotide decreased the induction of CYP1A and CYP1B1 by FICZ and by PCB126. Together, these results demonstrate that FICZ is a potent AHR agonist in zebrafish, inducing expression of multiple CYP1 genes largely through AHR2. Evolutionary conservation of the response to FICZ is consistent with a possible role as an endogenous signaling molecule acting through the AHR.     

Zebrafish Jak2a plays a crucial role in definitive hematopoiesis and blood vessel formation

We examined the role of zebrafish (Danio rerio) Jak2a, a homolog of mammalian Jak2, in the developing embryo by injecting in vitro synthesized Jak2a shRNA into zebrafish zygotes. Blood circulation was suppressed in Jak2a shRNA-injected embryos from 24 hours post fertilization (hpf) and all embryos died with enlarged pericardium, shortened body lengths, and defects in some vasculature within 8 days post fertilization. O-dianisidine staining of red blood cells revealed normal blood island formation with no circulating red blood cells. As in Jak2^−/− transgenic mice, expression of definitive Ba1 globin was significantly reduced in Jak2a knockdown embryos at 36 hpf, whereas expression of other hematopoietic markers, primitive be1 globin, gata-1, and scl, were unaffected. More importantly, blood vessel formation was disturbed in Jak2a knockdown embryos as revealed by alkaline phosphatase staining at 72 hpf. Thus, our data indicate that zebrafish Jak2a is important in both definitive hematopoiesis and blood vessel formation.