Figure 1

The effects of ethanol and cannabinoids on MHB and cardiac development. (A–E), MHB development was disrupted with 3–24 hpf 1.5% ethanol or 10 mg/L ACEA or 2.5 mg/L CP exposure (B,C,E), but not with 1.25 mg/L CP exposure ((D), arrow). (F–J), Obvious pericardial edema was only observed with exposure to 1.5% ethanol ((G), arrow), with no edema observed with any of the cannabinoid doses.
Figure 2

Effects of ethanol and cannabinoid exposure on the expression of cmlc2 in embryonic zebrafish heart. Compared to the cmlc2 expression in untreated embryos ((A), dorsal view, (F,F’) lateral view), only exposure to 1.5% ethanol disrupts the normal cmlc2 cardiac expression pattern ((B,G,G’), arrows) in 1 dpf and 3 dpf embryos. No effects of cannabinoid exposure on cmlc2 expression were observed at any concentrations of these agonists (C–E,H–J,H’–J’). Each experiment was conducted in triplicate with an n = 20, with 60/60 embryos showing a normal cmlc2 expression pattern with no treatment or cannabinoid agonist exposure, and 0/60 embryos showing a normal cmlc2 expression pattern with ethanol exposure.
Figure 3

The effects of ethanol and cannabinoid exposure on the expression of other cardiac markers in embryonic zebrafish heart. The expressions of nkx2.5 (A–E) and vmhc (F–J) were examined in 1 dpf heart following ethanol or cannabinoid exposure from 3–24 hpf. Compared to the marker expression in untreated embryos (A,F), only exposure to 1.5% ethanol disrupts the normal cardiac marker expression pattern (B,G), with no effects of cannabinoid exposure on cardiac marker expression observed at any concentrations of these agonists (C–E,H–J). Each experiment was conducted in triplicate with an n = 20, with 60/60 embryos showing a normal nkx2.5 or vmhc expression pattern with no treatment or cannabinoid agonist exposure, and 0/60 embryos showing a normal marker gene expression pattern with ethanol exposure.
Figure 4

The effects of ethanol and cannabinoid exposure on the expression of the cmlc2 cardiac marker in embryonic zebrafish heart following drug exposure from 10–24 hpf. The expression of cmlc2 was examined in 1 dpf (A–F) heart (dorsal view) and 3 dpf (G–L’) heart (ventral and lateral view) following ethanol or cannabinoid exposure from 10–24 hpf. Compared to the marker expression in untreated embryos (A,G,G’), only exposure to 1.5% ethanol disrupts the normal cmlc2 expression pattern (B,H,H’), with no effects of cannabinoid exposure on expression observed at any concentrations of these agonists (C–F,I–L’). Each experiment was conducted in triplicate with an n = 20, with 60/60 embryos showing a normal cmlc2 expression pattern with no treatment or cannabinoid agonist exposure, and 0/60 embryos showing a normal cmlc2 expression pattern with ethanol exposure.
Figure 5

Cannabinoid receptor expression in early zebrafish development. The expression pattern of cnr1 (A–C’) and cnr2 (D–F’) in 1 dpf, 2 dpf and 3 dpf embryos was analyzed by in situ hybridization. A robust expression was observed in the head region of embryos (A–F) with an apparent lack of expression in early heart (C,C’,F,F’).
Figure 6

Extracellular matrix signaling involving agrin and Shh is disrupted by ethanol exposure in early cardiac development. Cmlc2 mRNA expression was analyzed in dorsal views of 1 dpf embryos. (A), wild-type; (B), 0.5% ethanol exposure from 3–24 hpf; (C), 0.5 pmol agrin MO injection of embryos; (D), 1 pmol shh MO injection of embryos; (E), sub-threshold 0.1 pmol agrin MO injection; (F), 0.1 pmol agrin MO combined with 0.5% ethanol exposure; (G), sub-threshold 0.3 pmol shh MO injection; (H), 0.3 pmol shh MO combined with 0.5% ethanol; (I), 0.1 pmol agrin MO and 25 pg shhN183 mRNA injection of embryos; (J), 25 pg shhN183 mRNA injection rescues the cmlc2 expression pattern in embryos treated with 0.1 pmol agrin MO and 0.5% ethanol; (K), 0.3 pmol shh MO and 25 pg shhN183 mRNA injection of embryos; (L), 25 pg shhN183 mRNA injection rescues cmlc2 expression pattern in embryos treated with 0.3 pmol shh MO and 0.5% ethanol.
Figure 7

Cmcl2 mRNA decreases, resulting from MO and ethanol exposures in 1 dpf embryos. * Significantly different from control p < 0.01; ** p < 0.025; *** Significantly different from MO + EtOH, p < 0.025.
Figure 8

Cmlc2 mRNA expression in 3 dpf zebrafish heart following MO and EtOH treatments. Ventral and lateral views of embryos are shown. (A,A’), wild-type; (B,B’), 0.5% EtOH exposure from 3–24 hpf; (C,C’), 0.5 pmol agrin MO injection of embryos; (D,D’), 1 pmol shh MO injection of embryos; (E,E’) 0.1 pmol agrin MO injection; (F,F’), 0.1 pmol agrin MO combined with 0.5% EtOH exposure; (G,G’), 0.3 pmol shh MO injection; (H,H’), 0.3 pmol shh MO combined with 0.5% EtOH; (I,I’), 0.1 pmol agrin MO and 25 pg shhN183 mRNA injection of embryos; (J,J’), 25 pg shhN183 mRNA injection rescues cmlc2 expression pattern in embryos treated with 0.1 pmol agrin MO and 0.5% EtOH; (K,K’) 0.3 pmol shh MO and 25 pg shhN183 mRNA injection of embryos; (L,L’), 25 pg shhN183 mRNA injection rescues cmlc2 expression pattern in embryos treated with 0.3 pmol shh MO and 0.5% EtOH.
Figure 9

Cmcl2 mRNA decreases, resulting from MO and ethanol exposures in 3 dpf embryos. * Significantly different from control p < 0.01; ** p < 0.025; *** Significantly different from MO + EtOH, p < 0.01.
Acknowledgments
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