Figure 4

Cannabinoids impair Sonic Hedgehog signaling. (a) Effects of different concentrations of CBD, CP 55,940 and HU-210 on Smoothened agonist, SAG, stimulation of Gli1-mediated firefly luciferase expression in ShhLight2 cells. All data are the mean (±SEM) ratio of Gli1-mediated expression of firefly luciferase to constitutive renilla luciferase from 6 samples conducted in triplicate in two independent experiments, normalized as a percent inhibition of the maximal SAG effect. (b) Effects of different concentrations of CP 55,940 on Gli1-mediated firefly luciferase induced by a maximally effective SAG concentration (300 nM) in ShhLight2 cells. All data are the mean (±SEM) ratio of Gli1-mediated expression of firefly luciferase to constitutive renilla luciferase from 5 samples performed in triplicate in a single experiment. (c) Effects of different concentrations of CBD (yellow), CP 55,940 (green) and HU-210 (black) on Gli1-mediated alkaline phosphatase expression induced by a maximally effective SAG concentration (100 nM) in C3H10T1/2 cells. All data are mean (±SEM) alkaline phosphatase expression from 3 replicate samples from a single experiment, normalized as a percent inhibition of the SAG effect. (d) Shh pathway gene expression as measured by qRT-PCR in mouse rostral neural tube, 24 hours following GD 8 exposure to the CB vehicle or CP 55,940 (0.25 mg/kg). Shh, Gli1, and Gli2 are normalized to the housekeeping gene 18 s and portrayed as log₂ fold change from vehicle. All samples were measured in triplicate and the data are portrayed from a single experiment. Individual embryos are portrayed as open or filled circles (n = 9 embryos/5 litters for vehicle for Shh, or 10 embryos/5 litters for Gli1 and Gli2 vehicle; n = 10 embryos/4 litters for CP 55,940), while means (±SEM) are portrayed as open or filled bars. *p < 0.05, **p < 0.01 using two-sided Student’s t-tests. Individual embryos are marked by different fill colors. (e) The incidence of small eyes and midbrain/hindbrain boundary defects in zebrafish embryos receiving exposure to Shh mRNA (N183) + fish water vehicle (n = 40 for eye, 45 for MHB), CP 55,940 2.5 mg/L alone (n = 32 for eye, 34 for MHB), Shh mRNA (N183) + CP 55,940 2.5 mg/L (n = 32 for eye, 41 for MHB), CP 55,940 5.0 mg/L alone (n = 54 for eye, 57 for MHB), Shh mRNA (N183) + CP 55,940 5.0 mg/L (n = 53 for eye, 46 for MHB), 0.5% alcohol alone (n = 41 for eye, 45 for MHB), Shh mRNA (N183) + 0.5% alcohol (n = 30 for eye and MHB), 0.5% alcohol + CP 55,940 2.5 mg/L (n = 35 for eye, 45 for MHB), Shh mRNA (N183) + 0.5% alcohol + CP 55,940 2.5 mg/L (n = 34 for eye, 45 for MHB). **p < 0.01, ***p < 0.001 vs Shh mRNA (N183) + vehicle, ^^p < 0.01, ^^^p < 0.001 vs corresponding CP 55,940 treatment in absence of Shh mRNA using a Chi-square test. Data are expressed as the percent of zebrafish with defects (number of affected zebrafish/total number of zebrafish *100) from a single experiment.