Effects of MO-mediated CB1R knockdown on GnRH3 neurons in zebrafish embryos. (A) Western blot analyses of protein extracts from uninjected or z-cb1r MO-injected zebrafish embryos. A significant depletion of the CB1R protein was detected in the treated embryos. (B) Real-time qPCR analyses of z-cb1r and three z-hox genes on RNA samples extracted from uninjected (open bars) or z-cb1r MO-injected (solid black bars) embryos. Expression is shown relative to the expression of the housekeeping gene mRNA (z-β-actin). The abundance of the control RNA is set as 1. A depletion of the z-cb1r mRNA in embryos injected with z-cb1r MO is observed, as compared to control ones. No change in the expression of z-hox genes was observed, indicating unaltered developmental progression. (C–E) Representative images of EGFP GnRH3 neurons and projections in the nasal and the basal forebrain regions of zebrafish embryos, at the age 72 hpf. The observation plane is the same as in Figure 2. Scale bar is reported in C. Red arrows indicate EGFP+ neurons, white arrows indicate altered EGFP+ projections, and white asterisks indicate altered organization and fasciculation at the anterior commissure. Below each micrograph, the bright-field images show the general morphology and growth of the corresponding whole animal, with no difference between the three conditions. (F) Quantification of the number of EGFP+ neurons in the nasal region of uninjected (open bars), scrmbl MO-injected (solid grey bars) or z-cb1r MO-injected (solid black bars) embryos. A significant decrease was observed in embryos injected with the z-cb1r MO, compared to control ones. (G) Quantification of the misguidance and altered commissural phenotype, expressed as % of the number of embryos observed. A significant increase was observed in embryos injected with the z-cb1r MO, compared to control ones. Data are expressed as means ± SEM from three independent experiments. ** = p < 0.01; *** = p < 0.001. AC, anterior commissure.
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