Fig. 6

Haploinsufficiency in ribosome biogenesis-related genes heightens vulnerability to alcohol-induced craniofacial deficits in zebrafish embryos. (A) Zebrafish embryos injected with nonsense MO, or MOs against RPL5A, RPL11, RPS3A, NOLC1, p53, or MDM2 had no or modest deficits in the size or shape of cranial cartilage elements as compared with no-MO controls. Low-dose alcohol-only also did not worsen cranial development. The combination of low-dose alcohol and morpholinos directed against genes that promote ribosome biogenesis (rpl5a, rpl11, rps3a, nolc1) and p53/MDM2 signaling (mdm2) synergized to worsen cranial development and, in some instances, ablated cranial cartilage. Similarly, low-doses of the RNA polymerase I inhibitor CX5461 modestly affected cranial cartilage and synergized with low-dose alcohol to eliminate facial cartilage. Conversely, high-dose alcohol (boxed panels) caused craniofacial reductions that were prevented by morpholinos directed against p53. All views are ventral at 4 dpf with equivalent magnification. (B) Depiction of morphometry measurements, and quantification of cranial length, Meckel area, and ceratohyal area. Units are arbitrary and normalized to the mean of the uninjected controls. Open bars, no-alcohol; shaded bars, alcohol at the low dose or high dose as indicated. All values are mean ± SD with 4–14 embryos per treatment as detailed in Methods. Data analyzed using two-way analysis of variance followed by post hoc analysis using Holm-Sidak multiple comparisons (morpholino, compound) or within-group comparisons (alcohol vs. no-alcohol). * Different at p≤0.001 from its exposure-matched control for either nonsense-MO vs. gene-morpholino or no-compound vs. compound-treated. # Different at p≤0.001 from its treatment-matched control, Alcohol vs. no-Alcohol.