Figure 3

Expression of PDK1^G380D in zebrafish embryos affects embryogenesis. (A) Mitochondria with membrane potential were labeled using a Mitotracker labeling assay and the quantification was performed by counting the positively stained cells (see inset for close up on positive cells) in an area of 300 × 350 µm in the trunk of the embryo (white box). (A’) Mitotracker-positive cells shown in higher magnification. A significant decrease in the number of cells with labeled mitochondria was found in pdk1-MO + PDK1^G380D when compared to control and pdk1-MO + PDK1^wt embryos (n = 40–45 per group, **** p < 0.0001). (B) Expression of mutant PDK1 in zebrafish embryos resulted in a significant decrease in spontaneous movements (coils) when compared to the others experimental groups (n = 108–120 embryos per group, ** p = 0.003, *** p = 0.004, **** p < 0.0001). (C) Neuronal differentiation was assessed by labelling embryos with acetylated-a-tubulin (neurons, in green) followed by quantification of the neuronal projections in the tectum (top view). We found a significant decrease in the number of projections crossing the white dotted line of embryos injected with pdk1-MO + PDK1^G380D when compared to control embryos (n = 30 embryos per group, * p_{control vs. pdk1-MO} = 0.011 and p_{control vs. pdk1-MO + PDK1wt} = 0.0214, **** p < 0.0001). (D) Apoptosis was increased in experimental groups when compared to control embryos, detected by Western blot, but it does not correlate with the severity of the other phenotypes analyzed (quantification of cleaved Caspase-3 normalized to total protein, n = 4 protein extracts per group). (E) PS10, a compound known to inhibit the phosphorylation of PDH-E1α upstream of PDKs, inhibits the rescue by PDK^wt, and supports the hypomorphic nature of PDK1^G380D (n = 52–70 embryos per group, **** p < 0.0001).