Figure 3

dgat2 mutant embryos are still capable of triacylglycerol synthesis in the YSL.A, TopFluor C11 fatty acid was mixed with canola oil and microinjected into the yolk mass of dgat2^sa13945 mutants and siblings at 3 dpf. B, representative image of the TopFluor C11 signal in a dgat2^sa13945 zebrafish yolk sac 4 h after injection; scale = 200 μm. C and D, lipids were extracted from TopFluor C11 injected embryos and separated by thin layer chromatography (20 pooled embryos per sample, wild-type and dgat2^sa13945/+ embryos were combined). C, lipid classes were identified using TopFluor-labeled lipid standards. Arrow indicates TAG synthesized from TopFlour C11 in vivo. (CE: cholesterol ester, TAG: triacylglycerol, DAG: diacylglycerol, MAG: monoacylglycerol; PL: phospholipid; ∗ denote autofluorescent lipids in the fish lysate). D, the quantity of lipids in each class was quantified and expressed as a percent of total fluorescent lipids; N = 6 experiments, mean ± SD, Two-way ANOVA, not significantly different, p = 0.9929. Note, it appears that the TopFluor C11 is unable to be incorporated into cholesterol esters. E, large lipid droplets were manually dissected out of dgat2^sa13945 larvae at 6 dpf (40–50 LDs from 30 – 40 larvae). Lipids in LDs or an equivalent volume of media surrounding dissected larvae were separated by multistep gradient HPLC and detected with a charged aerosol detector (CAD) in picoamperes (pA) as described in (75). The lipid droplet samples contain large quantities of triacylglycerols and cholesterol esters. Traces are representative of three independent experiments.