Figure 5

Dgat1 is not responsible for the residual triacylglycerol synthesis in the dgat2 mutants.A, in situ hybridization for dgat1a and dgat1b expression at 3 and 6 dpf in wild-type AB embryos. dgat1a and dgat1b are not detected in the YSL, but are expressed in the intestine; images are representative of all embryos from three experiments at each stage (n = 4–six embryos per probe per experiment); Scale bars = 200 μm. B, quantitative RT-PCR for dgat1a and dgat1b expression in wild-type, dgat2^sa13945/+, and dgat2^sa13945 embryos at three dpf (N = 5–6 clutches; 10 pooled fish per sample/genotype, mean ± SD, One-way ANOVA, p = 0.0887 dgat1a, p = 0.8773 dgat1b). C, depiction of the dgat1a and dgat1b gene structure highlighting the nature and locations of the c770 and c773 CRISPR/Cas9 mutations (GRCz11; dgat1a ENSDARG00000103503, transcript 201 (ENSDART00000158946.2), dgat1b ENSDARG00000054914, transcript 201 (ENSDART00000077185.5)). D, quantitative RT-PCR for dgat1a and dgat1b expression in dgat2^sa13945versus dgat1a^c770;dgat1b^c773;dgat2^sa13945 triple mutant embryos at 3 dpf (N = 3 independent experiments; n = 9 fish per genotype; mean ± SD, unpaired t test, ∗∗∗p < 0.001). E, examples of yolk opacity phenotypes in dgat1a^c770;dgat1b^c773;dgat2^sa13945 triple mutant embryos at 3 dpf; Scale = 500 μm. F, embryos from in-crosses of dgat1a^c770/+;dgat1b^c773/+;dgat2^sa13945 parents were imaged and scored at three dpf for the degree of yolk opacity, binned into the four noted categories prior to genotyping and expressed as a percent of total embryos per genotype (N = 4 independent experiments, n = 22–89 fish per genotype). G, dgat2^sa13945 embryos were co-injected at the 1-cell stage with CMV:dgat1a-FLAG, CMV:dgat1b-FLAG and CMV:EGFP-CAAX plasmids, or CMV:EGFP-CAAX alone as a control. Bright-field images were obtained of all the embryos that expressed EGFP-CAAX in the YSL at 3 dpf; representative images of embryos from the two treatment groups (top, Scale = 500 μm). Images were assessed for the degree of yolk opacity, binned into the four noted categories of yolk opacity as noted in F and expressed as a percent of total EGFP-positive embryos/treatment group (n = 77 EGFP-CAAX and n = 77 dgat1-FLAG embryos pooled from three independent experiments, Chi-square test p < 0.0001).