Supplementary Figure 3: GstE14 functions in cholesterol metabolism.

A. Cuticle preparation of Df(GstE14) and spo mutant embryos incubated in Schneider’s medium supplemented with either 20E, ecdysone or cholesterol during mid-embryogenesis. Incubation with Schneider’s medium alone (mock) was used as control. All three compounds significantly suppressed embryonic lethality, as well as rescued epidermal differentiation, that is cuticle differentiation and trichome formation, for Df(GstE14) mutants. In contrast, spo mutants were rescued by the exogenous addition only of 20E and ecdysone, but not by cholesterol, consistently with the documented requirement of spo activity for the transformation of 7-dehydro-cholesterol to ketodiol³³. Scale bar, 100 μm. B. Schematic representation of the successive steps of the biosynthetic pathway leading to ecdysone production from dietary sterols. As deduced from rescuing experiments, GstE14 activity is required for the very early stages of the pathway, since its lack can be rescued by cholesterol. C. High cholesterol diet of parental flies suppresses the embryonic lethality of GstE14 mutants, allowing a marked increase in life span. Df(GstE14)/CyoDfdYFP and spo/TM3DfdYFP heterozygous flies were fed for two days with high cholesterol diet, or regular food medium for control, and transferred to egg collection devices. Parental high-cholesterol diet led to the survival of approx 10% of Df(GstE14) mutants, which hatched into viable L1 larvae. The experiments have been made four times independently. The total number of mutant embryos analysed is 422 individuals for GstE14 and > 1,000 for spo. Rescued larvae displayed no obvious morphological defects when compared with wild type larvae. Although these animals remained alive for several days (up to 7 days), they failed to proceed for pupariation, or even larval stage transitions, and instead remained long-lived L1 larvae as deduced from the examination of mouth hooks, a phenotypical marker of larval stages. Arrows highlight the number of mouth hook teeth in wild type, which displays a characteristic increase across larval stages. The chart plot means values, for three independent experiments. Errors bars are s.d., scale bar is 25 μm. D. Inactivation of GstE14 impinges on whole body cholesterol levels, both in embryos and in larvae. The sterol content of Df(GstE14) mutant embryos, and larvae driving UAS–dsRNA–GstE14 (line #1: HMJ21555; line #2 v1018884) in the ring gland (phm-Gal4) was assessed using a commercial assay. When compared with wild type controls, GstE14 embryos display higher levels of sterol (P value = 0.0028). The same was true for phm > dsRNA–GstE14larvae (P value = 0.0006), showing that GstE14 activity in the ring gland is required for maintaining proper cholesterol levels. Extracts were made from hand-counted embryos or larvae, with 1 to 5 independent samples of the same genotype per experiment. All experiments have been repeated independently three times. The graph shows all data points. Statistical tests used two-tailed Mann Whitney tests, error bars are s.d. (blue), means are indicated by a red dotted line.