Figure 5

GFP-LD distribution in early embryos. (A, B) Western analysis of wild-type (WT) and GFP-LD expressing (GL) animals. GL samples are from females carrying both a GFP-LD transgene and the matα4-Gal4-VP16 driver or from embryos laid by these females. GFP-LD was detected with anti-GFP antibodies; corresponding WT samples demonstrate specificity of detection. (A) GFP-LD is present in both ovaries and embryos. (B) GFP-LD is expressed at similar levels in Phase I, II, and III embryos. Tubulin serves as loading control. (C, D) GFP-LD expressing embryos examined live by confocal microscopy. In otherwise wild-type embryos, GFP-LD is present in distinct puncta that accumulate around the central yolk (C). In embryos mutant for Halo, GFP-LD puncta accumulate just under the nuclei (D); these puncta are shifted outwards relative to the embryos in C. Top: whole-embryo view. Bottom: detail of the embryonic periphery. The embryos in C and D are age-matched (top: early Phase II; bottom: mid Phase II). Scale bars in D represent 60 μm (top) and 10 μm (bottom), respectively. (E) GFP-LD expressing embryos imaged live by epifluorescence microscopy. Comparison to a wild-type embryo demonstrates that most of the signal is due to GFP-LD. Global distribution of GFP-LD in Phases I, II, and III mimics the distribution of lipid droplets at these embryonic stages (Fig. 3A); in particular, GFP-LD is spread throughout the periphery in Phase III if endogenous Klar β is present, but not if it is absent. (F) Even when expressed in the absence of endogenous Klar β, GFP-LD is present in discrete puncta (scale bar = 6 μm). (G) Triglyceride levels in wild-type and GFP-LD expressing embryos (0-1.5 hrs old) are very similar. Error bars represent the standard deviation from two different experiments.