Increasing scFv-FP expression allows translation detection at later stages. (A) Schematic of UASp-scFv-msGFP2 construct and activation by the Gal4 protein. Ten UAS sequences (purple) are placed upstream of the P-promoter (blue) and scFv-msGFP2 (green) sequences. Gal4 proteins (yellow) will bind the UAS sequences to activate transcription of the scFv-msGFP2. (B) Schematic of the expression of the scFv-msGFP2 in nosGal4 > UASp-scFv-msGFP2 female strain during oogenesis until egg deposition. (C, left) Schematic of a sagittal view of a Drosophila embryo with twi expression pattern in purple. Anterior side is on the left, posterior on the right, dorsal on the top, and ventral side on the bottom. Black square represents the imaged area of the right panels. Single Z-planes of confocal images from smFISH with direct scFv-msGFP2 FP signal (green) and MS2 probes (red) on nosGal4 > UASp-scFv-msGFP2 x twi_suntag_MS2_CRISPR embryos in early, early-mid, and mid-late n.c. 14. Gray squares represent the zoomed images for each panel. Nuclei are counterstained with DAPI (gray, bottom images) for staging. Scale bars, 10 µm on the larger images, and 5 µm on the zoomed images. (D, top) Schematic of a Drosophila embryo on the ventral side with gastrulation furrow represented with invaginating cells. Black square represents the imaged area of the bottom panels. (Bottom) Single Z-planes of confocal images from smFISH with direct scFv-msGFP2 FP signal (green) and MS2 probes (red) on nosGal4 > UASp-scFv-msGFP2 x twi_suntag_MS2_CRISPR and scFv-msGFP2 x twi_suntag_MS2_CRISPR embryos at gastrulation stage and on the ventral side. Scale bars, 10 µm on the larger images, and 5 µm on the zoomed images. Note that translation dots are visible only with nosGal4 > UASp-scFv-msGFP2. (E, top) Schematic of a Drosophila embryo on the ventral side with gastrulation furrow represented with invaginating cells. Black square represents the imaged area of the bottom panels. (Bottom) Single Z-planes of confocal images from movie of scFv-msGFP2 x yw, scFv-msGFP2 x twi_suntag_MS2_CRISPR and nosGal4 > UASp-scFv-msGFP2 x twi_suntag_MS2_CRISPR embryos at gastrulation stage (furrow represented with gray dashed line). Scale bar, 10 µm (see related Supplemental Movie S20).
