In situ hybridization and image acquisition

WISH and FISH were performed as described previously (15, 28). Digoxigenin (DIG)–labeled probes were used for the alkaline-phosphatase chromogenic staining of WISH. In the staining shown in Fig. 3A and figs. S2 and S4A, signals were amplified with a combination of anti–DIG-POD (horse-radish peroxidase) (used at 1:1000 dilution; Roche, 11 207 733 910), dinitrophenyl (DNP)–tyramide (1:100 dilution; TSA plus DNP system, PerkinElmer), and anti–DNP-AP (1:100 dilution; Vector MB-3100). In FISH, DIG, fluorescein (Flu), and DNP probes were used in combination with DyLight488, AlexaFluor568, DyLight594, and DyLight680 tyramides. In cell labeling and eRNAi experiments, biotin-dextran introduced in cell clones was visualized using Cy5-streptavidin in fixed samples (GE Healthcare, PA45001). Most samples were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich) to visualize DNA.

Stained embryos were mounted on glass slides with spacers. WISH samples were observed using a stereomicroscope system as described above (SZX12 and C7780-10) and an Axiophoto 2 fluorescence microscope (Zeiss) equipped with an AxioCam HRc (Zeiss). FISH samples were observed using a TCS SPE confocal system (Leica).