Flies of the appropriate genotype were allowed to lay eggs on juice agar plates with a dollop of thick yeast paste for two hours at 25 °C. Embryos were then aged for 3.5 h at 25 °C to reach ES 10, 5.5 h at 25 °C to reach ES 11, 15 h at 18 °C to reach ES 12, 17 h at 18 °C to reach ES 13, 19.5 h at 18 °C to reach ES 14, 23 h at 18 °C to reach ES 15, 14 h at 25 °C to reach ES 16, and 16 h at 25 °C to reach ES 17. For staining with anti-Vasa antibody, staged embryos were collected in mesh baskets, dechorionated in 50% bleach, washed with 1× PBS + 0.1% TritonX-100 (PBTx), and fixed in 4% formaldehyde, 1.75 ml PEMS (100 mM PIPES pH 6.9, 1 mM EGTA, 2 mM MgSO4), and 8 ml heptane for 20 min. Vitelline membranes were removed by manual shaking in 100% methanol for 1 min.

For staining with multiple antibodies, we used an alternative fixation as follows: Staged embryos were placed in boiling Triton/Salt (0.7% NaCl, 0.04% TritonX-100) solution for 5 s then moved to cool Triton/Salt solution on ice for 15 min. Vitelline membranes were removed by manual shaking in 50% methanol:50% heptane for 1 min.

Embryos were rehydrated, washed with PBTx, and blocked with 5% normal goat serum in PBTx. The embryos were then incubated in primary antibodies overnight at 4 °C, washed again in PBTx and incubated with secondary antibody for two hours at room temperature. When applicable, embryos were placed in 1:500 Hoechst 33342 (H3570, Invitrogen):PBTx for 2 min. Embryos were washed in PBTx before being mounted onto slides in Fluoromount medium (SouthernBiotech, Birmingham, AL, USA) and were visualized using a confocal microscope (Zeiss LSM710, LSM780 and LSM800) or a Dragonfly spinning disc microscope for high resolution images.

For Supplementary Movie 1, Z sections of 0.2 μm each (57 sections in total) were taken. Z stack series were combined into a movie using Surfaces technology in the Imaris software (version 9.6.0).

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