Nitex mesh

The jelly coat of unfertilized eggs was removed by passing them three times through an 80 μm Nitex mesh (Genesee Scientific) to facilitate egg adhesion on protamine-coated glass-bottom dishes (MatTek Corporation). Unfertilized eggs were transferred to protamine-coated glass-bottom dishes for a maximum time of 15 min before microinjection and fertilization. Microinjections were performed using a FemtoJet 4 and Injectman 4 micromanipulator (Eppendorf). Injection pipettes were prepared from siliconized (Sigmacote; Sigma-Aldrich) borosilicate glass capillaries (1 mm diameter). Glass capillaries were pulled with a needle puller (P-1000; Sutter Instruments) and ground with a 30° angle on a diamond grinder (EG-40; Narishige) to obtain a 5–10 μm aperture. Injection pipettes were back-loaded with 2 μl before each experiment and were not reused. Injection volumes were generally less than 5% of the egg volume, ~2–5 pL. GST-GFP-NLS and GST-mCherry-NLS proteins were diluted to ~2.5 mg/ml in PBS before injecting. Injecting undiluted GST-GFP-NLS did not affect developmental progression, suggesting that these protein concentrations are not detrimental to the embryo. RNA was diluted to ~50 ng/μl in PBS before injecting. These concentrations were empirically selected to optimize imaging while not affecting developmental progression.