Imaging dish

Embryos were anesthetized with 4g/L MS-222 and mounted in 0.8% low melting point agarose in 0.5X E2 medium on a Mat-Tek imaging dish. Prim migration was followed between 36- 48 hpf by timelapse analysis using a 40X water objective on a Zeiss LSM780 confocal microscope in a climate-controlled chamber. Recorded time lapses were max- projected and stitched in ZEN. In situ hybridization images were obtained on a Zeiss Axiocam compound scope using 10X and 40X-water objectives. Image processing was performed in Fiji.

Gea2-expressing yeast plasmids were transformed into gea2Δ yeast strain (CFY1470) and grown at 30°C in selection media to an OD600 of 0.6. Cells were added to an imaging dish (MatTek), allowed to settle for 10 min, then washed with fresh media. Cells were imaged using a CSU-X spinning-disk confocal system (Intelligent Imaging Innovations) with a DMI6000 B microscope (Leica), 100×1.46 NA oil immersion objective, and a QuantME EMCCD camera (Photometrics), using with a 200 μs exposure time.

To examine clathrin-mediated endocytosis, we used Alexa 488-conjugated transferrin (ThermoFisher, Waltham, MA). After microinjections, embryos were incubated in FSW at 12 °C until blastula stage (26 hpf). The embryos were then incubated in Alexa 488-conjugated transferrin at a working concentration of 25 μg/mL in FSW and NucBlue Live Cell ReadyProbe (ThermoFisher, Waltham, MA) to label DNA for 15 min, according to manufacturer’s instructions. Embryos were then deciliated by incubating in 2X ASW and transferrin for 2 min to immobilize embryos for imaging, then washed in 25 μg/mL Alexa 488-conjugated transferrin in FSW for 5 min. Embryos were then placed on an imaging dish (MatTek, Ashland, MA) containing FSW and imaged with Zen software using a Zeiss LSM 710 confocal microscope (Zeiss, White Plains, NY). The laser power and gain were unchanged throughout the experiment.