Schematic workflow of optogenetic analysis. Embryos were dechorionated and exposed to APV and 470 nm light from 1 to 3 dpf. Embryos were double-transgenic for foxP2:egfp-caax; foxP2:Gal4. Some embryos carried UAS:ChR2-YFP (determined by post-hoc genotyping). If neuronal activity could rescue pathfinding, only triple-transgenic embryos would have normal midline pathfinding (arrow). Bottom pictures show in situ c-fos analysis of experimental embryos after 470 nm light exposure. Embryos carrying ChR2-YFP have increased c-fos expression (red arrow in region of foxP2.A.2 neurons).