Zebrafish GFP Reporter Lens Assay

Two transient lens assays were conducted during this study. In the first assay, we injected GFP reporter constructs into zebrafish eggs at the one-cell stage and counted the number of positive lenses in live embryos at around 52 hpf or more than 20 h after the onset of enhancer activity in the lens from the Fugu element [8 (link)]. This leaves enough time for the detection of a robust fluorescent signal in the lens. Prior to counting GFP-positive lenses, we discarded those embryos with the weakest GFP signal, but never more than 10% of the entire batch. We then randomly picked about 30–50 embryos (or 60–100 lenses) and determined GFP expression under a Leica MZ 16F dissecting microscope. The injections were repeated in total three times for each construct.