Head size measurements of 3dpf embryos were assessed using brightfield microscopy and quantified using the NIH ImageJ software package. To assess proliferation, PH3-stained embryos, images were taken using fluorescent microscopy along the z-axis and stacked to obtain a focused image spanning the full head. PH3-positive cells from the forebrain to hindbrain (directly behind the cerebellum) were then counted for quantification purposes using ImageJ. TUNEL staining was performed to measure apoptosis using Apoptag Red In Situ Apoptosis Detection Kit (Millipore). TUNEL-stained embryos were then imaged and quantified using the same technique as for proliferation. All experiments were replicated twice and aggregate data was compiled. Statistical differences between controls and treatment conditions for each phenotype were calculated using Student’s t-test.
