Image quantification: Retinal ganglion cell (zn-5) fluorescence

3-dimensional datasets were acquired of laterally oriented embryos. For each embryo, a maximum intensity projection (MIP) was generated in FIJI. The range of slices used for the MIP was determined such that the most distal slice was the one where the lens was just visible and the most proximal slice was just behind the lens. As schematized in Fig 8K, an elliptical ROI (area = 18000 μm²) surrounding the retinal ganglion cell region was defined. The center of the elliptical ROI overlapped the center of lens. The fluorescence intensity of zn-5 staining within the ROI was calculated as the mean gray value using the Measure function in FIJI. Similarly, the mean gray value of zn-5 in a rectangular ROI (area = 3300 μm²) placed in the brain, dorsal to the eye, was also measured. The mean fluorescence intensity of zn-5 in the eye was normalized to the fluorescence intensity of zn-5 in the region outside the eye.