Analysis of Growth

The angle of growth was examined by drawing a line from the center of the cell to the tip of each neurite and measuring the angle of this line relative to a line drawn for the original axis of the rod photoreceptor, which was determined as explained below. The angle of growth was measured only on the basal side (axon-bearing side) of the photoreceptor where the gradient of the guidance cue was applied directly and, therefore, most assured. The amount of growth was quantified with a modified Scholl analysis: by counting all neurites that had grown 25 μm from the center of the cell using NIH ImageJ. This analysis was performed on the entire cell circumference since processes can be initiated from any point along the cell surface. Using ImageJ, the length of every neurite was measured from the cell body. Angles were tested for significance using the Watson-Williams U test while number and length of neurites were tested using 1-way ANOVA and the Tukey-Kramer test.

The axis of the photoreceptors was determined by drawing a line drawn between the center of the ellipsoid and the nucleus. Changes in orientation after spritzing from the original orientation of the ellipsoid and nucleus relative to the micropipette were measured as changes in the angle of the axis from the original axis. Changes in orientation were tested using 1-way ANOVA and Tukey-Kramer. All data were expressed as the mean plus the standard deviation. Significance was considered to be achieved at P ≤ 0.05.