Figure 7.

Expression of hairpin against rab11a leads to ectopic process formation, reduction in outer segment length, and increased photoreceptor death. A, Representative images of tadpoles expressing various constructs. Yellow, mVenus; green, EGFP-rab11a; red, TUNEL; blue, DRAQ5. B, Representative dot blots from whole tadpole eye showing rab11a expression changes with hairpin expression. Expression was normalized to tubulin (data not shown). Hairpin-expressing tadpoles had a significant 35.5% reduction in total eye rab11a (n = 5–8 per group; one-way ANOVA, F_(3,22) = 4.038, p = 0.02; Tukey' HSD test for nontransgenic versus hairpin, p = 0.038. C, Quantification of TUNEL-positive cells and OS length. Rods expressing a hairpin against firefly luciferase had normal morphology. Rods expressing a hairpin against rab11a had shorter outer segments and significantly increased numbers of TUNEL-positive cells per section. Coexpression of EGFP-rab11a resistant to the hairpin rescued the effects. TUNEL-positive cells (n = 8–14 per group; one-way ANOVA, F_(3,39) = 6.031, p = 0.002; Tukey's HSD test: for rab11a hairpin versus nontransgenic animals, p = 0.001; for rab11a hairpin versus luciferase hairpin, p = 0.009; for rab11a hairpin versus rescue, p = 0.048; all other comparisons, p > 0.05). OS length (n = 6–14 per group): one-way ANOVA: F_(3,34) = 6.858, p = 0.001; Tukey's HSD test: for rab11a hairpin versus nontransgenic animals, p = 0.006; for rab11a hairpin versus luciferase hairpin, p = 0.017; for rab11a hairpin versus rescue, p = 0.002; all other comparisons, p > 0.05. D, TUNEL-positive processes (arrow) projecting from the ONL into the inner retina were observed in rab11a hairpin-expressing tadpoles. Yellow, mVenus. E, Rhodopsin staining of tadpoles expressing hairpin constructs. Arrowheads indicate inner segment or nuclear layer staining of rhodopsin. Yellow, mVenus; red, rhodopsin; blue, DRAQ5. Scale bars, 10 μm. *p < 0.05, **p < 0.01, ***p < 0.001.