Eye phenotypes induced by secreted anti-Pax6 single-chain antibody. (a-f) Eye phenotypes observed at 30 hpf after injection of secreted single-chain anti-Pax6 (spaP6) mRNA into one cell embryos. (a) Normal, (b) small eyes, (c) dissymmetric eyes, (d) single eye, (e) no eye, (f) cyclops. (g) Quantification (percentage) of phenotypes: n, normal; d, dissymmetric, reduced and absent eyes (pooled); c, cyclops. Comparison between secreted anti-Pax6 (spaP6, n = 221, red), secreted anti-En (spaEn, n = 251, blue) and non-secreted anti-Pax6 (aP6, n = 178, dark red). Differences between spaP6 and spaEn or aP6, as well as between aP6 and spaEn, are statistically significant (p < 0.001, χ2 tests). (h) Quantification of single-chain mRNA expression. Top: western blot of protein extracts from embryos injected with the indicated RNAs (no : no RNA); the arrow indicates a single-chain antibody signal at approximately 34 kDa, and the arrowhead the cross-hybridizing protein species used for calibration. Bottom: densitometric scans of western signals indicate aP6 expression (ratio 1:1 with the endogenous signal) is at least as high as spaP6 expression (ratio 0.7:1 with the endogenous signal).