Figure 4
Pax6 and Tuj1 immunoreactivity. Localization of Pax6 (a–e,k–o), and βIII tubulin (f–j,p–t) proteins in wild-type (left 4 panels) and Pax6-CRISPR (rest of the panels) embryos at E16.5. High magnification images of eyes (boxed area in a–j) are shown in (k–t). Serial coronal sections were immunolabeled with anti-Pax6 and anti-βIII tubulin (Tuj1) antibodies. (a,k) In the E16.5 normal retina, expression of Pax6 is downregulated and restricted to retinal ganglion cells of the inner neuroblastic layer and to the innermost cells of the outer neuroblastic layer. (b,g) Oblique section from the T1#6 embryo confirming that both eyes were categorized appropriately as class 1 phenotype. (f,p) In the E16.5 normal retina, βIII tubulin is intensely detected in the inner neuroblastic layer. (l,q) Retinal development has delayed and the retina is not layered yet. Pax6 protein is detected all over the retina (l). βIII tubulin is restricted to the innermost thin domain where differentiating retinal ganglion cells reside (q). (n) The anti-Pax6 antibody detects abnormal Pax6 protein in which 3 amino acids are inserted in the PD. (u) Percentage of mutated and wild-type genotypes found in each embryo. Mut-Tr, mutations encoding truncated Pax6 proteins; Mut-IF, in-frame mutations; Wt, wild-type. Detailed results are shown in Fig. S5 and Table S4. (v) Relationship between the percent Pax6-positive retinal area and the percent truncated Pax6 mutations. The percent Pax6-positive retinal area showed an apparent negative correlation with the percent truncated Pax6 mutations, but it was not statistically significant (rs = −0.620; P = 0.056). (w) Similarly, the estimated area (mm2) of Tuj1-positive retina was positively correlated with that of Pax6-positive retina (rs = 0.830; P = 0.003). Scale bars: 500 μm in (a–j) and 100 μm in (k–t).
