Fig. 4: Ganglion cell loss and optic nerve atrophy in Pitx2^KI mutant mice.

A, B Retinal RGCs from 6-week-old (A) and 3-month-old (B) mice were labeled with anti-Brn3a, and the nuclei were counter-stained with DAPI. Scale bars: 25 μm. C Quantification of the Brn3a⁺ RGC number in GCL. Pitx2^KI mutant mice at 3 months of age exhibit slightly thinner inner and outer nuclear layers compared with WT mice (n = 6 per cohort). D Representative immunofluorescent enface images of enface view of a retinal flatmount from WT and mutants at 3 months of age labeled with anti-Brn3a for detecting RGCs. Scale bars: 50 μm. E Pitx2^KI mutants have significantly decreased numbers of RGCs per mm² (n = 8) relative to their control littermates (n = 10). F Fundus photograph at 5 months shows abnormal morphology of optic nerve head (arrowhead) in the eyes of Pitx2^KI mutant mice. G Representative images of histologic sections taken through nerve heads from Pitx2^WT and Pitx2^KI mice at 7 weeks and 3 months of age. The optic nerve head appeared abnormal in Pitx2^KI mice at both ages, with thinning of the nerve fiber layer (NFL) (red arrowheads) and optic nerve excavation is noted (asterisk). Black arrows indicate NFL entering the optic nerve (ON); V: Central blood vessel. Scale bars: 200 μm. H Quantitation and comparison of the NFL thickness near the optic nerve in 7-week and 3-month-old mice (n = 6 per cohort). I Position-matched images of the H&E staining (upper panel) and the immunofluorescent staining (lower panel) of retinal cross sections are shown. Retinal nerve fibers were labeled with neurofilament-L (NF-L), and the nuclei were counter-stained with DAPI. Scale bars: 25 μm. J Optic nerve atrophy was assessed in cross semithin sections of resin-embedded optic nerves from 5-month-old WT and mutant mice stained with toluidine blue staining. Scale bars: 100 μm. The panels on the right show high-magnification images of the boxed areas shown in the panels on the left. Scale bars: 10 μm. K Quantification of axon number in the optic nerve of 5-month-old mice (n = 10 per cohort). The Pitx2^KI mutants have significantly less axonal projections compared to Pitx2^WT mice. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. The statistics were calculated based on data from all tested mutants. The representative images of Pitx2^KI were from mutants with elevated IOPs. **P < 0.01; ***P < 0.001. #, no significant difference. The data are presented as mean ± SEM.