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Fig. 5 drish mutants show defects in the formation of the retinal ganglion cells, optic nerve and inner nuclear layer. (A, B, D, E) Confocal imaging of drish+/+ and drish−/− embryos processed for zn8 immunostaining at 2 dpf (A, B) and 4 dpf (D, E) to label RGC and optic nerve. The optic nerve appeared thinner in drish−/− mutant embryos compared to drish+/+ embryos at 2 dpf but no difference was observed at 4 dpf. Ventral view of the head region with anterior to the top is shown. Arrows denote the location where diameter measurements were taken. Asterisks in A and B denote retina. Note the reduced zn8 staining in the retina of drish−/− embryos. Scale bar, 50 μm. (C, F) Quantification of the optic nerve diameter at 2 and 4 dpf in individual drish+/+ and drish−/− embryos, respectively. Data pooled from two experiments, n = 20 (+/+) and 30 (−/−) at 2 dpf and n = 20 (+/+) and 33 (−/−) at 4 dpf. **p < 0.01, ns, not significant, Student's t-test. (G, H) Anti-caspase 3 immunofluorescence for apoptotic cells in drish+/+ and drish−/− embryos at 24 hpf. Maximum intensity limited projection of the eye area is shown. No apparent increase in apoptosis was observed in the mutant embryos (n = 20 for wt and 40 for mutant embryos, two replicate experiments). (I, J) H&E stained coronal sections of drish+/+ (I) and drish−/− (J) larval retina at 5 dpf were observed to analyze disruptions in granule cell layer, inner nuclear layer, photoreceptor layer, optic nerve pathfinding and optic chiasma formation. drish−/− larvae displayed normal morphology of retina with granule cell layer (GCL), inner plexiform layer (IPL), photoreceptor layer (PR), retinal pigmented epithelium (RPE), choroid (CH), optic nerve (ON) and optic chiasma (OC). However, the inner nuclear layer (INL) appeared thinner in the mutant larvae. (K–M) Quantification of cell number in granular, inner nuclear, and photoreceptor layers. No difference in the number of GCL and PR cells was observed between drish−/− and drish+/+ larvae. However, the number of cells in the inner nuclear layer (INL) of drish−/− retina was significantly reduced (Student's one-tailed t-test, p < 0.05) compared to drish+/+ retina (n = 5).