atoh7 mutants lack retinal ganglion cells and are blind. (A) Quantification of the number of nuclei in the GCL revealed a significant reduction in mutant fish. (B) The qPCR analysis showed a highly significant reduction of the ganglion cell specific marker islet2b mRNA in mutant retinae. (C) Expression analysis of islet2b using in situ hybridization shows specific expression (yellow dots) in the GCL of wildtype but not in mutant retinae. Nuclei were stained with DAPI (white). (D) Representative cross sections of wildtype and mutant retinae stained with H&E at the level of the optic nerve. In contrast to wildtype fish that form a regular optic nerve (upper panel), the optic nerve is absent in mutant retinae which rather contain an ectopic blood vessel connecting the subretinal space with the vitreous body (lower panel). (E) Calretinin (yellow), a marker for nerve fibers, is only detected in wildtype but not in mutant retinae. Nuclei were stained with DAPI (white). (F) Graphic representation of the velocity in anti-clockwise (+) and clockwise (?) direction of the eye movements of a representative wildtype (black line) and mutant (red line) fish depending on the stimulus (grey line). Wildtype fish track the stimulus reliably at low spatial frequencies in both directions but movements become more random at high spatial frequencies. Only random eye movements could be observed in mutant fish. (G) The optokinetic response (OKR) was measured with varying spatial frequencies (black-white, 30% contrast, 0.2/0.3/0.4 cycles per degree [cpd]). Whereas atoh7+/+ and atoh7± achieved comparable gains dependent on the spatial frequency (values), no OKR could be observed in mutants. Scale bars represent 250 µm in overviews and 25 µm in close-ups. Statistics: all data are represented as mean ± SD, t-test with multiple comparisons, *P < 0.05; **P < 0.01; ***P < 0.001, or ****P < 0.0001. cpd, cycles per degree; GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; ONL, outer nuclear layer; OPL, outer plexiform layer; RNFL, retinal nerve fiber layer; RPE, retinal pigment epithelium.
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