Fig. 9

Autophagy and CALM modulate photoreceptor degeneration in zebrafish.

(a) rho::GFP-tau fishes were incubated from 3 to 9 d.p.f. in either dimethyl sulphoxide (DMSO) or rapamycin (i) or from 3 to 7 d.p.f in EM alone or 10 mM ammonium chloride or 100 nM Wortmannin (ii). Images through the central retina at 9 d.p.f. (top panel; (i)) reveal rod degeneration (arrow) throughout the retina in control (DMSO-treated) larvae, whereas rod photoreceptors are present throughout the retina, particularly in the central region following treatment with rapamycin (arrow). Images taken through the central retina at 7 d.p.f. (bottom panel; (ii)) show normal photoreceptors in the marginal zones (arrows) and only limited numbers in the central region. NH₄Cl exacerbates degeneration-photoreceptors are absent from the central retina and reduced/absent from marginal zones (arrows). Sections were stained with anti-rhodopsin (1D1) antibody. GFP labels whole rod photoreceptors, whereas rhodopsin is present in the rod outer segment. GFP co-localizes with the red rhodopsin label in all experimental conditions. Scale bars, 50 µm. Quantification of rod photoreceptor degeneration (n=10 larvae per group; ***P<0.001; ###P<0.001, two-tailed unpaired t-test). Error bars are mean ±s.d. (b) Full-length CALM electroporation into rho::GFP larvae did not cause degeneration., while full-length CALM electroporation into rho::GFP-tau larvae exacerbated photoreceptor degeneration. Central retina sections in the region of the optic nerve head are presented. Scale bars, 50 µm. Data represent the ratio (in %) of the intensity of the GFP signal between CALM-HA electroporated eye versus control eye for five individuals per transgenic line. ***P<0.05; two-tailed t-test. Error bars are mean ±s.d. (c) NH₄Cl treatment of rho::GFP-tau immediately after unilateral CALM electroporation caused photoreceptor degenerationon the control (non-electroporated) side but did not alter the degeneration caused by CALM electroporation. Rapamycin treatment of rho::GFP-tau immediately after unilateral CALM electroporation rescued photoreceptors on the control (non-electroporated) side but not on the CALM-electroporated side. To demonstrate that loss of GFP corresponds to loss of photoreceptors, sections were stained with anti-rhodopsin (1D1) antibody. Wilcoxon signed rank test was used to compare the left eye versus the right eye of the same fish; Mann–Whitney test was used to compare drug treatment in the right eye of different fishes. *P<0.05. Scale bars, 50 µm. Error bars are mean ±s.e.m.