|
Fig. 6 Filaments from zebrafish with Impdh1a mutations are larger and localized to synapses (K238E) or synapses and nuclei (D226N). (A) K238E Impdh1a mutant, D226N Impdh1a mutant, Tg WT or WT sibling zebrafish were crossed with TαCP:eGFP (cone cytosol, green), and retinal sections were stained with Hoechst (nuclei, blue) and a custom anti-Impdh1a antibody (magenta). K238E mutant zebrafish have larger filaments at cone synapses, and D226N mutant zebrafish have larger filaments at cone synapses and nuclei, whereas Tg WT zebrafish have diffuse cytosolic Impdh1a staining. Images show the cone layer. CB, cell bodies; OS, outer segment; SYN, synapses. Scale bars: 10 µm. (B) D226N Impdh1a mutant retinal sections expanded 5.5×. Nuclei (blue) and Impdh1a (magenta). B′ is a zoomed-in area of a cone synapse (orange dashed line box); B″ is a zoomed-in area of a Impdh1a-positive filament in cone nuclei (yellow dashed line box). Scale bars: 10 µm (B); 1 µm (B′,B″). All scale bars corrected by expansion factor (5.5). (C) K238E Impdh1a mutant (top) and WT sibling (bottom) zebrafish retinas stained with Hoechst (nuclei, blue) and a custom anti-Impdh1a antibody (magenta) at 4 months of age. The K238E mutant zebrafish retina has large filaments at the cone synapses and in cell bodies near the mitochondrial cluster, whereas the WT sibling retina has small Impdh1a filaments throughout the cell bodies and synapses. Images show the cone layer. Scale bars: 10 µm. (D) Transmission electron micrographs of cone synapses of D226N mutant, K238E mutant, Tg WT or WT sibling zebrafish. Zebrafish with Impdh1a D226N and K238E mutations have filamentous structures in synapses (orange arrowheads). Zebrafish eyes were taken prior to degeneration. Scale bars: 600 nm (D226N); 500 nm (others).