Fig. 5

Loss of impdh1a does not alter the expression of impdh1b or impdh2 and does not cause photoreceptor degeneration.A, schematic of impdh1a KO mutation, a G > T mutation at essential splice site between exon 5 and 6 resulting in a premature stop codon 11 amino acids after exon 5. B, primary sequence of impdh1a_tvX1 compared with impdh1a KO. Blue marks start of retained intron, purple marks premature stop codon in impdh1a KO. qPCR analysis of (C) impdh1a, (D) impdh1b, and (E) impdh2 transcripts at 11:00 AM in WT, HET, and impdh1a KO retinas. N = 6 for WT and heterozygous animals and N = 5 for KO animals. F, representative IHC images of 18 month old impdh1a WT and KO retinas with mitochondria (MTCO1) stained magenta. Cone photoreceptors express eGFP under the cone transducin promotor (TaCP:eGFP) and are stained with eGFP antibody to visualize cones (green). The nuclei are stained in blue. The images are maximum intensity projections of 20 stacks 0.3 μm per step. G, control demonstrating lack of Impdh1a protein at 18 months in impdh1a KO retinas. H, mitochondrial (MTCO1) staining of impdh1a WT and KO retinas showing normal mitochondrial localization and morphology in the KO retina at 18 months. I, cone nuclei quantification and (J) outer nuclear layer thickness (rod nuclei) comparing impdh1a WT and KO zebrafish retinas at 18 months. K, rod- (gnat1) and cone- (es1) specific transcripts in WT compared with KO. impdh2 transcript, which is not found in rod or cones, used as a control. N = 5 animals for WT and N = 3 animals for KO. L, whole retina mtDNA copy number after feeding or after 18 to 24 h fasting; N = 3 for all the samples. M, representative TEM images of 18 month old WT and KO retinas showing high resolution morphology of retinal layers (left and middle panel) and mitochondria (right) in 18 months impdh1a WT and KO retinas. impdh, inosine monophosphate dehydrogenase; tv, transcript variant. ns p > 0.05, ∗ p ≤ 0.05.