Abstract
Purpose :
Retinitis pigmentosa (RP) is an inherited retinal degeneration characterized by progressive rod photoreceptor death followed by cone cell loss. Mutations in the PDE6B gene have been linked to RP, however, the underlying molecular mechanisms are largely unclear. Here we characterized novel pde6b zebrafish mutant models to better understand the role of these mutations during disease pathogenesis and develop more effective treatments.
Methods :
To create zebrafish pde6b (human PDE6B homolog) mutants, CRISPR/Cas9 mutagenesis was utilized. Mutant founders carrying mutations predicted to interrupt gene function were crossed with Tg(rho:YFP-MTR) (gmc500) fish to enable rod photoreceptors labeling with YFP, facilitating rod cell visualization and quantification. Rod cell loss kinetics were determined by intravital confocal imaging and microplate reader assays from 4 to 8 days post-fertilization (dpf). Quantitative PCR (qPCR) detected both rod and cone cell marker genes. Immunofluorescence (IF) using 1d1 (rod), zpr1 (cone) and PCNA (proliferating cells) were conducted at different development stages to detect cellular changes of retina (8-day, 14-day, 1-month and 7-month-old fish). Three rod cell death pathways (parthanatos, necroptosis and apoptosis) were studied using chemical inhibitors.
Results :
Three frameshift alleles were identified: pde6bJH603B, pde6bJH603C, and pde6bJH603E. Time-series phenotyping at the early developmental stages showed that reductions of rod-YFP expression were highly associated with homozygous mutations. Confocal imaging confirmed rod cell loss in mutant retinas. qPCR further verified the decreased expression of rod genes and no change of cone genes in mutants. IF studies found: 1) rod cell loss began early and lasted till adulthood; 2) increased PCNA+ cells in the outer but not the inner nuclear layer suggested rod progenitor but not Müller glial cell proliferation; 3) cone cell number remained normal at the early stages, but gradually decreased starting from one month old. Cell death pathway inhibitors failed to show a protective phenotype.
Conclusions :
We successfully generated three novel zebrafish RP models carrying pde6b mutations and exhibiting severe rod photoreceptor degeneration, recapitulating key features of human RP. These new models will be valuable tools for mechanistic and therapeutic RP studies.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.