June 2023
Volume 64, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2023
Novel zebrafish genetic models of retinitis pigmentosa - pde6b mutants exhibit the rod photoreceptor degeneration
Author Affiliations & Notes
  • Liyun Zhang
    Johns Hopkins Medicine Wilmer Eye Institute, Baltimore, Maryland, United States
  • Xiangqian Shi
    Johns Hopkins Medicine Wilmer Eye Institute, Baltimore, Maryland, United States
  • Abi Cha
    Johns Hopkins Medicine Wilmer Eye Institute, Baltimore, Maryland, United States
  • Emma Liu
    Johns Hopkins Medicine Wilmer Eye Institute, Baltimore, Maryland, United States
  • Meera Saxena
    Johns Hopkins Medicine Wilmer Eye Institute, Baltimore, Maryland, United States
  • Jeff S Mumm
    Johns Hopkins Medicine Wilmer Eye Institute, Baltimore, Maryland, United States
  • Footnotes
    Commercial Relationships   Liyun Zhang None; Xiangqian Shi None; Abi Cha None; Emma Liu None; Meera Saxena None; Jeff Mumm None
  • Footnotes
    Support  NIH Grant R01EY032533
Investigative Ophthalmology & Visual Science June 2023, Vol.64, 4887. doi:
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    • Get Citation

      Liyun Zhang, Xiangqian Shi, Abi Cha, Emma Liu, Meera Saxena, Jeff S Mumm; Novel zebrafish genetic models of retinitis pigmentosa - pde6b mutants exhibit the rod photoreceptor degeneration. Invest. Ophthalmol. Vis. Sci. 2023;64(8):4887.

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      © ARVO (1962-2015); The Authors (2016-present)

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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.

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