Abstract
Purpose :
Zebrafish retinal pigment epithelium (RPE) are able to regenerate after genetic ablation and our previous work has identified several critical regulators of RPE regeneration, including Wnt signaling, mTOR signaling, and immune response factors. Despite these advances, our understanding of the mechanisms driving intrinsic RPE regeneration remains limited. To address this, we conducted a rapid in vivo functional screen of candidate regenerative loci to determine a requirement for RPE regeneration.
Methods :
Target genes were selected based on upregulation in regenerating RPE identified from bulk RNA-sequencing data generated from FACS-sorted eGFP+ RPE cells. Synthetic guide RNAs (gRNAs) targeting three non-overlapping regions of each candidate gene were generated. Each gRNA was injected, followed by headloop (HL) PCR to confirm mutagenesis efficacy. Having validated each gRNA set, one-cell stage embryos derived from rpe65a:nfsB-eGFP transgenic crosses were then injected with the three synthetic gRNAs-cas9 protein complex (RNP) per gene. Control embryos were injected with a set of scrambled RNPs. At 5 days post-fertilization (dpf), RNP injected larvae carrying the rpe65a:nfsB-eGFP transgene were exposed to 10mM metronidazole (MTZ) for 24 hours to ablate the RPE. At 4 days post-injury (dpi), genotyping of individual embryos was performed to confirm mutagenesis. RPE-specific immunostaining with ZPR2 was performed on ablated RNP injected and control larvae at 4dpi. RPE pigment recovery between injected and control groups was quantified using a MATLAB-based script to assess effects on RPE regeneration.
Results :
Mutagenesis rates were high for gRNAs, supporting the efficacy of this approach. Preliminary MATLAB-based quantification data showed significant pigment recovery differences (p<0.05) between il11a and scrambled RNP injected groups, as well as between ptgs2a and scrambled RNP injected groups.
Conclusions :
il11a and ptgs2a are putative pro-regeneration genes required for RPE regeneration and screening of additional candidate regenerative loci are underway.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.