Abstract
Purpose :
Immune-derived cues are critical mediators of complex tissue regeneration in zebrafish. This has been demonstrated in multiple contexts, including the retina; however, it is not known if the immune response influences regeneration of the retinal pigment epithelium (RPE). We have generated a novel RPE ablation paradigm and characterized RPE regeneration post-injury. Here we aim to utilize this model to identify immune components that promote zebrafish RPE regeneration.
Methods :
rpe65a:nfsB-GFP zebrafish were used as the in vivo ablation model. This transgene is RPE-specific and expresses nitroreductase (nfsB; a metronidazole (MTZ)-converting enzyme) fused to GFP. Mature RPE were ablated in 5 days post-fertilization (dpf) larvae by 10mM MTZ exposure for 24 hours. Eyes from MTZ+ and MTZ- control larvae were enucleated at 2, 4, and 7 days post-injury (dpi) and GFP-positive cells were isolated by fluorescence-activated cell sorting. cDNA libraries from sorted cells were generated, sequenced, and functional annotation analyses were performed on differentially expressed genes (DEGs). To confirm localization of immune cells (e.g. macrophages) to the RPE injury site, immunostaining and in vivo imaging were performed. Small molecule agonists and antagonists were used to interrogate signaling pathways enriched by functional annotation. For these experiments, larvae were treated for 24 hours prior to RPE ablation, then continuously until fixation, with compounds or a vehicle control. The extent of RPE regeneration was assessed by quantifying bromodeoxyuridine (BrdU) incorporation and pigment recovery.
Results :
Genes associated with cytokine-cytokine receptor interactions were enriched at early (2dpi; n=3) and intermediate (4dpi; n=3) regeneration stages. Systemic dampening of the inflammatory response using 5µM dexamethasone (n=14) showed significant decreases (p≤0.001) in proliferation and RPE regeneration when compared to 0.05% DMSO controls (n=16). Preliminary assessment of in vivo light-sheet imaging datasets from larvae at 0.5-4dpi (n=3) shows interesting macrophage morphology and localization differences between MTZ+ and MTZ- controls. Further, preliminary data from isolectin b4 immunostaining suggests that macrophages are present in the RPE injury site.
Conclusions :
Preliminary analyses support a pro-regenerative role for the immune system during RPE regeneration.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.