Abstract
Purpose :
Microglia are resident immune cells of the retina that can change their function in a disease state. While their transition between states in disease has been documented, their state specific functional roles in regeneration and how this transition is regulated remain unclear. Our lab has established a transgenic P23H rhodopsin zebrafish model of Retinitis Pigmentosa with degeneration of rod photoreceptors followed by regeneration of new rods. We hypothesize that microglia undergo transcriptomic and phenotypic changes that might promote rod regeneration.
Methods :
Analysis of whole retina single-cell transcriptomes from three P23H and WT zebrafish was performed using Seurat package. Unsupervised clustering was performed to identify microglia clusters. To visualize microglia/macrophages, retinal sections from adult P23H and WT fish were stained with antibody 7.4.C.4.
Results :
Immunostaining of retinal sections with 7.4.C.4 antibody revealed that microglia/macrophages in P23H retinas acquired a more ameboid shape compared to WT. We detected a higher number of microglia/macrophages in the ONL and subretinal space. Analysis of scRNA seq data identified three distinct microglia clusters in both WT and P23H. Gene expression changed in all clusters in P23H. Genes associated with cytokine/chemokine signaling (tnfrsf9b, traf1, ccl38.6) were upregulated in P23H microglia cluster 27, whereas expression of genes in pathways related to phagocytosis (p2ry12, lamp2) was increased in P23H microglia cluster 31. We found that expression of several lncRNAs, si:ch211-214p16.2, LOC103909107, LOC103910136, LOC103909099 and LOC110439915 was microglia cluster-specific. Furthermore, these were among the top differentially-expressed genes and were upregulated by 3.5 to 17.6 fold in P23H microglia compared to WT.
Conclusions :
Microglia/macrophages in regenerating retinas of P23H fish undergo transcriptional changes and migrate towards rod photoreceptors in the ONL and subretinal space. Some of these microglia acquire ameboid shape suggesting that they phagocytose dying rods. We also have identified upregulation of lncRNAs expression in P23H microglia. LncRNAs regulate many cellular processes and we suggest that they might drive microglia/macrophage’s transcriptional change to promote regeneration of rods. Further knock-down studies will reveal specific roles of the lncRNAs in retinal regeneration.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.