(A-D) Immunofluorescent images of p-S6 staining on transverse cryosections from MTZ⁺ DMSO-, rapamycin- or INK128-treated larvae at 2dpi. Nuclei (white), p-S6 (magenta) (E) p-S6 levels in the RPE layer were significantly decreased in rapamycin-treated and INK128-treated larvae when compared with DMSO-treated controls. (F) Schematic of the experimental paradigm showing the timeline for chemical treatments and ablation. (G-J) Immunofluorescent images of BrdU staining on transverse cryosections from MTZ^- DMSO-, rapamycin- and INK128-treated larvae at 9dpf and (L-O) from MTZ⁺ DMSO-, rapamycin- or INK128 -treated larvae at 4dpi. White arrowheads indicate BrdU⁺ cells in the RPE layer. Nuclei (white), BrdU (magenta). (K) Quantification of BrdU⁺ cells in the MTZ^- RPE layer showed no significant differences between DMSO-treated and inhibitor-treated larvae. (P) Quantification of BrdU⁺ cells in the RPE layer showed significantly fewer BrdU+ cells in MTZ⁺ rapamycin- and INK128-treated larvae when compared to DMSO-treated controls. (Q-T) Brightfield images of cryosections from MTZ⁺ DMSO-, rapamycin- or INK128-treated larvae at 4dpi. White arrows indicate the edges of pigment recovery. (U) Quantification of percent RPE recovery showed a significant impairment in pigment recovery in rapamycin- or INK128-treated larvae. p-values: ** ≤ 0.01, ***≤ 0.001, and **** ≤ 0.0001. Statistical information can be found in S9 Table. Dorsal is up and distal is left. Scale bar = 50μm.

(A) Schematic of the experimental paradigm showing the time points for MTZ ablation and sample collection on transgenic larvae (rpe65a:nfsB-eGFP). (B-H) Immunofluorescent images of p-S6 staining on transverse cryosections from unablated (MTZ⁻) and (I-O) ablated (MTZ⁺) larvae from 3hpi to 4dpi. White arrows indicate the p-S6⁺ eGFP^- cells in the RPE layer. White arrowheads indicate p-S6⁺ cells in the retinae. (J’-J”‘) High-magnification images showing the colocalization of p-S6 and eGFP at 6hpi, 12hpi (K’-K”‘) and 1dpi (L’-L”‘). Nuclei (white), eGFP (green), p-S6 (magenta). (P) Quantification of the p-S6⁺ area in the RPE layer revealed that expression was low in the RPE of unablated larvae but increased significantly in ablated larvae from 3hpi to 3dpi. p-values: ** ≤ 0.01, *** ≤ 0.001. Statistical information can be found in S9 Table. Dorsal is up and distal is left. Scale bar = 50μm.

(A,B) Immunofluorescent images of p-S6 staining on transverse cryosections from ablated mtor^+/+ and mtor^-/- at 2dpi. Nuclei (white), p-S6 (magenta). (C) Quantification of the p-S6 signal in the RPE layer revealed a significant decrease in MTZ⁺mtor^-/- larvae, when compared to MTZ⁺mtor^+/+ siblings. (D,E) Immunofluorescent images of BrdU staining on transverse cryosections from MTZ- (D,E) and MTZ⁺ (F,G) mtor^+/+ and mtor^-/- larvae at 9dpf/4dpi Nuclei (white), BrdU (magenta). (H) Quantification of BrdU⁺ cells in the RPE layer showed no significant differences between MTZ^-mtor^+/+ and mtor^-/- larvae, but a significant decrease in MTZ⁺mtor^-/- larvae when compared to MTZ⁺mtor^+/+ siblings. (I-J) Immunofluorescent images of ZPR2 staining on transverse cryosections from MTZ⁺mtor^+/+ and mtor^-/- larvae at 4dpi. Single channel (I’ J’) rpe65a:nfsB-eGFP images and (I” J”) ZPR2 images. Nuclei (white), eGFP (green), ZPR2 (magenta). White arrows highlight the edges of eGFP or ZPR2 recovery in the RPE. (K) Quantification of the eGFP expression recovery showed a significant decrease in MTZ⁺mtor^-/- larvae, compared to mtor^+/+siblings. p-values: * ≤ 0.05, ** ≤ 0.01. Statistical information can be found in S9 Table. Dorsal is up and distal is left. Scale bar = 50μm.

(A) Schematic of the experimental paradigm showing the timeline for chemical treatments and ablation. (B,C) Immunofluorescent images of p-S6 staining on transverse cryosections from MTZ⁺ DMSO- and MHY1485-treated larvae at 2dpi. Nuclei (white), p-S6 (magenta). (R) Quantification of p-S6 signal in the RPE layer showed a significant increase of p-S6 levels in MHY1485-treated larvae when compared to DMSO-treated controls. (D-O) Fluorescent images of BrdU immunostaining on cryosections from MTZ^- and MTZ⁺ DMSO- and MHY1485-treated larvae at (D-G) 7dpf/2dpi, (H-K) 8dpf/3dpi and (L-O) 9dpf/4dpi. White arrowheads highlight BrdU⁺ cells in the RPE layer. Nuclei (white), BrdU (magenta). (T) Quantification of BrdU⁺ cells in the RPE layer revealed significantly increased proliferative cells in the RPE of MTZ⁺ MHY1485-treated larvae when compared to DMSO-treated controls at 3dpi, but no significant differences between ablated MHY1485-treated larvae and DMSO-treated siblings at 2dpi or 4dpi. There were no significant effects of MHY1485 treatment on MTZ^- larvae across all time points. (P-Q) Fluorescent images of ZPR2 immunostaining on cryosections from MTZ⁺ DMSO- and MHY1485-treated larvae at 3dpi. White arrows highlight the edges of ZPR2 signal recovery. Nuclei (white), eGFP (green), ZPR2 (magenta). (S) Quantification of percent ZPR2 recovery showed a significant increase in MHY1485-treated larvae over DMSO-treated controls. p-values: * ≤ 0.05, ** ≤ 0.01, **** ≤ 0.0001. Statistical information can be found in S9 Table. Dorsal is up and distal is left. Scale bar = 50μm.

(A) Representative FACS plots from MTZ^- and MTZ⁺ samples to show gating parameters used to isolate GFP⁺ PI^- RPE cells (B) Volcano plots showing differentially expressed genes between MTZ⁺ rapamycin-treated and DMSO-treated groups at 2dpi and 4dpi. Dashed lines indicate the threshold criteria. Each dot represents an individual gene; red dots represent significantly upregulated DEGs, blue dots represent significantly downregulated DEGs, and black dots represent non-significant genes. (C) Pathway enrichment analysis on the significantly downregulated 2dpi and 4dpi DEGs. Numbers in parentheses are the gene counts enriched in each pathway. (D) Hierarchical clustering heatmap of the 15 genes enriched in the 2dpi immune system Reactome pathway across all four experimental groups showing these immune related genes are downregulated by rapamycin treatment. Heatmap legend represents log₁₀ (counts per million mapped reads; CPM).

(A) Schematic of the experimental paradigm showing the timeline for chemical treatments and ablation on transgenic larvae (mpeg1:mCherry;rpe65a:nfsB-eGFP). (B-E) Immunofluorescent images of mCherry staining on transverse cryosections from MTZ^- and MTZ⁺ DMSO- and rapamycin-treated larvae at 3dpi. Nuclei (white), mCherry (magenta). (F) Quantification of the mCherry signal in the RPE layer showed a significant increase in MTZ⁺ DMSO-treated larvae, when compared to MTZ^- DMSO controls. The percent of the RPE covered by mCherry⁺ cells was significantly reduced in MTZ⁺ rapamycin-treated larvae when compared to MTZ⁺ DMSO-treated controls. However, there was no significant difference between MTZ^- DMSO-treated and rapamycin-treated groups. p-values: * ≤ 0.05, ** ≤ 0.01. Statistical information can be found in S9 Table. Dorsal is up and distal is left. Scale bar = 50μm.

(A,J) Schematic of the experimental paradigm showing the timeline for chemical treatments and ablation on transgenic larvae (mpeg1:mCherry;rpe65a:nfsB-eGFP). (B-G) MTZ⁺ DMSO- and PLX3397-treated larvae at 2dpi. Single channel immunofluorescent images of mCherry (D,E) and p-S6 (F,G) are shown. (B’,D’,F’) are high-magnification images showing the colocalization of p-S6 and mCherry. Nuclei (white), eGFP (green), p-S6 (magenta), mCherry (yellow). (H) Quantification of mCherry signal in the RPE layer showed a significant depletion of mCherry⁺ macrophages/microglia in the RPE after PLX3397 treatment in MTZ⁺ larvae, compared to DMSO-treated controls. (I) Quantification of p-S6 levels in the RPE layer showed a significant decrease in PLX3397-treated larvae, when compared to DMSO-treated controls. (K-N) Fluorescent images of p-S6 immunostaining on cryosections from MTZ^- and MTZ⁺ DMSO- and Dex-treated larvae at 7dpf/2dpi. Nuclei (white), eGFP (green), p-S6 (magenta). (O) Quantification of p-S6 level in the RPE layer showed a significant increase in MTZ⁺ DMSO-treated larvae when compared to MTZ^- DMSO-treated control, but no significant differences between Dex-treated and DMSO-treated larvae from MTZ^- or MTZ⁺ groups. p-values: ** ≤ 0.01, *** ≤ 0.001. Statistical information can be found in S9 Table. Dorsal is up and distal is left. Scale bar = 50μm.

(A) mTOR activity is rapidly induced in damaged/regenerating RPE cells between 6hpi and 1dpi. (B) Activation of mTOR signaling in RPE cells regulates the expression of immune-related genes, including cytokines and chemokines. (C) mTOR activity is required for the recruitment of macrophages/microglia to the injured/regenerating RPE. (D) mTOR is also activated in macrophages/microglia recruited to the RPE post-injury and these cells are required to maintain mTOR activity in RPE cells during the later stages of the regenerative response. This maintenance occurs in an inflammation-independent manner.