Fig 1.
(A) Cartoon depicting the rpe65a:nfsB-eGFP transgene and treatment course of unablated embryos. (B) Transverse cryosections of an unablated 6dpf larva. (B,B’) After exposure to PTU between 1-5dpf, transgene expression is specifically restricted to mature RPE cells, with the brightest expression confined to the central two-thirds of the RPE. Arrowheads indicate apical microvilli. (B”) DIC images reveal RPE repigmentation and normal photoreceptor layer architecture. (C) Cartoon depicting the nitroreductase-mediated ablation paradigm: after washing out PTU, larvae were treated with MTZ for 24 hours. Within cells expressing the transgene, nfsB converts MTZ into a potent DNA crosslinking agent and induces cell death. (D,D’)Transverse cryosections of a 1dpi larva reveal significant disruption of eGFP+ cell morphology and disorganization in INL nuclear lamination. Arrows indicate delaminated and pyknotic nuclei. (D”) DIC images reveal a lack of RPE pigmentation and the marked disruption of photoreceptor layer architecture. Green = eGFP, blue = nuclei. Dorsal is up and distal is left. Scale bar = 40μm.
Fig 2.
Ablation of the RPE leads to degeneration of underlying photoreceptors.
(A-D) Transverse cryosections stained for TUNEL (red). Compared to untreated (A,C) larvae, ablated RPE were disrupted by 12hpi (B), and TUNEL+ cells appeared throughout the RPE and ONL at 24hpi (D). (E, F) Quantification of TUNEL+ cells/section in the RPE (E) and ONL (F) revealed a significant increase in the RPE by 12hpi and in the ONL by 18hpi. Significance determined using Mann-Whitney U test. * p≤0.05, ** p<0.005, *** p<0.0005. (G-I) Transverse sections of unablated 6dpf larvae stained for ZPR2 (G), ZPR1 (J), and F-Actin (M) (red). By 1dpi, ZPR2 is disrupted in a similar manner to eGFP (H), and ZPR1+ cones appear swollen and degenerated (K), and photoreceptor outer segment cytoskeletons become disorganized (N). By 3dpi, ZPR2 signal is absent from the central injury site (I) and PR morphology is notably degraded (L,O). Green = eGFP, blue = nuclei. Dorsal is up and distal is left. Scale bar = 40μm.
Fig 3.
TEM analysis confirms degeneration of the RPE, photoreceptors, and Bruch’s Membrane.
(A,B) TEM images of unablated 8dpf and (C,D) 3dpi eyes. Compared to unablated controls, the ONL and RPE is degenerated in ablated larvae, with large aggregates of debris notable in the RPE (C, arrow). Magnified views of BM reveal reduced BM thickness as well as obvious gaps (D, arrows). (E) Quantification of BM thickness reveals a significant reduction in BM thickness in ablated larvae (Student’s T-test, MTZ- n = 3 eyes, MTZ+ n = 4 eyes p<0.0001).
Fig 4.
RPE ablation results in defects in visual function.
(A-C) To measure the OKR, the right eye of ablated and unablated larvae was exposed to a rotating stimulus, and the position of the stimulated eye was recorded at 1dpi (A, n = 3 unablated, n = 4 ablated), 2dpi (B, n = 3 unablated, n = 12 ablated) and 3dpi (C, n = 3 unablated, n = 12 ablated). (D) Quantification revealed that ablated larvae had a significantly reduced stimulus tracking gain at 2dpi (Mann-Whitney U test, P = 0.0055). Recovered OKR was detectible by 3dpi (Mann Whitney U test, P = 0.1878).
Fig 5.
RPE regeneration initiates in the periphery and proceeds inward.
Transverse sections of unablated larvae stained for the RPE marker ZPR2 (A), R/G cone photoreceptor marker ZPR1 (B) and F-Actin (C) at 11dpf. Ablated eyes stained for ZPR2 (D,G,J,M), ZPR1 (E,H,K,N), and Phalloidin (F,I,L,O) at 4, 6, 7 and 14dpi. Green = eGFP, blue = nuclei, red = marker. eGFP+ RPE appears in the periphery at 4dpi (marked by arrows in D-F). As regeneration proceeds, eGFP+ RPE extends further toward the eye center, and the leading tip of the regenerated monolayer often consists of both immature and mature RPE (ZPR2+/eGFP- cells in G). PR morphology appears to recover in the periphery proximal to regenerated RPE. By 7dpi, ZPR2+ RPE is present throughout the RPE (J), and PR morphology begins to recover in the central injury site (K,L). By 14dpi, mature eGFP+/ZPR2+ RPE cells are present throughout the RPE (M), and PR morphology further improves in the central retina (N,O). Dorsal is up and distal is left. Scale bar = 40μm.
Fig 6.
Regenerated RPE appears normal 7 months post-injury.
Stitched-together confocal images of transverse cryosections of 7 month post-injury fish (C,D) and age-matched sibling controls (A-B). (A) Robust eGFP and ZPR2 expression exists throughout the central RPE. (B-B”) Within the central RPE, eGFP is strongly expressed in the RPE cell body and labels apical processes, most strongly toward the cell body, while ZPR2 labels the RPE cell body and apical processes closer to the outer limiting membrane. (C) In ablated fish, strong expression of eGFP and ZPR2 is evident throughout the retina. (D-D”), and a similar pattern of eGFP and ZPR2 expression is observed in the central injury site. Scale bar = 100μm.
Fig 7.
Longitudinal OCT analysis of RPE regeneration.
(A-C) Representative time series from a single (A) unablated and (B) ablated larva. Central edge of maximal RPE intensity marked with red line. (C) Quantification of the RPE signal (backscatter) from the dorsal periphery to optic nerve across unablated (blue) and ablated (red) larvae at 1dpi and 5dpi (error bars = SEM). The measured RPE was divided into quintiles (cartoon), and the area under the curve within each quintile was measured. At 1dpi, backscatter intensity in ablated RPE is significantly below unablated intensity in the 3 quintiles closest to the optic nerve, while at 5dpi, only the central-most quintile is significantly reduced (MTZ-: n = 10, MTZ+ n = 9, Student’s unpaired t test, **p<0.005, ***p<0.0005).
Fig 8.
TEM analysis of regenerated RPE.
(A,B) TEM images of unablated 19dpf and (C,D) 14dpi eyes (C) Organized photoreceptor outer segments are visible in the ablated photoreceptor layer, and a regenerated RPE is present. (E) Quantification of BM thickness. Student’s T-test reveals that BM thickness is not significantly different in ablated larvae * p<0.05. (MTZ- n = 3 eyes, 81 measurements; MTZ+ n = 3, 81 measurements).
Fig 9.
Regeneration of the adult RPE initiates in the periphery and proceeds centrally.
(A-E) Stitched-together transverse cryosections of unablated (A, n = 4), 3dpi (B, n = 4), 7dpi (C, n = 4), 14dpi (D, n = 3), and 35dpi (E, n = 4) adult eyes, as well as magnified insets of the dorsal peripheral RPE (A’-E’) and central RPE (A”-E”). Red = ZPR2, Green = eGFP, blue = nuclei. At 3dpi, RPE cell degeneration occurred in a large portion of the RPE (B, arrowheads), indicated by loss of eGFP and ZPR2 expression and disruption of overall morphology of the RPE (B’,B”). At 7dpi, increased colocalization of eGFP and ZPR2 defined the peripheral edge of the RPE injury site (C’,C, arrowheads) while the central RPE was absent eGFP and ZPR2 expression (C”). At 14dpi, ZPR2+/eGFP+ RPE reappears in the periphery (D’) and extends inward toward the injury center (D, arrowheads). Magnified images of the central injury site also reveal recovered ZPR2 localization to the apical processes (D”). At 35dpi, ZPR2+/eGFP+ RPE reappear throughout the injury site (E), and proper polarization of ZPR2 and eGFP colocalization has been reestablished throughout (E’-E”). (A-E) Scale bar = 100μm. (A’-E”) Scale bar = 40μm. (F) Quantification of percent RPE regeneration based on measurements of contiguous eGFP+/ZPR2+ expression revealed significant RPE degeneration at 3dpi and 7dpi when compared to MTZ- controls. RPE recovery occurred by 35dpi. Mann-Whitney U Test, * p<0.05.
Fig 10.
RPE regeneration involves a robust proliferative response.
(A-L) Transverse retinal sections of unablated (A-F) and ablated (G-L) larvae exposed to 24-hour BrdU pulses at various days post-injury. BrdU+ cells first appear in the periphery as early as 0-1dpi (arrow, G), and 1-2dpi (arrow, H). As regeneration proceeds, BrdU+ cells appear closer to the central injury site and at the inner tip of the regenerating RPE layer (arrows, I). BrdU+ cells then populate the injury site (arrows, J-L). (M-T) en face wholemount images of unablated (M-P) and ablated (Q-T) eyes from larvae exposed to BrdU between 3-4dpi. White arrowheads in (Q) and (Q, inset) indicate BrdU+/eGFP+ cells near the injury site. Yellow arrowhead in (S) and (T) indicate BrdU+ cells proximal to the injury site that are beginning to become pigmented. (Inset, T) Magnified image of BrdU+, pigmented cells. (U) Quantification of total number of BrdU+ cells/section in the RPE (U) and central retina (V) reveals an increase of BrdU+ cells in the RPE starting at 0-1dpi and peaking at 3-4dpi. Proliferation in the central retina significantly increased at 3-4dpi. Mann-Whitney U Test, * p<0.05, ** p<0.005, *** p<0.0005. Dorsal is up and distal is left. Scale bar = 40μm.
Fig 11.
Wholemount analysis of RPE cell proliferation and regeneration.
(A-J) en face wholemount images of unablpated (A-E) and ablated (F-J) larvae exposed to 2-hour EdU pulses immediately before fixation and staining for ZPR2 at various time points post-injury. (K) Cartoon depicting the 4 categories into which the RPE was divided, based on cellular pigmentation and intensity of ZPR2 expression: injury site cells peripheral RPE (pigmented, dimly ZPR2+), differentiated RPE (pigmented, ZPR2+), transition zone (lightly pigmented, ZPR2+ consisting of incompletely differentiated RPE extending into the injury site, and injury site (unpigmented, ZPR2-) (L) Quantification of the area of RPE comprised by each domain during regeneration reveals the degeneration of a large proportion of RPE rapidly after ablation, and that regeneration also rapidly occurs, with a transition zone appearing by 1dpi, and differentiated RPE reappearing in the periphery by 2dpi. The entire RPE is repopulated by differentiated RPE by 7dpi. (M-N) Transverse cryosections of unablated (M) and ablated (N) eyes at 1dpi. Magnified insets (M’,N’) reveal the presence of EdU+ cells in the RPE periphery in ablated retinae (N’ arrowhead). (O-R) Quantification of the density of EdU cells throughout regeneration in the peripheral RPE (O), Transition Zone (P), differentiated RPE (Q) and Injury Site (R) suggest that peripheral cells respond to injury by proliferating, that proliferation continues within newly-generated RPE and halts after regeneration is repopulated.
Fig 12.
Proliferative RPE contributes to the regenerated RPE monolayer.
(A-A”) Transverse sections from unablated larvae exposed to BrdU from 5-6dpf and pulsed with EdU for 2 hours before fixation at 8dpf. (B-B”) Transverse sections of ablated larvae exposed to BrdU from 0-1dpi and pulsed with EdU for 2 hours before fixation at 3dpi. (A’,B’) Magnified inset of BrdU/EdU. (A”,B”) Magnified inset of BrdU/EdU and DIC. Arrowheads in (B) highlight BrdU+ PRs that have integrated into the ONL. Arrow in (B’,B”) highlights a proliferative RPE cell, and arrowheads highlight unpigmented, previously-proliferative RPE-like cell in the injury site. (C) Quantification of BrdU/EdU+ and BrdU+ nuclei in the injury site. (D,E) Larvae exposed to BrdU 0-1dpi and fixed at 7dpi. (F-G) Larvae exposed to BrdU 3-4dpi and fixed at 7dpi. (H,I) Larvae exposed to BrdU 5-6dpi and fixed at 7dpi. (J) Quantification of the average number of BrdU+ cells per section. (K) Quantification of the location of individual BrdU+ cells relative to the center of the RPE. The line indicates the average location of BrdU+ cells, and the whiskers indicate standard deviation. (L,M) Quantification of BrdU+ cells that were labeled 0-1dpi within the RPE at 7dpi in ablated larvae. Analysis of eGFP+BrdU+ and GFP-BrdU+ cells in the RPE reveal that most BrdU cells in the RPE are eGFP+ at 7dpi. (C) Quantification of the location of individual BrdU+ cells relative to the center of the RPE indicates that eGFP+BrdU+ cells tend to be located toward the center and eGFP-BrdU+ localize toward the periphery. Mann-Whitney U Test, * p<0.05, ** p<0.005, *** p<0.0005. Scale = 100μm. Dorsal is up and distal is left. Scale bar = 40um.
Fig 13.
Pharmacological inhibition using IWR-1 impairs RPE regeneration.
(A-D) Transverse sections of lef1 or sense RNA expression in unablated 6dpf (MTZ-) and ablated 1dpi (MTZ+) larvae. lef1 is detected in and around the RPE in MTZ+ (B’) but not MTZ- larvae (A’). lef1: n>5; lef1 sense: n = 4. (E-J) Transverse sections of 4dpi ablated DMSO- (E,H; n = 10) and 15μM IWR-1-treated (F,I; n = 11) larvae exposed to a 24-hour pulse of BrdU from 3-4dpi. (E,F) Green = eGFP, blue = DNA, red = BrdU; white arrowheads highlight BrdU+ cells in the RPE. (G) Quantification of BrdU+ cells/section reveals that IWR-1 treatment significantly decreases the number of proliferative cells in the RPE at 4dpi (Student’s unpaired t-test, *** p<0.0001). Brightfield images (H,I) and quantification of percent RPE recovery/section (J) shows a significant delay in recovery of a pigmented monolayer in IWR-1 treated larvae (Student’s unpaired t-test, *** p<0.0001). (I) Black arrowheads indicate the central-most edge of the regenerating RPE. Transverse sections of 4dpi ablated DMSO- (K; n = 6) and 15μM IWR-1-treated (L; n = 5) larvae stained for ZPR2 (red). Green = eGFP and blue = nuclei. ZPR2 staining overlaps with a thick, heavily pigmented regenerated RPE monolayer. (L) Arrowheads indicate the central-most edge of the regenerating RPE. In IWR-1-treated larvae, ZPR2 staining is not observed central of the rim of pigment indicating a lapse in RPE regeneration, not a pigmentation deficiency. Dorsal is up and distal is left. Scale bars = 40μm.
Fig 14.
Model of RPE regeneration in larval zebrafish.
(A) nfsB-eGFP is specifically expressed in mature RPE in the central two-thirds of the eye. (B) Application of MTZ leads to apoptosis (red) of RPE and PRs. (C) RPE ablation leads to degeneration of PRs and Bruch’s Membrane (dotted line). (D) Unablated RPE in the periphery begin to proliferate and extend into the injury site (blue). (E) As regenerated eGFP+ RPE appear in the periphery, the RPE can be divided into 4 zones: peripheral RPE, differentiated RPE, transition zone, and injury site. (E, inset) Regenerated differentiated RPE appears in the periphery proximal to the unablated peripheral RPE, and contains proliferative cells adjacent to the transition zone. The transition zone consists of still-differentiating RPE cells and proliferative cells. The injury site is comprised of unpigmented proliferative cells that do not express any RPE differentiation markers. (F) Regeneration of a functional RPE layer and Bruch’s Membrane is complete by 14dpi.