Retinoic acid signaling mediates peripheral cone photoreceptor survival in a mouse model of retina degeneration
- Department of Genetics and Ophthalmology, Howard Hughes Medical Institute, Blavatnik Institute, Harvard Medical School, United States
Figures
Figure 1 with 4 supplements
Cones in the rd1 mouse model survive long-term in regions with active RA signaling.
(A) Schematic of the timeline of rod and cone photoreceptor death in the rd1 mouse model. (B) rd1 retinal flatmount IHC against Cone Arrestin (CAR) at P40. (C) Retinal flatmount IHC against CAR for …
Figure 1—figure supplement 1
Long-term cone survival despite complete rod loss.
Representative images of smFISH against Arr3 (middle panels) and Nrl (right panels), cone and rod markers, respectively, in WT (top panels) and P366 rd10 (bottom panels) retinas, counterstained with …
Figure 1—figure supplement 2
Cone and MG enrichment in CAR+ FACS-isolated cells.
Relative expression of rod (Rho, Nrl), cone (Arr3, Rxrg), and MG (Glul) markers in CAR+ and CAR- FACS-isolated populations from CD1 mice as detected by ddPCR.
Figure 1—figure supplement 3
Aldh1a1 is expressed and RA signaling is active in peripheral MG.
IHC against B-gal, Aldh1a1, and GS in adult RARE-LacZ (non-degenerating) retinas showing the central (top panels) and peripheral (bottom panels) regions. Scale bar; 100 µm.
Figure 1—figure supplement 4
RA signaling activity is detected only in MG and a subset of ChAT+ amacrine cells.
IHC against B-gal and retina cell type specific markers, including CAR (cones), Chx10 (bipolar cells and MG), Brn3a (RGCs), Iba1 (microglia), GS (MG), and ChAT (amacrine cells) in P100 rd1; …
Figure 2 with 2 supplements
RA is necessary for peripheral cone survival.
(A) Schematic of the experimental design. Aldh Flox mice were injected with tamoxifen to create a conditional knockout (cKO) of the RA synthetic enzymes at P10, and the retinas were harvested at …
Figure 2—figure supplement 1
P4 subretinal injection of AAV-ShH10Y-CMV-GFP leads to retina-wide infection of MG and photoreceptors.
IHC against GFP in sections and flatmounts of P40 rd1; RARE-LacZ retinas infected with AAV-ShH10Y-CMV-GFP after P4 subretinal injection. Scale bars; 500 µm (left panel), 100 µm (right panel).
Figure 2—figure supplement 2
Cyp26a1 is overexpressed upon infection with AAV-ShH10Y-CMV-Cyp26a1.
smFISH against Cyp26a1 using SABER FISH in P40 rd1; RARE-LacZ retina sections infected with AAV-ShH10Y-CMV-Cyp26a1 and AAV-ShH10Y-CMV-GFP. Scale bar; 100 µm.
Figure 3 with 2 supplements
Constitutive RA signaling is sufficient for cone survival in the central retina.
(A) Schematic of the experimental design. Retinas of rd1; RARE-LacZ mice were electroporated with bpCAG-BFP with or without the CAG-RARa-VP16 construct at P0 or P1 and harvested at P40. (B) …
Figure 3—figure supplement 1
Cyp26a1 expression is upregulated upon RARa-VP16 overexpression.
smFISH against BFP and Cyp26a1 using SABER in P40 rd1; RARE-LacZ retina sections electroporated with CAG-RARa-VP16. Scale bar; 100 µm.
Figure 3—figure supplement 2
Cone quantification strategy in gain-of-function experiments.
Schematic of the area of cone quantification in the dorsal retina. Red dots represent surviving cones, and the green region is the area of electroporation. A circle with a radius of 1.5 mm was used …
Figure 4
ALDH1A1 is enriched in the peripheral retina of humans.
(A) Relative expression level of ALDH1A1 compared to loading control, TBP, in the center and periphery of the human non-RP retinas (Center: n = 5, Periphery: n = 5, Two-way ANOVA, P = 0.0146). …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (Mus musculus) | RARE-LacZ | Jackson Laboratories | 008477 | |
| Genetic reagent (Mus musculus) | CAG-CreER | Jackson Laboratories | 004682 | |
| Genetic reagent (Mus musculus) | Pdgfra-Cre | Jackson Laboratories | 013148 | |
| Genetic reagent (Mus musculus) | Aldh1a1, 2, 3 Flox/Flox | PMCID: PMC5363406 | ||
| Recombinant DNA reagent | Rho-Cre | PMCID: PMC1764220 | ||
| Recombinant DNA reagent | Cralbp-Cre | PMCID: PMC1764220 | ||
| Recombinant DNA reagent | CAG-RARa-VP16 | This Study | Expression of RARa-VP16 from CAG promoter | |
| Recombinant DNA reagent | CALNL-RARa-VP16 | This Study | Cre-dependent expression of RARa-VP16 from CAG promoter | |
| Recombinant DNA reagent | TRE-RARa-VP16 | This Study | Doxycycline-dependent expression of RARa-VP16 | |
| Recombinant DNA reagent | CAG-rtTA | This Study | Expression of rtTA from CAG promoter | |
| Recombinant DNA reagent | AAV-CMV-Cyp26a1 | This Study | AAV expression plasmid encoding Cyp26a1 from CMV promoter | |
| Antibody | (Rabbit polyclonal) anti-Cone Arrestin | Millipore Sigma | AB15282 | (1:3000) |
| Antibody | (Chicken polyclonal) anti-B-galactosidase | Aves Labs | BGL1010 | (1:3000) |
| Antibody | (Goat polyclonal) anti-B-galactosidase | AbD Serotec | 4600–1409 | (1:3000) |
| Antibody | (Mouse monoclonal) anti-Brn3a | Millipore Sigma | MAB1585 | (1:500) |
| Antibody | (Rabbit polyclonal) anti-Iba1 | GeneTex | GTX100042 | (1:500) |
| Antibody | (Mouse monoclonal) anti-Glutamine Synthetase | Millipore Sigma | MAB302 | (1:3000) |
| Antibody | (Goat polyclonal) anti-ChAT | Millipore Sigma | AB144P | (1:500) |
| Antibody | (Chicken polyclonal) anti-GFP | Aves Labs | GFP-1020 | (1:3000) |
| Antibody | (Mouse monoclonal) anti-Sox2 | R&D Systems | AF2018 | (1:500) |
| Antibody | (Rabbit polyclonal) anti-Opn1mw | Millipore Sigma | AB5405 | (1:500) |
| Antibody | (Goat polyclonal) anti-Aldh1a1 | Abcam | 9,883 | (1:1000) |
| Sequence-based reagent | Mm Arr3 RNAscope Probe | Advanced Cell Diagnostics | 486,551 | |
| Sequence-based reagent | Mm Nrl RNAscope Probe | Advanced Cell Diagnostics | 475,011 |
Additional files
-
Supplementary file 1
Genotyping and ddPCR primers and oligonucleotide sequences for SABER FISH probes.
- https://cdn.elifesciences.org/articles/76389/elife-76389-supp1-v1.xlsx
-
Transparent reporting form
- https://cdn.elifesciences.org/articles/76389/elife-76389-transrepform1-v1.pdf
