Retinal landscape of WT and P23H retinas.

A) Immunostaining for PCNA (yellow), rhodopsin (green), and Flag tag (P23H rhodopsin transgene; magenta) in adult WT retina. B) UMAP projection of the different cell clusters identified through SC-RNA Seq. C) Immunostaining for PCNA (yellow), rhodopsin (green), and Flag tag (magenta) in adult P23H retina. Orange arrows point to off-target labeling of green cone photoreceptor outer segments by Retp1 antibody. Blue arrows point to deformed outer segments of rod photoreceptors. Note that the ONL of the P23H retina is much thinner than WT retina. D) UMAP projection of the different cell clusters identified through SC-RNA Seq. P23H retinas have a unique cluster identified as new rods (circled in red). ONL=outer nuclear layer (photoreceptors), OPL= outer plexiform layer (photoreceptor synapses), INL= inner nuclear layer (horizontal, bipolar, amacrine and Müller cells), IPL= inner plexiform layer (synapses), RGC= retinal ganglion cells, AC= amacrine cells, BPC= bipolar cells, HZC = horizontal cells, RPE = retinal pigmented epithelial cells, MGC= Müller glial cells, RPC= retinal progenitor cells, NPC= neurogenic progenitor cells.

Genes used to identify new rod cluster.

Identification of progenitor cell types in the retina.

A) Violin plot of fam60al, a gene that is uniquely and highly expressed in both progenitor cell clusters (RPC, cluster 12; NPC, cluster 13). B) Violin plot of aurkb, a gene that is uniquely and highly expressed only in the retinal progenitor cell cluster. C) Detection of fam60al and aurkb transcripts through in-situ hybridization in adult Zf retina. D) Detection of fam60al transcripts through in-situ hybridization. Fam60al is detected in both the ONL and INL regions of the retina. E) Detection of aurkb transcripts through in-situ hybridization. Aurkb is detected predominantly in the ONL region of the retina.

BrdU pulse-chase in adult Zf retina.

A) Timeline schematic of experimental procedures. B) Rhodopsin and BrdU labeling in WT A/B retina collected on the 3rd day of BrdU injections. C) Rhodopsin and BrdU labeling in WT A/B retina collected 7 days after the last BrdU injection. D) 3-D image of WT A/B retina collected on the 3rd day of BrdU injections. E) Rhodopsin and BrdU labeling in P23H retina collected on the 3rd day of BrdU injections. F) Rhodopsin and BrdU labeling in P23H retina 7 days after the last BrdU injection. G) 3-D image of P23H retina collected on the 3rd of BrdU injections. H) Quantification of the number of BrdU positive cells at each timepoint. Data points represent individual animals. **** p < 0.0001; n = 5 animals/condition.

Trajectory analysis of WT and P23H SC-RNA seq data.

A) Expression score analysis and pseudotime analysis of the progenitor cell populations found in the WT data. B) Expression score analysis and pseudotime analysis of the neurogenic progenitor cell cluster in the P23H data. C) Expression score analysis and pseudotime analysis of the retinal progenitor cell cluster in the P23H data. D) Expression score analysis and pseudotime analysis of rod photoreceptor maturation. The higher the expression score and lower the pseudotime, the more likely the predicted trajectory will happen.

DrivAER prediction of master regulators involved in rod photoreceptor regeneration.

A) Transcription factors regulating genes involved in proliferation of RPCs. B) Transcription factors regulating genes involved in differentiation of RPCs into new rods. C) Transcription factors regulating genes involved in maturation of new rods. D) E2f1, e2f2, and e2f3 are predicted to play an important role in the proliferation of progenitor cells in the ONL. Prdm1a is predicted to play an important role in the differentiation of progenitor cells into rod photoreceptors. Sp1 is predicted to play an important role in the maturation of newly formed rod photoreceptors.

Effects of e2f’s 1-3 and aurkb knockdowns on RPC proliferation.

A) Timeline schematic of experimental procedures. B) BrdU labelling of control retina. C) BrdU labelling of retina injected with a cocktail of e2f1, e2f2, and e2f3 Vivo-morpholinos. D) BrdU labelling of retina injected with the aurkb Vivo-morpholino. E) TUNEL staining of control retina. F) TUNEL staining of retina injected with a cocktail of e2f1, e2f2, and e2f3 Vivo-morpholinos. G) TUNEL staining of retina injected with the aurkb Vivo-morpholino. H) Quantification of the number of BrdU positive cells found within each treatment. Both e2f’s 1-3 and aurkb knockdown significantly reduced the number of BrdU-positive proliferating progenitor cells. I) Quantification of the number of TUNEL positive cells found within the ONL in each treatment. Neither e2f’s 1-3 nor aurkb knockdown altered the number of TUNEL-positive cells in the ONL. Data points represent individual animals. * p < 0.05; n = 5 animals/condition.

Vivo-Morpholinos for targeted gene knockdowns.

Biological processes affected by Vivo-morpholino knockdown of regulators of RPC proliferation.

A) Biological processes downregulated (red; left) and upregulated (green; right) after knockdown of E2Fs 1-3. B) Biological processes downregulated (red; left) and upregulated (green; right) after knockdown of AurkB. Data are derived from two replicate samples per condition.

Biological processes downregulated by e2f1s1-3 knockdown.

Biological processes upregulated by e2fs1-3 knockdown.

Biological processes downregulated by aurkb knockdown.

Biological processes upregulated by aurkb knockdown.

Effects of prdm1a knockdown on RPC proliferation and rhodopsin expression in adult P23H retina.

A) Timeline schematic of experimental procedures. B) Rhodopsin expression and BrdU labelling of control retina. C) Rhodopsin expression and BrdU labelling of retina injected with the prdm1a Vivo-morpholino. D) Quantification of BrdU labelling and rhodopsin expression for each treatment. While the number of BrdU-positive proliferating progenitor cells did not change, prdm1a knockdown reduced rhodopsin expression in the ONL. ** p < 0.05; n = 5 animals/condition.

Colocalization of cells that are BrdU-positive and express rhodopsin.

A) Timeline schematic of experimental procedures. B) Rhodopsin expression in a control P23H retina. B’) BrdU labelling in a control retina. B’’) Colocalization of cells that are BrdU-positive and express rhodopsin. C) Rhodopsin expression in a P23H retina after prdm1a knockdown. C’) BrdU labelling in a P23H retina after prdm1a knockdown. C’’) Colocalization of cells that are BrdU positive and express rhodopsin. D) The number of mislocalized cells found in the INL that expressed rhodopsin increased significantly after prdm1a knockdown. E) The number of BrdU-positive cells did not change after prdm1a knockdown. F) The fraction of BrdU-positive cells that also expressed rhodopsin was significantly reduced after prdm1a knockdown. *** p < 0.001, * p < 0.05; n = 4 animals/condition. G) Biological processes downregulated after Prdm1a knockdown at Day 3 (orange;left) and Day 6 (cyan;right) of collection. Data are derived from two replicate samples per condition.

Biological processes downregulated by prdm1a knockdown (collection at 53 hpii).

Biological processes downregulated by prdm1a knockdown (collection on Day 6).

Check for progenitor cell differentiation into other cell types after prdm1a knockdown.

A) BrdU labelling of P23H retina collected 4 days after prdm1a knockdown. B) Immunostaining for red/green cones in P23H retina collected 4 days after prdm1a knockdown. C) Immunostaining for bipolar cells in P23H retina collected 4 days after prdm1a knockdown. D) Colocalization of A, B, and C show that BrdU-positive cells do not express cone arrestin or pkcα. E) BrdU-positive cells do not express Blue Opsin. F) BrdU positive cells do not express UV opsin.

Primary and secondary antibodies used in this study.

HiPlex probes used in this study.

Dotplot analysis of the trajectory genes controlled by e2f1, e2f2, and e2f3.

The list of genes from the trajectory that were predicted to be controlled by the e2f’s 1-3 has been illustrated in a Dotplot to show their average expression levels (color scale) and fraction of cells expressing the gene (dot size) within each cluster of the P23H dataset.

Dotplot analysis of the trajectory genes controlled by prdm1a.

The list of genes from the trajectory that were predicted to be controlled by prdm1a has been illustrated in a Dotplot to show their average expression levels (color scale) and fraction of cells expressing the gene (dot size) within each cluster of the P23H dataset.

Dotplot analysis of the trajectory genes controlled by sp1.

The list of genes from the trajectory that were predicted to be controlled by sp1 has been illustrated in a Dotplot to show their average expression levels (color scale) and fraction of cells expressing the gene (dot size) within each cluster of the P23H dataset.