Glycolysis expression during development and accelerated development in Pten-cKO retinas.

(A) UMAP plot of scRNA-seq data collected from wild-type retinas between E11 and P14 (Clark et al., 2019). Stages of data collection are color-coded. Transcript distribution of Cdk1, a marker of proliferating RPCs, and Hk1 and Pgk1, glycolytic enzymes, showing enriched Pgk1 expression in earlier-staged RPCs.

(B) Schematic illustration of the bell curve-like distribution of rod photoreceptor differentiation and glycolytic activity, which peak at the same period during development.

(C) E18.5 retinal cross sections immunostained with PAX6, PTEN, and VSX2.

(D) Schematic of the strategy used to generate Pten-cKO animals.

(E) Immunostaining of P0 Pax6-Cre-GFP and Pten-cKO retinas, showing GFP expression in RPCs throughout the retina, with higher levels in the periphery in the ciliary marginal zone (CMZ).

(F) Retinal cross sections of wild-type and Pten-cKO animals at P14 injected with BrdU at P4, showing immunostaining with PTEN (green) and BrdU (red) in wild-type and Pten-cKO retinas.

(G) Quantification of the number of BrdU cells in the gcl, inl and onl. Plots show means ± SEM. N=3 biological replicates/genotype, all with 3 technical replicates. P-value calculated with unpaired t-test. onl, outer nuclear layer; inl, inner nuclear layer; gcl, ganglion cell layer.

(H) Graph showing differentiation pattern during retinal development in Pten cKO and wild-type animals.

onbl, outer neuroblast layer; inl, inner nuclear layer; gcl, ganglion cell layer; le, lens; cmz, ciliary marginal zone. Scale bar 50μM in C, 400μM in E and 100μM in F. Figure 1B was created using BioRender.com. Figure 1D was created using BioRender.com. Figure 1H was created using BioRender.com.

Glycolytic gene expression and activity are elevated in P0 Pten-cKO retinas.

(A) Heat map of bulk RNAseq data comparing gene expression in P0 Pten-cKO (N=5) and wild-type (N=4) retinas.

(B) Volcano plot of DEGs with Log2FC=2, showing both down-regulated (to the left) and up-regulated (to the right) genes. Venn diagram shows 667 up-regulated genes and 408 down-regulated genes in P0 Pten-cKO retinas.

(C) KEGG pathway enrichment related to DEGs, showing an enrichment of up-regulated glycolytic pathway genes in P0 Pten-cKO retinas (arrow).

(D) Schematic of glycolysis pathway, showing the intracellular enzymes involved in metabolizing glucose into lactate. Also shown is the Slc16a3 symporter, which extrudes lactate and H+ to increase intracellular pHi. Figure 2D was created using BioRender.com.

(E) Normalized RPKM (reads per kilobase million) values for glycolytic gene expression in P0 wild-type and Pten-cKO retinas. Plots show means ± SEM. N=4 biological replicates for wild-type and N=5 for Pten-cKO retinas. P-values calculated with Wald test, with a Benjamini-Hickberg correction for multiple comparisons.

(F) Normalized glycolytic gene expression from qPCR of P0 Pten-cKO (N=3) and wild-type control (N=3) retinas. Plots show means ± SEM. N=3 biological replicates/genotype, all with 3 technical replicates. P-values calculated with unpaired t-test.

(G) Seahorse assay on P0 wild-type and Pten-cKO RPCs cultured in vitro. Points of treatment with oligomycin (O), FCCP (F), and Rotenone/Antimycin A (R/A) are indicated. Left plot shows elevated extracellular acidification rate (ECAR) in Pten-cKO RPCs, while plot to the right shows no significant effect on oxygen consumption rate (OCR). Plots show means ± SEM. N=3 biological replicates for Pten-cKO and N=8 biological replicates for wild-type, all with 3 technical replicates. P-values calculated with unpaired t-test (GraphPad Prism, USA).

(H) Lactate assay on P0 wild-type and Pten-cKO retinas. Plots show means ± SEM. N=3 biological replicates/genotype, all with 3 technical replicates. P-value calculated with unpaired t-test.

Glycolytic inhibition reduces proliferation and photoreceptor differentiation in P0 retinal explants.

(A) Schematic of retinal explant experiments, showing treatment with pharmacological inhibitors (2DG or GPI), and the labeling of proliferating RPCs (30 min BrdU) or birthdating of newborn retinal cells (24 hr BrdU).

(B) Schematic of glucose uptake replaced by 2DG, which leads to the accumulation of 2-DG-6P that cannot be further metabolized by the glycolytic pathway.

(C) P0 retinal explants treated with 5mM or 10mM 2DG for 1 day in vitro (DIV), showing BrdU immunolabeling and the number of BrdU+ cells after a 30 min labeling with BrdU.

(D) P0 retinal explants treated with 5mM or 10 mM 2DG for 1 day in vitro (DIV), showing BrdU immunolabeling and the number of BrdU+ cells after a 24-hr labeling with BrdU, including co-labeling with Ki67 to calculate the p-fraction (%BrdU+Ki67+cells/total Ki67+ cells).

(E-G) P0 retinal explants treated with 5 mM or 10 mM 2DG for 1 day in vitro (DIV) and immunostained for Vsx2 (E), CC3 (F) or CRX (G).

(H) P0 retinal explants treated with glycogen phosphorylase inhibitor (GPI) at 12.5 μM and 25 μM for 24 hr and immunostained for BrdU after a 30 min incubation at the end of the treatment period.

Plots in C-H show means ± SEM. N=3 biological replicates/treatment, all with 3 technical replicates. P-values calculated with one-way ANOVA with Tukey post hoc test. Scale bars= 25 μM in C, E and G, and 50 μM in D, F and H. Figure 3A was created using BioRender.com. Figure 3B was created using BioRender.com.

Promotion of glycolytic flux with a cytoPFKB3-GOF mouse model accelerates photoreceptor differentiation and outer segment maturation.

(A) Schematic showing function of PFKB3 as an activator of PFK1 through the conversion of fructose-6-phosphate to fructose-2,6-bisphosphate.

(B) Schematic showing generation of an RPC-specific cytoPFKB3-GOF mouse model.

(C) Lactate assay performed on P0 wild-type and cytoPFKB3-GOF retinas.

(D,E) Birthdating experiments performed by injecting BrdU into pregnant females at E12.5 and harvesting retinas at P7. BrdU immunostaining of P7 wild-type and cytoPFKB3-GOF retinas (D) and quantification of the %BrdU+ cells in each of the nuclear layers (E).

(F) Rhodopsin immunostaining of P7 wild-type and cytoPFKB3-GOF retinas. An expansion of outer segment (OS) area is indicated by the arrow and quantified in the graph.

(G) Bulk RNAseq mining of photoreceptor outer segment genes de-regulated in P0 Pten-cKO retinas.

(H) Schematic of mode of action of 2DG.

(I) Rhodopsin immunostaining of P7 wild-type and Pten-cKO retinas, either without treatment or following the administration of 2DG between P0-P7. The expanded OS area in Pten-cKO retinas is indicated with an arrow. Quantifications of photoreceptor OS areas are shown in the graph.

Plots show means ± SEM. N=3 biological replicates/treatment group except C, with cytoPFKFB3-GOF (N=3) and wild-type (N=2), all with 3 technical replicates. P-values calculated with t-tests in C and F, and one-way ANOVA with Tukey post hoc test in E and G. Scale bars 100 μM in E and 50 μM in F and G. gcl, ganglion cell layer; inl, inner nuclear layer; onl, outer nuclear layer; os, outer segments. Figure 4A was created using BioRender.com. Figure 4B was created using BioRender.com.

Reducing intracellular pH (pHi) inhibits RPC proliferation and differentiation.

(A) Schematic showing function of Slc16a1/3 symporters in extruding lactate and H+ out of the cell to increase pHi during elevated glycolysis. Also depicted is the impact of acidic pH and oligomerizing PKM to prevent its activity.

(B) Schematic of retinal explant experiments, showing treatment with a pharmacological inhibitor (CNCn) of Slc16a symporters or growth in different pH media, and the labeling of proliferating RPCs (30 min BrdU) or birthdating of newborn retinal cells (24 hr BrdU).

(C) P0 retinal explants treated with 5mM CNCn for 1 day in vitro (DIV) followed by a 30 min BrdU pulse, showing immunolabeling and the quantification of BrdU+ RPCs.

(D) P0 retinal explants incubated in media buffered to pH6.5, pH7.4 or pH8.0 for 1 day in vitro (DIV), showing BrdU immunolabeling and the number of BrdU+ cells after a 30 min labeling with BrdU.

(E) P0 retinal explants incubated in media buffered to pH6.5, pH7.4 or pH8.0 for 1 DIV, showing BrdU immunolabeling and the number of BrdU+ cells after a 24-hr labeling with BrdU, including co-labeling with Ki67 to calculate the p-fraction (%BrdU+Ki67+cells/total Ki67+ cells).

(F-H) P0 retinal explants incubated in media buffered to pH6.5, pH7.4 or pH8.0 for 1 DIV and immunostained for Vsx2 (E), CC3 (F) or CRX(G).

(I) P3 retinal explant electroporated with pHluorin-tdTomato, imaged after 2DIV to show electroporated cells (tdTomato+) have varying levels of 488nm emission from pHLuorin (green).

Plots show means ± SEM. N=3 biological replicates/treatment group, all with 3 technical replicates. P-values calculated with t-tests in C, and one-way ANOVA with Tukey post hoc test in D,E,F-H. Scale bars= 25 μM in C, D, F and H, and 50 μM in E, G and I. Figure 5A was created using BioRender.com. Figure 5B was created using BioRender.com.

Wnt signalling is a downstream mediator of Pten and glycolysis.

(A) Schematic illustration of canonical Wnt signaling pathway.

(B) UMAP plot of scRNA-seq data collected from wild-type retinas between E11 and P14 (Clark et al., 2019). Transcript distribution of Wnt7b, Lef1 and Dkk1 Wnt genes.

(C) Normalized RPKM (reads per kilobase million) values for Wnt pathway-associated genes in P0 wild-type (N=4) and Pten-cKO (N=5) retinas.

(D) Western blot of β-catenin expression, normalized to β-actin levels, in P0 retinal explants treated with 5mM and 10mM 2DG or in P0 retinal explants cultured in media buffered to pH8.0, pH7.4 or pH6.5 (E), all for 24 hrs.

Plots show means ± SEM. N=3 biological replicates/treatment group, all with 3 technical replicates. P-values calculated with t-tests in C, and one-way ANOVA with Tukey post hoc test in D, E. Figure 6A was created using BioRender.com.

Wnt signaling modulate retinal proliferation and differentiation.

(A-C) P0 retinal explants treated with 5μM or 10μM FH535 to block Wnt signaling for 1 day in vitro (DIV), followed by immunostaining for BrdU after a 30 min pulse (A), CRX (B) or CC3 (C).

(D) Immunostaining P14 wild-type and Ctnnb1-cKO retinas with rhodopsin. Asterisk points to ONL rosette in Ctnnb1-cKO retina. Graph showing quantification of OS area.

(E) Summary of the main conclusions of the study, showing that elevated glycolytic flux increases pHi and Wnt signaling.

All experiments included three biological replicates for each group. Statistical analysis was done using one-way ANOVA with Tukey post hoc test used in A-C and unpaired t-test in D. Scale bars= 50 μM in A-D. gcl, ganglion cell layer; inl, inner nuclear layer; onl, outer nuclear layer; os, outer segments. Figure 7E was created using BioRender.com.