Fig. 1

Intravitreal injection of Noggin or anti-sFRP2 stimulates proliferation in the ciliary epithelium and increases primary sphere-forming retinal stem cell number. A Schematic of the intravitreal injection paradigm followed by endpoint IHC. Mice received one intravitreal injection per day for three days while EdU was delivered via the drinking water continuously until Day 4. Mice were euthanized for histochemical analysis on Day 4 or Day 31. Injections consisted of PBS control, Noggin or anti-sFRP2 at indicated concentrations. B-G Fluorescence images of EdU labeling in the CE of eye sections from mice treated with PBS, anti-sFRP2 or Noggin at Day 4 and Day 31. B, E EdU labeling in PBS-treated eyes. C, F EdU labeling in Noggin-treated eyes. D, G EdU labeling in and anti-sFRP2-treated eyes. Hoechst stain was used to label all nuclei. White arrows indicate EdU⁺ cells. Straight dashed line indicates the border between the ciliary epithelium and the retina. H-J Ki67 immunostaining and EdU labeling at the Day 4 timepoint in the CE of eye sections from mice treated with PBS, anti-sFRP2 or Noggin. h PBS-treated eyes. i Anti-sFRP2-treated eyes. j Noggin-treated eyes. White arrows indicate EdU⁺ cells. Green arrows indicate Ki67⁺ cells. Yellow arrows indicate EdU + Ki67 co-labeled cells. Hoechst stain was used to label all nuclei. Dashed line box indicates high magnification inset. K Quantification of the proportion of EdU + Ki67 co-labeled cells relative to the total number of EdU-labeled cells in the CE at Day4 and Day 31. N = 3–5 mice per group. L-O Quantification of EdU-labeled cells in the CE and NR normalized by regional area in eye sections from mice treated with the indicated conditions. l Day 4 anti-sFRP2. (p = 0.004; N = 3–6 mice per group). M Day 31 anti-sFRP2. (p = 0.015; N = 3 mice per group). N Day 4 Noggin. (p =  < 0.001; N = 3–6 mice). o Day 31 Noggin; N = 3 mice per group. P Quantification of EdU-labeled cells in the CE and NR normalized by area in eyes treated with PBS, Noggin, anti-sFRP2 or both Noggin + anti-sFRP2 at Day 4. (p < 0.001; N = 3–6 eyes per group). Q Schematic of the intravitreal injection paradigm followed by endpoint clonal RSC sphere forming assay from primary CE. Mice received one intravitreal injection per day for three days. On Day 10, mice were enucleated, and the CE was dissected for a subsequent 7-day clonal sphere growth assay in vitro. R, S Quantification of RSC sphere frequency normalized to naïve un-injected control. R anti-sFRP2 dose–response. (p < 0.0001; N = 10–16 eyes per group). (S) Noggin dose–response. (p < 0.001; N = 5–16 eyes per group). Each data point represents a single eye. Statistics: One-way ANOVAs with Holm-Sidak posthoc tests. Data are means ± SEMs. * =  p < 0.05