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Fig. 3 | Stem Cell Research & Therapy

Fig. 3

From: Activation of adult mammalian retinal stem cells in vivo via antagonism of BMP and sFRP2

Fig. 3

Tamoxifen induction of reporter expression in Msx1-CreERT2;Rosa26-tdTomato mice labels the ciliary epithelium and CE-derived RSC spheres. A Schematic of Msx1-CreERT2;Rosa26-tdTomato mouse lines and transgene induction paradigm. Four consecutive daily intraperitoneal tamoxifen injections were followed by endpoint clonal RSC sphere assay and immunohistochemistry (IHC) two weeks after the injection period. B-E Fluorescence microscopy of eye sections and RSC spheres from the eyes of Msx1-CreERT2;Rosa26-tdTomato mice two weeks post-tamoxifen injection. B-D Msx1-Cre+ mice. C-E Littermate control Msx1-Cre−mice. A lower magnification image is shown compared to panel D to demonstrate that there were no positive spheres in a broad field of view. CE = ciliary epithelium, NR = neural retina. F The Percentage of RSC spheres with Msx1-tdTomato expression derived from tamoxifen-treated Msx1-CreERT2;Rosa26-tdTomato mice. Each data point represents the average % labeled RSC spheres per well for a single mouse. Red dashed line indicates between mouse average. N = 4 mice. G Schematic of Msx1-CreERT2;Rosa26-tdTomato transgene induction followed by fluorescence activated cell sorting (FACS) and subsequent clonal RSC sphere assay. H-J Representative FACS gating plots for CE cells derived from Msx1-CreERT2;Rosa26-tdTomato mice following tdTomato reporter induction. N p = non-pigmented; p = pigmented; tdT +  = tdTomato positive; tdT- = tdTomato negative. K-M The proportions of each FACS subpopulation. K The proportion of pigmented and non-pigmented cells. L The proportion of Msx1-tdTomato+ and Msx1-tdTomato− cells within the pigmented CE population. (p < 0.001; N = 4 FACS experiments). M The proportion of Msx1-tdTomato+ and Msx1-tdTomato− cells within the non-pigmented CE population. (p = 0.005; N = 4 FACS experiments). N The number of RSC spheres that formed 7 days after sorting for each FACS subpopulation. N = 3 FACS experiments. o-s Representative images of RSC sphere assay output wells for each sorted subpopulation 7 days following FACS. O Unsorted CE cells. Two images stitched together (indicated by the dashed line). P Pigmented Msx1-tdTomato+ CE cells. Two images stitched together (indicated by the dashed line). The white arrowhead indicates a sphere that has partial expression of tdTomato. Q Pigmented Msx1-tdTomato− CE cells. R Non-pigmented Msx1-tdTomato+ CE cells. S Non-pigmented Msx1-tdTomato− CE cells. White arrows indicate single Msx1-tdTomato expressing cells. Dashed line box indicates high magnification inset. Statistics: t-tests. Data are means ± SEMs. * = p < 0.05

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