Figure 6
The short-term effects of Notch inhibition on RPC reporters. (a) Confocal images of vertically sectioned E5 chick retinas co-electroporated with CAG::Nucßgal, ThrbCRM1::GFP or VSX2ECR4::GFP, and with or without CAG::MAML-DN. The retinas were cultured for 8 h post-electroporation. The scale bar shown in the bottom right picture denotes 40 µm and applies to all images. All images are oriented with the scleral side of the retina at the top of the image. (b) Flow cytometry quantification of the percentage of VSX2ECR4, ThrbCRM1 and VSX2ECR4/ThrbCRM1 reporter-positive cells within all the electroporated cells. Dissociated chick retinal cells electroporated ex vivo at E5 with the co-electroporation control CAG::iRFP with or without CAG::MAML-DN. The retinas were cultured for 8 h post-electroporation. The Shapiro–Wilk normality test was used to confirm the normal distribution. Mann–Whitney test was used to test significance in VSX2ECR4 quantification. A two-tailed student’s t-test was used to test significance in ThrbCRM1 and double-positive quantifications. (c) Flow cytometry quantification of the percentage of VSX2ECR4, ThrbCRM1 and VSX2ECR4/ThrbCRM1 reporter-positive cells within all the electroporated cells. Dissociated chick retinal cells electroporated with CAG::iRFP ex vivo at E5 and cultured for 20 h. The Shapiro–Wilk normality test was used to confirm the normal distribution. *** signifies p < 0.001 with a two-tailed student’s t-test. Each point represents one biological replicate. (d) Quantification of the percentage of VSX2, Otx2 and double marker-positive cells within the electroporated cell population from cell counting. Sectioned chick retinas electroporated ex vivo at E5 with the co-electroporation control CAG::Nucßgal with or without CAG::MAML-DN. The retinas were cultured for 20 h and immunostained with VSX2 or Otx2 antibodies. The Shapiro–Wilk normality test was used to confirm the normal distribution. * signifies p < 0.05, *** signifies p < 0.001 with a two-tailed student’s t-test. Each point represents one biological replicate. The columns represent mean, and the error bars represent standard deviation.
