Figure 7
Notch inhibition leads to a delayed increase in cones relative to cone/HC RPCs. (a) Schematic of experimental set-up. Retinas were electroporated with VSX2ECR4::PhiC31 and CAG::stopGFP responder plasmid to label multipotent RPCs (mpRPC)s and their descendents. In addition, ThrbCRM2::TdT was co-electroporated to identify a subset of cones. CAG::MAML-DN was included in experimental conditions and omitted in control conditions. CAG::iRFP was included as a co-electroporation control (not shown). Retinas were assessed 2 to 5 days later by flow cytometry to measure GFP, TdT, and iRFP activity. Non-electroporated cells are shown in blue, lineage-traced cells other than cones in green, non-lineage traced ThrbCRM2+ cells in magenta and lineage traced ThrbCRM2+ cells in orange. (b) Flow cytometry quantification of the percentage of VSX2ECR4 lineage traced cells, ThrbCRM2::TdTomato reporter-positive cells or ThbrCRM2::TdTomato reporter-positive cells with VSX2ECR4 activity (double positive) within all the electroporated cells using VSX2ECR4 lineage trace strategy with the PhiC31 responder plasmid shown in Fig. 4b. Dissociated chick retinal cells electroporated ex vivo at E5 with the co-electroporation control CAG::iRFP, VSX2ECR4::PhiC31 and its responder plasmid, ThrbCRM2::TdTomato, and with or without CAG::MAML-DN. The retinas were cultured for 2 or 3 days post-electroporation. A two-tailed student’s t-test was used to test significance. The Shapiro–Wilk normality test was used to confirm the normal distribution. * signifies p < 0.05, ** signifies p < 0.01, *** 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. (c) Same as (a) but with 4 or 5 days culture.
