Figure 4

Figure 4

Ectopic expression of the full-length Crb2a promotes RPC proliferation. (A) Schematic of Crb2a function in the regulation of multiple signaling pathways. (B,C) Schematic of the (B) Crb2aFL and (C) Crb2aEXT transgene protein structures. HSE indicates the presence of an 8× repeat of the bi-directional heat-shock element to drive GFP and transgene expression simultaneously. (D–F) Representative images assessing levels of EdU incorporation within transgene clones (GFP), induced with a 30-min heat-shock at 24 hpf, after a 12-h EdU pulse from 36 to 48 hpf in (D) Control (WT background; H2a-mCherry; nlsGFP), (E) Crb2aFL (WT background; Crb2aFL) overexpression, or (F) Rab11aDN/Crb2aFL (Rab11aDN background; Crb2aFL) retinas. (G) Quantification of the percentages of cell cycle exit (EdU negative) within retinal sections. Listed n-values indicate the number of individual embryos counted for each genotype. Data represent the mean percent of cell cycle exit across clones, with error bars indicating SEM. * Indicate p < 0.05 after Tukey's multiple comparisons tests of a One-way ANOVA (p = 0.0002). (H–K) Representative images (>3 embryos assessed/genotype) of transgene overexpression of (H,I) Crb2aFL or (J,K) Crb2aEXT in either (H,J) WT or (I,K) Rab11aDN backgrounds, assessing localization of Crb2a transgene expression (HA tag). White boxes represent regions of highlighted in high magnification insets. Arrows in panel I represent Crb2aFL accumulation in puncta, suggesting accumulation of the transgene within non-plasma membrane associated focal puncta when expressed in the Rab11aDN background. Scale bars represent 50 μm.