(A–C) Confocal micrographs of vertical sections of retinas electroporated with the OTX2ECR2::GFP reporter and respective CRISPR complex (control or with guide) at E5 and cultured for 2 days and immunostained simultaneously with panPOU4F, ISL1 and LHX1 along with GFP. White arrows show GFP-positive cells that are not positive for any of the three markers. (D–F) Confocal micrographs of whole mount retinas electroporated with the OTX2ECR2::GFP reporter and respective CRISPR g2 at E5 and cultured for 2 days and immunostained simultaneously with panPOU4F and ISL1, along with GFP. White arrows show cells that are not positive for the markers, while the yellow arrow shows a marker positive RGC. (G) Dot plot presenting representative markers for cluster New. The size of the dot corresponds to the percentage of cells expressing the marker (x-axes) in each cluster (y-axes). The blue color intensity represents the average of the expression level. (H–L) Lineage tracing of the OTX2ECR2 element shows predominantly RGCs in the OTX2CRISPR retina, compared to the PR-rich CTRL. OTX2ECR2 element drives the expression of PhiC31 recombinase, that, upon recombination of the attachment sites, results in GFP expression in all cell with past and present activity of the OTX2ECR2 element. OTX2ECR2 lineage-traced cells in the outermost half of a whole mount CTRL retina (H) and in the innermost half of the whole mount OTX2CRISPR retinas (J, K). (I) Micrograph of a CTRL WM retina imaged in the GCL shows no OTX2ECR2 lineage traced cells are amongst the β-Galactosidase (electroporation control marker) positive ones. (L) Quantitative analysis of whole retina thickness in both CTRL and mutant samples. Bar graph represents percentages of GFP/POU4F double-positive cells, from total number of GFP-positive cells. Datapoints represent a technical replicate from each retina counted (three technical replicates for each of the three biological replicates). (M–N) Contribution of OTX2 during the cell fate specification in the developing chick retina. (M) OTX2 is necessary for the generation of two PRs types, as well as one type of cone homotypic progenitor cell (chPCs), repressing the generation of subtypes of RGCs and HCs. (M). OTX2 activates genes required for formation of PRs, inhibiting regulators of RGC (POU4F2/3, DLX1/2), HC (ONECUT, PROX1) and PAX6 expression. WM, whole mount, OR, outer retina, GCL, ganglion cell layer.