Validation of mutant line and cell type analysis parameters. (A) Representative images of Six3:cre activity. Six3:cre was crossed to the Cre-dependent reporter Ai14. Cre-positive (red) cells overlapped with the RGC marker RBPMS (green) at P3. (B and C) Representative images (B) and quantification of (C) LKB1 immunoreactivity (magenta) in control Stk11^F/F animals and Stk11^F/F; Six3-Cre animals at P3. Co-staining with DAPI and RGC marker RBPMS (cyan) shows that LKB1 is lost from the retina generally and from RGCs specifically (arrows). (C and D) Representative images show that mistargeted cells (anti-RBPMS, green) are positive for additional RGC markers (anti-Brn3a, magenta, C) and negative for markers of cycling progenitors (Ki67, magenta, D) in LKB1^RET mice at P0 (arrows). (E) Representative quadrant demarcation lines (white doted lines) utilized for quantification of the numbers and location of RGC nuclei relative to their distance from the apical retina surface. These quadrants were demarcated as apical (quadrant one; Q1), upper middle (quadrant two; Q2), lower middle (quadrant three; Q3), and basal (quadrant four; Q4) retinal quadrants. Each quadrant thus represents 25% of the retina at each time point. The cellular composition of these quadrants change slightly over development as retinal layers matures (DAPI, blue). (F) Quantification of GCL thickness at P3. There is no significant increase in the average thickness of GCL of RGC nuclei in LKB1^RET mice relative to controls. Data are represented as the mean ± the SEM. nonsignificant (n.s.), nonparametric Mann–Whitney rank-sum U test. Source data are available for this figure: SourceData FS1.