(A) Schematic of outer plexiform layer (OPL) development from P3 to P14. The outer retina contains developing rods (yellow) and cones (green) that extend axons beginning at P1. Cones form contacts with horizontal cell interneurons (blue) at P3, where nascent synapse patches emerge at sites of contact (red circles), followed by synaptogenesis beginning at P5. This corresponds with OPL formation. Rods begin to form synaptic connections with horizontal cells by P8, and bipolar cells also begin to become integrated at this time (orange, rod bipolars; dark green, cone bipolars). Ribbon synapse formation is complete and OPL sublamination begins by P14. (ONBL, outer neuroblast layer; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer). (B–D) In control animals, the nascent OPL is first visible at P3 when it appears as small discontinuous gaps in the nuclear staining (DAPI, grey; arrowheads point to patches). These patches are distinct from gaps caused by horizontal cell bodies (magenta stars). In Lkb1-Ret animals, these OPL patches are small and located closer to the apical surface (B) and are reduced in number (C, n = 322 control cells and n = 252 Lkb1-Ret cells). N = 4 control and N = 4 Lkb1-Ret animals. At P5, nascent patches converge in control animals to generate a single continuous layer, forming the OPL. In Lkb1-Ret animals, the OPL was discontinuous and patches were misaligned and located closer to the apical surface (D n = 487 control cells and n = 339 Lkb1-Ret cells). This resulted in a marked decreased in total OPL area (E). N = 4 control and N = 8 Lkb1-Ret animals. Scale bars = 25 µm. Data are represented as a distribution of the distance of patches from the apical surface (B,D, ***p<0.001, unpaired two-tailed Student’s t test) or as the mean ± the s.e.m. (E, **p<0.01, non-parametric Mann-Whitney Rank Sum U-test).