Fig. 2: L75Pfs retinal organoids display an overabundance of S-opsin expressing cells at the expense of rods compared to WT organoids.

a, b Confocal images from stage 3 organoids (i.e., presence of photoreceptor outer segments) showing a single layer of cones with few S-cones (green) in WT organoids (a) versus an abundance of S-cones (green) distributed throughout the ONL in L75Pfs organoids (b). ML-cones are shown in orange. Scale bars = 25 μm. c–n Photoreceptor characterization of WT and L75Pfs retinal organoids. Bright field (c, i) and confocal (d–h and j–n) images showing S-opsin+/ARR3+ cones (k, l) distributed throughout the ONL of L75P(fs) organoids that do not express the rod marker NR2E3 (m) (a transcription factor whose expression is controlled by NRL). This finding is in contrast to WT organoids that display ordered expression of cones (e, f) along the outermost ONL with a multicellular layer of NR2E3+ rod nuclei (g) internal to the cone layer, as well as an overall low number of S-opsin+ (e) cones. Scale bars: c, I = 250 microns; d–h and j–n = 25 μm. o, p Quantification of photoreceptors in confocal images of stage 3 organoids from 3 WT lines and 3 L75Pfs clones. o, p Quantification of photoreceptors in confocal images of stage 3 organoids from 3 WT lines and 3 L75Pfs clones. o NR2E3+ rod and ARR3 + cone abundance as a percentage of total nuclei in the ONL: 15 images from 5 organoids per line or clone were counted. p ML- and S-cone abundance as a percentage of total ARR3+ cones in the ONL: 11 WT images from 4 organoids per line and 15 L75Pfs images from 5 organoids per clone were counted. q RT-qPCR from stage 2–3 organoids showing a reduction in rod transcripts and an increase in S-opsin transcripts in L75Pfs organoids relative to WT organoids. p < 0.005, Mann–Whitney test.