Fig 2: RO-RPE interaction enhances the outer segment number at the connection site.(a) For live-cell observation of RoCs, hiPSC-RPE was labeled with a pJG-IRBP-GFP viral vector prior co-culture (green); hiPSC-derived RO-RPCs were labeled with PNA lectin Alexa Fluor 568 (red). RO-RPE interaction site is illustrated as an orthogonal view (b) Orthogonal view of RO (Day 181) and RPE co-cultured for 7 days in the RoC and subsequently stained in situ for rhodopsin (green) and phalloidin (red). c) Distance between GFP-labeled RPE and PNA lectin-stained segment tips in a) was assessed by measurement using orthogonal images (n = 12 chip compartments). (d) Representative cryosection from 7 days co-cultured day 260 ROs and hiPSC-RPE. Sections were immunostained with ROM1 (green; outer segment marker), phalloidin (white; cytoskeleton) and rhodopsin (red; rods). (e) Electron microscopic image of a day 190 organoid facing RPE cultured in the RoC. (f) Representative electron microscopic images of inner and outer segments on the surface of day 181 ROs cultured for 7 days in f1) the RoC, f2) the RoC without RPE and f3) dish-cultured ROs. Black arrows indicate outer segments identified by stack formation. (g) Exemplary high magnification image of an outer segment-like structure containing organized membrane disks found in day 181 ROs cultured for 7 days in the RoC with RPE. (h) Number of segments/100 µm RO circumference comparing RoC, RoC without RPE and dish cultured RO. In the RoC, only the RPE facing side was analyzed (n = 3 RoC, 4 RoC w/o RPE and 3 dish cultured ROs were analyzed). Bars indicate (a-b) 40 µm, (d) 40 µm, (e-f) 5 µm, (g) 1 µm. Blue: DAPI. IS = inner segment, OS = outer segment. Error Bars: S.E.M. *p-value<0.05.10.7554/eLife.46188.019Figure 6—source data 1.Source data for Figure 6c.10.7554/eLife.46188.020Figure 6—source data 2.Source data for Figure 6h.

Fig 3: Outer segment peeling generates inner segment-enriched mouse retina samples. (A) Diagram of the mouse retina OS peeling method. Mouse retina slices are iteratively peeled from filter paper 8 times to remove rod outer segments (OS, yellow) from the inner segment (IS) layer (magenta). (B) Western blot analysis comparing control full retina samples vs. retinas after OS peeling/IS-enriched retinas; both from Rho-GFP/+ mice. 2% of total volume from lysates from 1 retina (either control or peeled) were used from each condition for SDS-PAGE. Molecular weight marker sizes are indicated in kDa. Immunolabeled bands from antibodies targeting OS proteins, including NbGFP-A647, Rho-C-1D4 and ROM1 (black arrow points to the ROM1 monomer band), were reduced in peeled lysates, whereas IS proteins STX3 and Rab11a were roughly equal demonstrating IS enrichment. Tubulin immunoblotting was performed as a loading control. (C) Control full retina slices and (D) IS-enriched retinal slices from Rho-GFP/+ mice were fixed and stained for resin embedding, ultrathin sectioning and TEM ultrastructure analysis. TEM images were pseudocolored to point out key rod structures as follows: OS = yellow, IS = magenta, connecting cilia (CC)/basal bodies = blue. Single rod examples are magnified from both conditions to emphasize intact CC ultrastructure and the preservation of the IS plasma membrane (magenta arrows). The locations of the distal appendages (DAPs) and rootlet are annotated in (D). (E) Z-projection SIM images of Rho-GFP/+ IS-enriched retinas immunolabeled with NbGFP-A647 (magenta) and centrin antibody (green). Fluorescence in the single rod example corresponding to the OS, IS and CC are annotated. (F) Control SIM image of a WT IS-enriched retina immunolabeled and imaged as in (E) to demonstrate NbGFP-A647 labeling specificity. Scalebar values match adjacent panels when not labeled.