Nanotube‐like processes facilitate material transfer between photoreceptors

A

MIP image of wild‐type (wt) retina transplanted with P8 Nrl.Gfp^+/+ CD73⁺ MACS‐enriched photoreceptors; dashed box indicates ROI subjected to 3D deconvolution and volume reconstruction (right) and shows ^PhNT‐like connection between donor and host photoreceptor at the level of host inner segment. Additional images show 3D segmentation of z = 0 and z = 38 focal planes, and reveal the ^PhNT; green = GFP, blue = nuclei; Scale bar = 10 µm in MIP and 5 µm volume reconstruction.

B, C

Assessment of properties of ^PhNT‐like processes between donor and host photoreceptors (n = 19 ^PhNT‐like processes; N = 10 eyes) showing (B) diameter and (C) length.

D

MIP image of wt retina transplanted with P8 Nrl.Gfp^+/+ x myrRFP^/+ photoreceptors; showing wt host ONL photoreceptor labelled with cGFP (green) but not with myrRFP (red); Scale bar = 50 µm.

E

Quantification and statistical analysis of material transfer events following subretinal transplantation of live (cGFP = 3,775 ± 804, myrRFP = 436 ± 145) or UV‐treated (cGFP = 1.3 ± 2.5, myrRFP = 4.3 ± 5.7) Nrl.Gfp^+/+x myr‐Rfp ^+/+ photoreceptors into wt hosts (N = 8 and N = 4 retinae, respectively); one‐way ANOVA, parametric, two‐tail, Tukey’s multiple comparisons; graph shows mean ± SD.

F–H

Assessment of potential for transfer of donor Gfp and Gnat1 mRNA during transplantation using RNAscope™. (F) Representative MIP images of Gnat1 ^−/− eyes transplanted with Nrl.Gfp^+/+ donor cells and processed in situ for Gfp mRNA (red) and Gnat1 mRNA (magenta); blue = nuclei; ROIs indicated in (F) show GFP^+ve donor cell mass (CM) and GFP^+ve cells within Gnat1 ^−/− host outer nuclear layer (ONL). G, H Quantification of Gfp and Gnat1 mRNA (mean fluorescence intensity per cell), respectively (N = 3 retinae); one‐way ANOVA, non‐parametric, two‐tailed analysis showed no significant differences in signal intensity for either Gnat1 or Gfp mRNA between GFP^+ve and GFP^−ve host photoreceptors.

I

Manipulation of actin signalling alters material transfer in vivo: (far left) MIP image showing GFP^+ve host photoreceptor (green) expressing Rod α‐transducin (red) after transplantation of retinal cells transduced with lenti‐CAG‐P2A.Gfp (transduction control) into Gnat1 ^−/− recipient; scale bar = 10 µm; adjacent images show Gnat1 ^−/− eyes transplanted with P2 retinal cells transduced with (left) plenti‐CAG‐P2A.GFP (green), or (middle) plenti‐CAG‐RhoA‐P2A.GFP (green), or (right) plenti‐CAG‐ΔΝRac1‐P2A.GFP (green); green = GFP, blue = nuclei; Scale bar = 20 µm.

J

Quantification of the number of GFP⁺ cells in Gnat1 ^−/− host ONL, (N = 5 retinae per condition); plenti‐CAG‐P2A.GFP (2,328 ± 234), plenti‐CAG‐RhoA‐P2A.GFP (668 ± 481), plenti‐CAG‐ΔΝRac1‐P2A.GFP (547 ± 306); one‐way ANOVA, non‐parametric, Dunn’s multiple comparisons test. Graph shows mean ± SD.

Figure 6. Transplanted photoreceptors form PhNT processes with host photoreceptors and transfer cytoplasmic and membrane proteins but transfer of mRNA was not detected.

Figure 6. Transplanted photoreceptors form ^PhNT processes with host photoreceptors and transfer cytoplasmic and membrane proteins but transfer of mRNA was not detected.