Nanotube-like processes facilitate material transfer between photoreceptors (2021)
Figure 6

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

a, MIP image of wildtype (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 labeled with cGFP (green) but not with myrRFP (red); Scale bar = 50µm;

e, quantification and statistical analysis of material transfer events following subretinal transplantation of live (cGFP = 3775 ± 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 ± S.D.

f-h, Assessment of potential for transfer of donor Gfp and Gnat1 mRNA during transplantation using RNAscopeTM. 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 (2328 ± 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.

Data information: CM = cell mass; All eyes were processed 3 weeks post-transplantation. RPE- Retinal Pigment Epithelium; ONL - outer nuclear layer; INL - inner nuclear layer; GCL- Ganglion Cell Layer. Graphs show mean ± S.D.