Fig. 5: HBEGF-TNF-treated HROs show glial scar formation. | Nature Communications

Fig. 5: HBEGF-TNF-treated HROs show glial scar formation.

From: HBEGF-TNF induce a complex outer retinal pathology with photoreceptor cell extrusion in human organoids

Fig. 5

a Schematic: retinal dyslamination and glial pathologic changes investigated (10-days HBEGF-TNF (HT) treatment). b Transmission electron microscopy of epoxy-embedded HRO sections with HT treatment/without (CTRL): images of the most apical HRO regions show photoreceptors (P) with inner segments (PIS) that contain mitochondria and are interconnected with Müller glia (MG). Pseudocolors: cell junctions (red lines), MG (blue). N = 1 independent experiment (N) with n = 3 HROs. c Correlative-light electron microscopy: in control HROs, ACTB immunostaining, visualized by gold particles (black dots, 10 nm gold), was restricted to cell junctions (c1, 2: arrowheads) of the OLM and apical microvilli of MG. Conversely, prominent electron-dense, filamentous structures spanning between cell junctions (arrowheads) were observed in HT-HRO: these were confirmed to be actin filaments (ACTB+, c3–5). Extruded cell nuclei (EC) stained with DAPI are frequently observed in HT-HROs, but not controls. N = 1 independent experiment (N) with n = 3 HROs. d En-face images of HRO wholemounts with/without HT treatment (10 days) immunostained for MG (SLC1A3 + RLBP1) and photoreceptor (RCVRN) markers. Quantitative analysis of marker area: MG expand at the apical surface level of HT-HROs while PIS diminish, indicative of glial scar formation. Graphs: circles represent individual HROs (n) derived from N = 1 independent experiments (N) with n = 9; 3 ROIs averaged/n; two-sided Student’s t test; *P < 0.0001. Graphs (mean ± SD) and statistics over n. e Tokuyasu cryosections stained with RCVRN (10 nm protein A gold) to identify photoreceptors (P). MG processes (RCVRN-negative, pseudocolored in blue) are thickened upon HT treatment, indicating hypertrophy (N = 1). Filaments (arrowheads) were observed in MG processes of HT-HRO. N = 1 independent experiment (N) with n = 3 HROs. TJ tight junction, AJ adherens junction. Scale bars: b, c3 5 µm, c1, 4 1 µm, c2, 5 200 nm, d 10 µm, e1, 2 500 nm. Source data are provided as a Source Data file.

Back to article page