Fig. 3: HBEGF-TNF treatment induces photoreceptor cell extrusion and photoreceptor inner segment (PIS) defects. | Nature Communications

Fig. 3: HBEGF-TNF treatment induces photoreceptor cell extrusion and photoreceptor inner segment (PIS) defects.

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

Fig. 3

a Live-imaging of HROs. b Differential interference contrast. c Spinning disk confocal microscopy. b, c HT-HROs and controls (CTRL) live in culture (Supplementary Data 3 and Movies 2–4). SiR-DNA cell nuclei live-dye labeling (acutely applied for imaging). b En-face imaging: ectopic nuclei appeared after 5 days in HT-HRO on its surface, but not in controls (optical plane positioned at the PIS level). Independent experiments: n = 10 HROs (Supplementary Data 3). c Cross-sectional image series from Supplementary Movie 3: cell nuclei move out of the apical organoid boundary onto its surface (yellow line). Cell nuclei in the outer retina appear round in controls and before extrusion, and transiently became hourglass-like shaped during extrusion. Independent experiments of n = 8 HROs (Supplementary Data 3). d HT-HROs compared to CTRL analyzed by scanning (SEM) electron microscopy (EM) of (1−2, 1’−2’), transmission EM (TEM) of epoxy-embedded HROs (3, 5, 3’, 5’−6’), and correlative-light electron microscopy (CLEM) of ultrathin cryosections (4, 4’): RCVRN (photoreceptors, P), SLC1A3 (Müller glia, MG), and DAPI (extruded (*) cell nuclei) staining, visualized by immunofluorescence and gold particles (black dots, 10 nm gold). N = 1 independent experiment (N) with n = 3 (TEM) and n = 5 (SEM) HROs (n) analyzed. e1–5 TEM image series: photoreceptors (P, green pseudocolor) during or after extrusion (10-days HT). Cell junctions (OLM): red labeled. Photoreceptor nuclei: asterisks. Double asterisk: a dead nucleus (e5). e6–9 CLEM of HT-HROs (e6, e7: 10-days HT; e8, e9: 20 days HT) immunolabeled for RCVRN (e6, 7) or ARR3 (e8, 9) and DAPI confirms that photoreceptors become extruded. e6 Extruded cells (asterisks) and e7 a photoreceptor in the process of being squeezed through the OLM, indicated by hourglass-shaped nucleus and position of cell junctions (white dashed line (e6), red pseudocolor (e7)), are visible. Boxed area: higher magnification in (e7). e8, 9 PIS (green) of a cone (ARR3 labeled) in HT-HRO. e TEM and CLEM: each N = 1, n = 3 HROs/N. Mitochondria (M). Outer (ONL)/inner (INL) nuclear layer. Scale bars: b 25 µm, c, e4, e6 10 µm, d1, 1’ 50 µm, d2–4, 2’−4’; ROI of d2, 2’; e1–3, e5 5 µm, d5, 5’−6’; e5, 7, 8 2 µm, ROI of d4’ 500 nm, e9 200 nm.

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