PMC

Translocation of Tα in rd17 retinas. AAV-expressed rod Tα and cone Tα was observed by immunostaining in both rods (arrows) and cones (arrow heads) in rd17 retinas after treatment with AAV5-CBA-rod Tα (A) or AAV5-CBA-cone Tα (B) vectors. Cones were labeled with PNA (green); rod or cone Tα was labeled red by rod or cone-specific antibodies. (A) Localization of vector-expressed rod Tα in cones paralleled that of endogenous cone Tα. In the dark, AAV-expressed rod Tα was observed only in the OS in both rods and cones. Under light conditions, rod Tα was observed in both rod OS and IS, whereas rod Tα remained only in cone OS. (B) Localization of vector-expressed cone Tα in rods was similar to that of endogenous rod Tα. In the dark, cone Tα was observed only in OS of both rods and cones. Under light conditions, exogenously expressed cone Tα was redistributed into rod IS, whereas the majority of cone Tα remained localized to cone OS. (Scale bar, 20 μm.)

a, Schematic illustration of the two-step reprogramming method to generate rod photoreceptors. b–j, Characterization of MG-derived rod differentiation through Initial (b–d), Intermediate (e–g), and Terminal (h–j) stages. Arrowheads: cell soma. Arrows: rod outer segments. Double arrows: synaptic terminals. Scale bar: 25 μm. Experiments were repeated 6 times independently with similar results. k–m, Quantification of MG-derived rod differentiation at 1 (k), 2 (l), and 4 weeks (m) after the second injection of ShH10-GFAP-mediated gene transfer of Otx2, Crx, and Nrl for rod induction. Data are presented as mean ± SEM, n = 7 retinas. n and o, Lineage analysis of MG-derived rod photoreceptors. (n) Untreated MG fate mapping mice with MGs labeled by tdTomato. (o) Treated MG fate mapping mice using the two-step reprogramming method. Arrowheads: rod soma. Arrows: rod outer segments. Scale bar: 20 μm. Experiments were repeated 4 times independently with similar results. p–t, Quantification of MG-derived rod photoreceptors in the Dorsal (p), Nasal (q), Temporal (r), and Ventral (s) quadrants of retinal flat-mount preparations at 4 weeks after the second injection for rod induction. Scale bar: 20 μm. Experiments were repeated 4 times independently with similar results. Data in (t) show mean ± SEM, n = 4 retinas. Control measurements were combined across quadrants.

Characterization of rod cell death. (A, B, D) One-μm optical sections of isolated rod cells fluorescently labeled with AO and EtdBr obtained by confocal microscopy. (C) Nomarski optics image of an isolated rod cell treated with high K⁺ Ringer's solution demonstrating rod cell morphology after loss of membrane integrity. (A) Live rod cells had green (AO), homogeneously stained chromatin. (B) Early apoptotic rod cells had green (AO), condensed chromatin. (C) Rod cells that had lost membrane integrity could be identified by their rectangular, elongated ellipsoid. *, ellipsoid; N, nucleus. (D) Late apoptotic cells with permeant membranes had red EtdBr, condensed chromatin. (Bar = 10 μm.) (E) Percent change in the number of apoptotic rod cells after treatment with light and 10 μM 9-cis-retinal. Photoactivation of reconstituted isorhodopsin increased apoptotic rod cell death by 49%. In n = 5 experiments from two animals, a total of 83 apoptotic rod cells was counted; *, P < 0.01, means ± SEM. (F) Percent change in the number of apoptotic rod cells after treatment with 2.5 μM β-ionone. β-ionone treatment increased apoptotic rod cell death by 46%. In n = 9 experiments from three animals, a total of 120 apoptotic rod cells was counted; *, P < 0.01, means ± SEM. (G) The effect of β-ionone on rod cell death was blocked by preincubation and cotreatment with 1 μM DEVD-fmk. In n = 11 experiments from four animals, a total of 4,417 rod cells was counted; *, P < 0.01 compared with control; **, P < 0.01 compared with β-ionone treated; unpaired t test, means ± SEM.

A-A'. Fluorescent and phase contrast images showing the absence of any TUNEL labeling (red) in a wild-type retina prior to heat-shock. B-B'. Fluorescent and phase contrast images showing the absence of TUNEL labeling (red) in a Tg(hsp70:dn-fgfr1) retina prior to heat-shock. C-C'. Fluorescent and phase contrast images showing the absence of TUNEL labeling (red) in a wild-type retina after 10 days of daily heat-shock. The white bar depicts the normal thickness of rod inner and outer segments. D-D'. Fluorescent and phase contrast images showing TUNEL-positive nuclei in the outer nuclear layer (red) of a Tg(hsp70:dn-fgfr1) retina after 10 days of daily heat-shock. The white bar depicts the normal thickness of rod inner and outer segments, showing rod outer segment degeneration in transgenic retinas. E. Rhodopsin immunolocalization to rod outer segments in a wild-type retina after 10 days of daily heat-shock. F. Rhodopsin immunolocalization to rod outer segments in a Tg(hsp70:dn-fgfr1) retina after 10 days of daily heat-shock, showing degenerated and disorganized outer segments. G. Graphic depiction of the average number of TUNEL-positive nuclei in wild-type and Tg(hsp70:dn-fgfr1) retinas after either 0 or 10 days of daily heat shock. ROS = rod outer segments; RIS = rod inner segments; ONL = outer nuclear layer; INL = inner nuclear layer; GCL = ganglion cell layer. Scale bar: Panel A = 50 μm (A-D'); Panel E = 50 μm (E-F).