Cell death markers double labeled with 5mC (+) or 5hmC (+) photoreceptors in <i>rd1</i> mouse retinas.

<p>Retinas from P14 <i>rd1</i> mice were double-labeled with TUNEL stain (A, D and G) and 5mC (B and E) or 5hmC (H) with central (A–C, G–I) and peripheral retina (D–F) shown accordingly. Discrete signals for cCaspase3 (J) and 5mC (K) labeling show virtually no overlap in signals (L). Overlap in signals are indicated by white arrows as shown in panels C, F and I. Cells labeled with one marker but not the other are indicated with dark vertical arrows. ONL = outer nuclear layer; INL = inner nuclear layer; GCL = ganglion cell layer. Scale bars = 50 µM.</p

Other retinal degeneration models with elevated 5mC levels.

<p>(A) P24 rhoGFP homozygous mice with sporadic PR loss with 5mC staining (magenta) in the ONL (green = GFP in rods). (B–C) Subcellular localization of 5mC (magenta; B) enveloped within a photoreceptor cell body expressing GFP (green; C). (D–F) 5mC staining of a wild type one month-old mouse retina explant undergoing progressive photoreceptor degeneration after 4, 7 and 9 days in vitro (DIV), respectively. ONL = outer nuclear layer; INL = inner nuclear layer; GCL = ganglion cell layer. Scale bars = 20 µM (A) and 100 µM (F).</p

Comparison of cCaspase3 and TUNEL co-labeling with 5mC in the developing chicken retina.

<p>(A) Low magnification of 5mC and cCaspase3 staining at E11. (B) Nuclear counterstaining of a low magnification section. (C) 5mC and (D) cCaspase3 staining. Colocalization of 5mC (+) and cCaspase3 (+) cells indicated by white arrows overlaid in (E). Dark arrows indicate 5mC (+) cells that are cCaspase3 (−). (F) 5mC and (G) TUNEL staining at E11. (H) Co-localization indicated by white arrows. TUNEL (+) but 5mC (−) indicated by a dark arrow. ONL = outer nuclear layer; INL = inner nuclear layer; IPL = inner plexiform layer; GCL = ganglion cell layer. Scale bars = 100 µM in A–B; 75 µM in C–H.</p

5mC expression in the ONL of the <i>rd1</i> mouse retinal degeneration model.

<p>(A1, B1) 5mC labeling and (A2, B2) Hoechst labeling of the central and peripheral retina, respectively, of an <i>rd1</i> mouse at P14 during the peak of rod degeneration. (C) Age-matched retinas from wild-type control and (D) <i>rd1</i> mice, respectively, at P14 with Hoechst (blue) and 5mC (green) staining (arrows). (E) 5mC (green) and cone marker PNAL (magenta) staining co-localize (arrows) in a subset of cones in the remnants of the ONL. ONL = outer nuclear layer; INL = inner nuclear layer. Scale bars = 100 µM (B2) and 35 µM (C).</p

Detection of 5hmC during photoreceptor degeneration in the <i>rd1</i> mouse.

<p>(A–E) <i>Rd1</i> or (F–J) wild-type mouse retinas, P9 through P13, were labeled with antibodies against 5hmC (A–J). White arrows indicate 5hmC (+) cells in the ONL, dark arrows indicate occasional (+) cells in the INL. (K) Quantification of percent positive cells in the outer or inner retina for wild-type (+/+) and <i>rd1</i> are plotted as a function of days of age. (L) 5hmC (+) cells in the ONL with decreased staining for intercalating DNA dyes including Hoechst. The inset is the same image taken at a significantly higher gain. Black squares = +/+ ONL, black triangles = +/+ inner retina, orange circle = <i>rd1</i> ONL, orange triangle = <i>rd1</i> inner retina. Values are mean ± sem. ns = not significant, ****p<0.0001 versus wild type. ONL = outer nuclear layer; INL = inner nuclear layer; GCL = ganglion cell layer. Scale bar (A) = 25 µM.</p

5mC accumulation in the early developing chicken embryo.

<p>(A1) A diagram indicating the relative cross sectioned region of tissues studied (lowercase letters correspond to the respective panels below). (A2) A representative embryo at E3.5. (B) 5mC deposits in neurons lining the midbrain optic tectum and (C) a region corresponding to the developing heart. (D) The region adjacent to the neural tube (nt), notochord (nc) and aorta (a). (E1 & F1) Hoechst nuclear counterstained retinal sections correspond to sections in E2 and F2, respectively. (E2) 5mC in the developing retina, lens and surrounding mesenchyme. (F2) 5hmC in the developing retina. Arrows indicate 5mC (+) and 5hmC (+) cells. Scale bars = 100 µM in D, 75 µM in E2 and 50 µM in F2.</p

5mC (+) cells in the developing chicken retina.

<p>(A) The morphogenic furrow at E4 with a dense patch of 5mC staining (see arrows) (B) A magnified image of 5mC staining in the morphogenic furrow in a serial section taken at higher magnification. (C) 5mC (+) cells at E7, (D) E11, (E) E13 and (F) E20 are indicated by arrows when present. RPE = retinal pigment epithelium; NE = neuroepithelium; ONL = outer nuclear layer; INL = inner nuclear layer; GCL = ganglion cell layer. Scale bars = 75 µM in A and 200 µM in C–F.</p

Detection of 5mC and PAR in the outer nuclear layer of degenerating <i>rd1</i> retinas.

<p>(A–C) P14 <i>rd1</i> or (D–F) wild type mouse retinas were labeled with antibodies against 5mC and PAR. Panels A and D are co-labeled with Hoechst nuclear counterstain to indicate the respective retinal layer where 5mC exists. White arrows (B–C, E–F) indicate cells in which both antigens were detected. Dark arrows indicate cells that were labeled with 5mC but not PAR. RPE = retinal pigment epithelium, ONL = outer nuclear layer; OPL = outer plexiform layer, INL = inner nuclear layer; GCL = ganglion cell. Scale bar (L) = 50 µM.</p

Information on primary antibodies used in this study (See also Materials and Methods).

<p>M = mouse monoclonal; R = rabbit polyclonal; S = sheep polyclonal.</p