Figure 1. Light scattering by retinal nuclei reduces with chromocenter number during development.

(A) Longitudinal section showing the path of light through the mouse retina, including the rod nuclei dominated outer nuclear layer (ONL). Ganglion cell layer (GCL), inner nuclear layer (INL) and outer nuclear layer (ONL) and the inner and outer segments (IS and OS). (B1) (top) Downregulation of the lamina tether LBR (yellow) enables fusion of mobilized chromocenters and thereby an architectural inversion of mouse rod nuclei. (bottom) FISH images of rod nuclei stained with DAPI (blue) showing the dense chromocenters, LINE rich heterochromatin (H4K20me3, magenta) and SINE rich euchromatin (H3K4me3, green) (B2) DAPI section of WT mouse retina in comparison to a Rd1/Cpfl1-KO mouse retina showing the presence of only the inner retina. (B3) Quantification of image transmission shows that the inner retina alone (Rd1/Cpfl1-KO, N = 5) transmits approximately 50% more image detail than the full retina (N = 11), suggesting significant image degradation in the thick outer nuclear layer. (C) FACS scattering profiles comparing retinal neurons, cortical neurons and N2a neuroblastoma cells showing lower light scattering properties of retina neurons. (Inset) Volume-specific light scattering is significantly reduced in the retinal cell nuclei. (D, E) FACS scatter plot for isolated retinal nuclei from WT developmental stage week three pup (P25) and adult mice demonstrating stronger large angle scattering by the P25 nuclei. (F) Histogram of side scattering in adult and P25 retina depicting a higher side scattering for the developing retinal nuclei. (G) Sorting of developmentally maturing nuclei according to different side scattering signal. Insets show representative examples of Hoechst stained nuclei in the corresponding sort fractions. The rectangles represent sorting gates for microscopy analysis. (H) Quantification of reduced scattering with chromocenter number is sufficiently explained by a wave optical model of light scattering n = 38 nuclei. (Error bars in (H) show s.d.) Scale bars (A) - 10 μm. (B1), G - 5 μm, (B2) – 50 µm.

Figure 1—figure supplement 1. Heterochromatin in mouse rod nuclei exhibits unusual dense packing.

Retinal cells of transgenic mice expressing GFP (green) under rod-specific Nrl promoter (A; Akimoto et al., 2006) and under control of the ROSA26 promoter (B; Ivanova et al., 2005). In inverted rod nuclei, the chromatin of the central chromocenter (arrows) and the surrounding shell of LINE-rich heterochromatin (arrowheads) is packed so densely that free molecules of GFP do not penetrate into these nuclear regions. In contrast, loosely packed euchromatin in the peripheral nuclear shell (empty arrowheads) allow GFP penetration. In conventional nuclei, exemplified by ganglion and bipolar cells, the entire nucleoplasm, regardless to chromatin nature, is penetrable for GFP with chromocenters showing slightly less permeability (arrows). Nuclei are counterstained with DAPI (magenta). Single confocal sections.

Figure 1—figure supplement 2. Reorganization of rod nuclear architecture in the course of postnatal retinal development (A) and in transgenic rods expressing LBR (B, C).

(A) Difference in nuclear architecture of terminally differentiated rods (adult,) and photoreceptor progenitors (P6) is highlighted by GFP (green) expressed under Nrl promoter and freely distributed through nucleoplasm and cytoplasm. During first 4–6 weeks of postnatal development, conventional nuclear architecture of rod progenitors (arrow), characterized by multiple chromocenters adjacent to the nuclear periphery, is gradually rearranged into inverted one of fully mature rods (arrow) with a single central chromocenter surrounded by LINE-rich heterochromatin. (B, C) Rod nuclei ectopically expressing LBR (green) in adult TG-LBR retina have conventional nuclear organization with chromocenters adjacent to the nuclear lamina (B) and euchromatin occupying the nuclear interior (C). Nuclear lamina is stained with antibodies to lamin B (B) and euchromatin is highlighted by H4K5ac staining (C). Note that only proportion of rods in TG-LBR retina express LBR and thus maintain conventional nuclei (arrows). Nuclei of rods not expressing LBR are lacking peripheral tethers of heterochromatin and ultimately undergo inversion (arrowheads). Nuclei are counterstained with DAPI (magenta). Single confocal sections.