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. 2019 Dec 11;8:e49542. doi: 10.7554/eLife.49542

Figure 2. Developmental arrest of chromocenter fusion increases light scattering from rod nuclei in measurements and tissue simulations.

(A1) Schematic of the normal rod nuclear WT development and inversion arrested nuclei by LBR overexpression. (A2) EM images illustrating different electron densities in the euchromatic and heterochromatic phase underlying their refractive index (RI) differences (scalebar 5 μm). (mid-top) Immunostaining of overexpressed of LBR tethers (yellow), and high-density heterochromatin (DAPI, magenta). (mid- bottom) Heterochromatic chromocenters (DAPI, magenta) and euchromatin (H4K5ac, green) (B) Chromocenter number distribution in LBR overexpressing rod nuclei is drastically different from WT mice, and similar to a developing WT pup (P14). (C) Side scattering assessed by FACS for TG-LBR retina nuclei is higher than that of WT nuclei and comparable to that of a WT P14 nuclei with similar chromocenter numbers. Note the shift of peak value upon LBR overexpression. (D1) 3d RI distribution mapped onto anatomically faithful volumetric ONL images. WT inverted architecture (right, top) and early developmental state (left) (simulation). (D2) (top) Differential simulations of light propagation in the ONL, using same positions and shapes of about 1750 nuclei, but varying chromatin distributions. (bottom) Maximum projection illustrating greater proportions of scattered light (angles > 30 deg) in the ONL with multiple chromo-centered nuclei. (E) Quantitative analysis of this data. (F) Angle weighted volume-specific scattering strength for nuclei models evaluated by Mie scattering theory. (G) Excess scattering occurring in multi-chromocenter nuclei models. (H) Chromocenters scattering reconstituted in an emulsion of silica spheres in glycerol-water mixture. Pictograms reflect accurate number ratio of spheres.

Figure 2.

Figure 2—figure supplement 1. Distribution of chromocenters in nuclei of adult rods transgenitically expressing LBR in comparison to nuclei of P14 WT rods.

Figure 2—figure supplement 1.

Exemplified areas of ONL in adult TG-LBR (upper raw) and P14 WT (bottom raw) retinas. Note that only a proportion of rods in transgenic retina express LBR (see also Figure 1—figure supplement 2). Nuclei of rods expressing LBR exhibit conventional chromatin arrangement with chromocenters adjacent to the nuclear envelope (arrows); rod nuclei not expressing LBR remain inverted with one central chromocenter (arrowheads). P14 WT rod nuclei have still conventional nuclei, although exhibit signs of ongoing inversion with massive chromocenter fusion. Immunostaining for lamin B is used to outline nuclear border (green, left panel); immunostaining for LBR is used to highlight transgenic rods (green, right panel); nuclei are counterstained with DAPI (magenta). Single confocal sections.
Figure 2—figure supplement 2. Transgenic expression of LBR does not influence rod photoreceptor structure.

Figure 2—figure supplement 2.

Retina areas with LBR-positive and LBR-negative (asterisks) rod clones demonstrating unaltered stratification. The three photoreceptor layers, represented by photoreceptor perikarya (ONL), cytoplasm of inner segments (IS) and dendritic ends of outer segment (OS), are preserved in TG-LBR retina. Immunostaining of LBR (green); nuclear counterstain with DAPI (blue). Maximum intensity projections of 5–7 µm confocal stacks.