The nuclear positions of short chromosome segments of different gene densities belonging to either the A or B compartment were studied using FISH with a cocktail of BAC probes on retinal cryosections at six developmental stages: P0, P6, P13, P21, P28 and adult (AD; 3.5 months). For the analysis of BAC signal distribution, three stages were considered: P0, with conventional nuclei of rod progenitors; P13, with rod nuclei in a transient state of inversion; and adult, with fully inverted rod nuclei. Cells with conventional nuclear organization in the inner nuclear layer (INL) of adult retina were used as a control. Between 100 and 120 alleles per chromosomal region were analysed. a, Immuno-FISH experiment showing how FISH signals were classified according to their localization in the three major nuclear zones. EC, euchromatin; HC, heterochromatin; cHC, constitutive heterochromatin. Definitions of these three types of chromatin have been previously published1. BAC 12 maps to the most peripheral euchromatic shell of the rod nucleus stained with anti-H3K4me3 antibody. This nuclear zone is adjacent to the nuclear periphery and contains the genic part of the mouse genome (see Supplementary Fig. 2). BACs 2 and 11 are located in the heterochromatic zone of the nucleus encircling the chromocentre and stained with anti-H4K20me3 antibody. Thus, classification of BAC signals based on DAPI staining is justified by immunostaining of histone modifications and enables the signal distribution analysis described in b–d. Top, localization of BAC signals (blue, white arrows) and histone modifications (green) in DAPI-counterstained nuclei (red). Numbers in the lower left corners indicate the BAC numbers (for their coordinates, see Methods). Bottom, greyscale images of DAPI and positions of the BAC signals (red arrows) represented by false-coloured mask. b–d, Analysis of BAC signal positions after FISH with BAC cocktail probes mapping to selected chromosome regions. Top, schematics of the chromosome regions on MMU1 (b), MMU2 (c) and MMU6 (d). The coloured segments differ in their gene content and assignment to either the A or B compartment. The striped boxes with numbers below indicate the BACs used for FISH. Bottom left, graphs showing the distribution of the segments within rod nuclei at the three developmental stages and adult cells of the inner nuclear layer. The bars represent the proportion of signals in each nuclear zone: adjacent to constitutive heterochromatin (dark grey), within heterochromatin (light grey) and within euchromatin (white). Bottom middle, schematics showing typical segment distribution of the studied regions. Bottom right, representative nuclei after three-colour (b) or four-colour (c, d) FISH. The images are maximum-intensity projections of short (1.4–2-μm) stacks. False colours assigned to segments correspond to the colour code used in each panel. The experiment was repeated twice. For an example of the localization of a single gene and its movement together with chromatin of the A compartment during nuclear inversion, see Supplementary Fig. 3.