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First published online 16 August 2005
doi: 10.1242/jcs.02500

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Distinctive nuclear organisation of centromeres and regions involved in pluripotency in human embryonic stem cells

¹ MRC Human Genetics Unit, Crewe Road, Edinburgh, EH4 2XU, UK
² Roslin Institute, Roslin BioCentre, Midlothian, EH25 9PS, UK

View larger version (36K): [in a new window]   Fig. 1. The radial distribution of HSA18 and 19 in hES cells. (A) hES cell nuclei, counterstained with DAPI (blue) and hybridised with chromosome paints for HSA18 or 19. (B) Distribution of HSA18 and 19 hybridisation signals within the nucleus of H1 ES cells analysed by erosion of 2D images into five concentric shells from the edge (1) to the centre (5) of the nucleus. The mean (±s.e.m.) proportion of hybridisation signal, normalised to the amount of DAPI signal, is shown for each shell (n=50). (C) Analysis of HSA18 and 19 hybridisation signals within 3D-preserved hES cell nuclei. Graphs are the distributions of the centres of the HSA18 and 19 territories, along the fractional radius of each nucleus, along the x, y and z-axes (n=20). Bar, 5 µm.

View larger version (37K): [in a new window]   Fig. 2. Radial distribution of 6p, 12p, OCT4 and NANOG in ES cells. (A) Interphase hybridisation of BAC probes containing OCT4 or NANOG (red), and chromosome paints for either 6p or 12p (green), within the nuclei of human ES cells counterstained with DAPI (blue). (B) Distribution of HSA6p and 12p hybridisation signals within the nucleus of ES cells, by erosion of 2D images into 5 concentric shells from the edge (1) to the centre (5) of the nucleus. The mean (±s.e.m.) proportion of hybridisation signal, normalised to the amount of DAPI signal, is shown for each shell (n=50). C) Distribution of hybridisation signals from OCT4 or NANOG-containing BACs within the nucleus of ES cells, by erosion of 2D images into five concentric shells from the edge (1) to the centre (5) of the nucleus. The proportion of hybridisation signals, normalised to the amount of DAPI signal, is shown for each shell (n=50). Bar, 5 µm.

View larger version (21K): [in a new window]   Fig. 3. Intrachromosome territory organisation of NANOG and OCT4. (A) Position (mean±s.e.m.) in µm, relative to the inside, edge or outside CTs, for loci including NANOG and OCT4, as well as loci flanking OCT4, in the nuclei of hES cells () and LCLs () (n=100). Negative values indicate localisation outside the CT. The map of the genomic regions around OCT4 and NANOG (according to NCBI build 35) is shown below. Genes present in the BACs used are highlighted in bold. (B) Histogram of the distribution of FISH signals from a BAC containing OCT4, relative to the edge of the chromosome 6p CT, in nuclei from hES cells (open bars) and LCLs (filled bars). Negative distance indicates localisation outside the visible limits of the CT (n=100).

View larger version (36K): [in a new window]   Fig. 4. Centromere and telomere localisation in hES cells. (A) Localisation of centromeres (CREST, red), and nucleoli (Ki67, green) in single image frames, taken at 0.75 µm intervals, through the z-axis of hES cell nuclei counterstained with DAPI (blue). Note the absence of centromeres from the nuclear periphery. (B) Mean (±s.e.m.) proportion of centromeres per cell that are associated with the nuclear periphery (left), the nucleolus (middle), or neither of these nuclear compartments (right), in H1 ES cells (open bars), LCLs (filled bars) and fibroblasts (hatched bars) (n=20). The mean (±s.e.m.) distribution of (C) centromeres and (D) telomeres through the z-plane from the top (0) to the bottom (1) of nuclei from ES, LCL and fibroblast cells (n=20). Bar, 5 µm.

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