The architecture of chicken chromosome territories changes during differentiation

BACKGROUND: Between cell divisions the chromatin fiber of each chromosome is restricted to a subvolume of the interphase cell nucleus called chromosome territory. The internal organization of these chromosome territories is still largely unknown. RESULTS: We compared the large-scale chromatin structure of chromosome territories between several hematopoietic chicken cell types at various differentiation stages. Chromosome territories were labeled by fluorescence in situ hybridization in structurally preserved nuclei, recorded by confocal microscopy and evaluated visually and by quantitative image analysis. Chromosome territories in multipotent myeloid precursor cells appeared homogeneously stained and compact. The inactive lysozyme gene as well as the centromere of the lysozyme gene harboring chromosome located to the interior of the chromosome territory. In further differentiated cell types such as myeloblasts, macrophages and erythroblasts chromosome territories appeared increasingly diffuse, disaggregating to separable substructures. The lysozyme gene, which is gradually activated during the differentiation to activated macrophages, as well as the centromere were relocated increasingly to more external positions. CONCLUSIONS: Our results reveal a cell type specific constitution of chromosome territories. The data suggest that a repositioning of chromosomal loci during differentiation may be a consequence of general changes in chromosome territory morphology, not necessarily related to transcriptional changes

Rabl's model of the interphase chromosome arrangement tested in Chinise hamster cells by premature chromosome condensation and laser-UV-microbeam experiments

In 1885 Carl Rabl published his theory on the internal structure of the interphase nucleus. We have tested two predictions of this theory in fibroblasts grown in vitro from a female Chinese hamster, namely (1) the Rabl-orientation of interphase chromosomes and (2) the stability of the chromosome arrangement established in telophase throughout the subsequent interphase. Tests were carried out by premature chromosome condensation (PCC) and laser-UV-microirradiation of the interphase nucleus. Rabl-orientation of chromosomes was observed in G1 PCCs and G2 PCCs. The cell nucleus was microirradiated in G1 at one or two sites and pulse-labelled with 3H-thymidine for 2h. Cells were processed for autoradiography either immediately thereafter or after an additional growth period of 10 to 60h. Autoradiographs show unscheduled DNA synthesis (UDS) in the microirradiated nuclear part(s). The distribution of labelled chromatin was evaluated in autoradiographs from 1035 cells after microirradiation of a single nuclear site and from 253 cells after microirradiation of two sites. After 30 to 60h postincubation the labelled regions still appeared coherent although the average size of the labelled nuclear area fr increased from 14.2% (0h) to 26.5% (60h). The relative distance dr, i.e. the distance between two microirradiated sites divided by the diameter of the whole nucleus, showed a slight decrease with increasing incubation time. Nine metaphase figures were evaluated for UDS-label after microirradiation of the nuclear edge in G1. An average of 4.3 chromosomes per cell were labelled. Several chromosomes showed joint labelling of both distal chromosome arms including the telomeres, while the centromeric region was free from label. This label pattern is interpreted as the result of a V-shaped orientation of these particular chromosomes in the interphase nucleus with their telomeric regions close to each other at the nuclear edge. Our data support the tested predictions of the Rabl-model. Small time-dependent changes of the nuclear space occupied by single chromosomes and of their relative positions in the interphase nucleus seem possible, while the territorial organization of interphase chromosomes and their arrangement in general is maintained during interphase. The present limitations of the methods used for this study are discussed

Positional changes of pericentromeric heterochromatin and nucleoli in postmitotic Purkinje cells during murine cerebellum development

Previous studies revealed changes of pericentromeric heterochromatin arrangements in postmitotic Purkinje cells (PCs) during postnatal development in the mouse cerebellum (Manuelidis, 1985; Martou and De Boni, 2000). Here, we performed vibratome sections of mouse cerebellum (vermis) at P0 (day of birth), at various stages of the postnatal development (P2-P21), as well as in very young (P28) and 17-months-old adults. FISH was carried out on these sections with major mouse satellite DNA in combination with immunostaining of the nucleolar protein B23 (nucleophosmin). Laser confocal microscopy, 3D reconstructions and quantitative image analysis were employed to describe changes in the number and topology of chromocenters and nucleoli. At all stages of postnatal PC development heterochromatin clusters were typically associated either with nucleoli or with the nuclear periphery, while non-associated clusters were rare (<1% at P0 to P21 and about 3% in adult stages). At P0, about 2-4 nucleoli and 7-8 pericentromeric heterochromatin clusters were variably located within PC nuclei. The relative volume of heterochromatin clusters associated with the nucleoli (about 50%) was roughly equal to the volume of clusters associated with the nuclear periphery. Positional changes of both nucleoli and centromeres towards the nuclear center occurred between P0 and P6. At P6 the average number of chromocenters per PC nucleus had decreased to about five. In agreement with previous studies, one or occasionally two nucleoli were noted at the nuclear center surrounded by major perinucleolar heterochromatin clusters. The relative volume of these perinucleolar clusters increased to about 84%, while the volume of clusters in the nuclear periphery decreased to about 15%. At subsequent postnatal stages, the arrangement of most pericentromeric heterochromatin around a central nucleolus was maintained. In adult animals, however, we observed a partial redistribution of heterochromatin towards the nuclear periphery. The average total number of pericentromeric heterochromatin signals increased again to about ten. The volume of heterochromatin associated with the nuclear periphery roughly doubled (30%), while the volume of the perinucleolar heterochromatin decreased correspondingly. Copyright (C) 2004 S. Karger AG, Basel

Towards many colors in FISH on 3D-preserved interphase nuclei

The article reviews the existing methods of multicolor FISH on nuclear targets, first of all, interphase chromosomes. FISH proper and image acquisition are considered as two related components of a single process. We discuss (1) M-FISH (combinatorial labeling + deconvolution + widefield microscopy); (2) multicolor labeling + SIM (structured illumination microscopy); (3) the standard approach to multicolor FISH + CLSM (confocal laser scanning microscopy; one fluorochrome - one color channel); (4) combinatorial labeling + CLSM; (5) non-combinatorial labeling + CLSM + linear unmixing. Two related issues, deconvolution of images acquired with CLSM and correction of data for chromatic Z-shift, are also discussed. All methods are illustrated with practical examples. Finally, several rules of thumb helping to choose an optimal labeling + microscopy combination for the planned experiment are suggested. Copyright (c) 2006 S. Karger AG, Basel