Figure 2

FISH with a chromosome 1 paint probe (red) and the lysozyme gene domain probe (green/yellow). (a) FISH on metaphase chromosomes. The chicken lysozyme gene domain is located on the short arm of chromosome 1. Note that the library probe mix used gives particularly strong signals at the centromeres (arrows). (b-g) 3D-FISH on structurally preserved nuclei. For each cell type, single confocal sections of one nucleus are shown. In b-f, nuclear outlines were drawn after the DNA counterstain which was omitted from the figure to avoid obstruction of the territory signals. In addition to the cLys domain signals, centromeres are in focus in some of the sections (arrows). (b) Multipotent myeloid precursor cell. (c) Myeloblast. (d) Macrophage without LPS-activation. (e) Macrophage with LPS-activation. On the right hand side, a threshold of 80 was applied to the territory signal of the central image to visualize disaggregation into several objects. While usually only few objects are present in any given focal plane, in this particular example the breakup is well recognizable. The algorithm applied in the calculations works on 3D-stacks, however. The macrophage cell line is aneuploid (see Methods), the cells shown in d and e have three territories with chromosome 1 material, each containing a cLys signal. (f) Erythroblast. (g) An additional section of the erythroblast shown in f visualizes a cluster of chromosome 1 material (red in left image) in a central nuclear area (arrow) with low DNA-counterstain but associated with a brightly stained region (see main text). Such clusters were less pronounced when only paint probes from early DOP-PCR-amplification rounds were used (see Methods for details). DNA counterstain is blue in left, gray in right image. Scalebar 5 μm for b-g. Whereas in precursor cells the lysozyme gene domain signal was found nearly always inside the territory, in differentiated cells more external positions were frequent. Note that the multipotent myeloid precursor cell has a relatively small nucleus and nuclear volume is increased in the further differentiated cell types.