Interphase nucleus organization: new concepts and techniques

Abstract

The studies of early cytologists demonstrated that metaphase chromosomes were not simply aggregations of dispersed interphase chromatin. Well before the turn of this century it had been observed that the positions of mitotic chromosomes correlated with their respective positions in preceding cell divisions. Since these early observations there has been a consistent interest in assessing the relative order of chromosomes in nuclei, especially in interphase, and elaborating on the importance of any observed order with reference to gene expression. A major problem in analysing interphase nuclei has been the difficulty in visualizing chromosomes at this stage in the cell cycle. In recent years significant progress has been made in the rapid analysis of nuclei at metaphase as well as interphase and this was the subject of the workshop. A. J. Hilliker presented the studies from his laboratory on the analysis of relative chromosome positions in cells, in Drosophila melanogaster, which are destined to differentiate into salivary gland cells. The assay measuring the relative positions of chromosomes included irradiating larvae at a stage when progenitor salivary gland cells have undergone their last cell division, to cause cross-linking between chromosome regions that are juxtaposed. The actual positions of the cross-links could be accurately measured cytologically by studying the polytene chromosomes of the fully differentiated salivary gland cells. These cytogenetic studies indicated that the only chromosomal regions that were cross-linked are those from'the same arm of a chromosome. Essentially no crosslinks were found between different chromosome arms leading Hilliker to argue that chromosomes were in clearly defined domains even in interphase when they are in an apparently diffuse stage. This conclusion was also clear from the studies presented by F. M. Waldman using in situ hybridization techniques on human B cell nuclei. Waldman, Pinkel, and Gray have studied the relative positions of specific human chromosomes using fluorescence to detect repetitive, chromosome-specific DNA probes followed by optical sectioning microscopy and computerized image analysis. In the B lymphocytes analysed, the positions of the repetitive sequences on chromosomes Y, 1 , 4 , and 9 were located nearer to the periphery of the nucleus than would be expected from random placement. Probe positions did not appear to alter in the transition from G1 to S phase. The technique of optical sectioning was discussed in more detail by M. Rykowski. The OM1 optical microscope system discussed by Rykowski was designed by John Sedat, David Agard, and colleagues at UCSF. The system combined the use of a cooled CCD (charge-couple device) camera with computational image analysis to yield high quality, three-dimensional (3-D) images of a variety of biological specimens using conventional fluorescence microscopy. The linearity and wide dynamic range of the CCD camera was demonstrated using an in situ hybridization analysis of Drosophila polytene chromosome band 3C7 (the location of the Notch gene) to allow the observation of very fine cytological features. The use of conventional UV light rather than laser light allowed maximum flexibility in choice of fluorochromes with minimal photobleaching. It was evident that the parallel development of computer programmes for image enhancement, analysis, and interpretation is critical to the continued study of the 3-D arrangements of chromosomes in nuclei. J. L. Oud presented studies using the confocal scanning laser microscope, a system that is quite different from the one described by Rykowski. Oud and his colleagues did not use in situ hybridization techniques to identify chromosomes since the cells of interest to them were anaphase cells from Crepis capillaris. In these cells the three chromosome pairs could be readily identified. To estimate the extent to which chromosomes are ordered, 76 Feulgen-stained root-tip anaphases were analysed and the observed chromosome order was compared with that expected from a random ordering model. The work demonstrated a significant surplus of one of the arrangements with a juxtaposition of the two chromosomes with a nucleolar organizer region. Oud suggested that the nucleolus was an important factor in determining the final 3-D arrangement of chromosomes in nuclei. In conclusion, the workshop provided participants with a unique opportunity to see the major techniques available for analysing 3-D arrangements of chromatin-chromosomes in nuclei. It was clear that chromosome domains exist and that the relative arrangement of these domains is dynamic. Observations such as those from Crepis on the preferred juxtaposition of Nor-chromosomes left open the possibility that in specific tissues certain chromosomes may be located in specific positions. Such specific positioning may be limited to defined stages in the cell cycle.