Three-dimensional analysis of DNA replication foci: a comparative study on species and cell type in situ

Abstract 

Chromatin morphology of interphase nuclei in most cell lines of quail (Coturnix coturnix japonica) and chick (Gallus gallus domesticus) embryos shows typical interspecies differences. This intrinsic marker has been used in quail/chick chimerisation experiments, where also differences between cell types were noted. We asked whether similar differences between species and between cell types could be observed in S phase nuclei in situ. In this report, we used bromodeoxyuridine (BrdU) pulse labelling and anti-BrdU immunofluorescence to detect DNA replication foci in the nuclei of identified cells. In the central nervous system of 5- to 7-day-old quail and chick embryos, mesoderm-derived cells with strikingly different morphology and topographical distribution were studied: endothelial, i.e. polarised cells forming continuous tubes, and macrophages, i.e. non-polarised, ameboid or ramified individual cells. Using confocal microscopy, replication foci in the nuclei were assessed quantitatively and three-dimensional visualisations were produced. We consistently observed that: (1) chick, but never quail, nuclei displayed completely confluent replication sites, independent of cell type, and (2) macrophages, but not endothelial cells, had distinct perinucleolar replication sites, independent of species. We thus demonstrate a new relationship between cell type and spatial arrangement of DNA replication sites, and conclude that interspecies differences of chromatin distribution are conserved throughout S phase. Our results strongly recommend that work done on nuclear structure in vitro should not be extrapolated without reservation to cells in vivo.