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.

DNA replication and nuclear architecture

“…A great variability of size (0.2± 0.4 mm) and number (500±1,500) of foci was described for early S-phase [Tomilin et al, 1995]. As an example, foci observed in embryonic quail cells were larger than in embryonic chick cells and also less con¯uent [Cossmann et al, 2000]. Moreover, foci in primary ®bro-blasts are limited in number at the onset of Sphase compared to immortalized cells [Kennedy et al, 2000].…”

“…At the level of¯uorescence microscopy resolution, the term of replication foci is de®ned as solid bodies or dots resulting from the detection of the labeled nucleotides incorporated into newly synthesized DNA [Cossmann et al, 2000]. Such foci are often considered as replication sites resulting from the clustering of small replicons [Berezney et al, 2000].…”

Pericytes in the mature chorioallantoic membrane capillary plexus contain desmin and α-smooth muscle actin: relevance for non-sprouting angiogenesis

“…However, it may become possible to study identiWed, unsynchronized cells in situ during their S-phase due to their chromatin patterns, as we have done before using the BrdU technique (Cossmann et al 2000). Thus, after suYcient training by a human expert of a selflearning classiWcation algorithm (using the hints provided by additional markers), the goal of identifying several cell types with only one DNA stain appears to be a realistic one.…”

Neuroectodermal origin of brain pericytes and vascular smooth muscle cells

“…3, or QH1 and ␣SMA, not shown) was achieved by a sequential staining protocol as described before for QH1 and bromodeoxyuridine (Cossmann et al, 2000). In view of the excellent reproducibility of immunofluorescence patterns, we are confident that the absence of graft mesoderm-derived, QCPN-positive, QH1-negative cells in the host brain reliably indicates the inability of ECs to transdifferentiate into vSMCs in the CNS.…”

“…Figures were assembled with Adobe Photoshop for publication. Additional methodological details on immunofluorescence and imaging have been previously published (Kurz et al, 1996Cossmann et al, 1997Cossmann et al, , 2000.…”