Cell proliferation in the gastrulating chick embryo: a study using BrdU incorporation and PCNA localization

Abstract

Cell proliferation in the gastrulating chick embryo was assessed using two independent techniques which mark cells in S phase of the mitotic cycle: nuclear incorporation of bromodeoxyuridine (BrdU) detected immunocytochemically and immunolocalization of proliferating cell nuclear antigen (PCNA). Computer-reconstructed maps were produced showing the distribution of labelled nuclei in the primitive streak and the cell layers. These distributions were also normalized to take into account regional differences in cell density across the embryo. Results from a 2 hour pulse of BrdU indicated that although cells at caudal levels of the primitive streak showed the highest incorporation, this region showed a similar proportion of labelled cells to the surrounding caudal regions of the epiblast and mesoderm when normalized for cell density. The entire caudal third of the embryo showed the highest proportion of cells in S phase. Cells of Hensen's node showed a relatively low rate of incorporation and, although the chordamesoderm cells showed many labelled nuclei, this appeared to be a reflection of a high cell density in this region. Combining this result with results from a 4 hour pulse of BrdU permitted mapping of cell generation time across the entire embryo. Generation times ranged from a low value of approximately 2 hours at caudal levels of both the epiblast and mesoderm, to an upper value of approximately 10 hours in the rostral regions of the primitive streak, in the mid-lateral levels of the epiblast and in the chordamesoderm rostral to Hensen's node. Cells at caudal regions of the primitive streak showed a generation time of approximately 5 hours. Taking into account that cells are generally considered to be continuously moving through the primitive streak, we conclude that cell division, as judged by generation time, is greatly reduced during transit through this region, despite the presence there of cells in S phase and M phase. Immunocytochemical localization of PCNA-positive nuclei gave generally similar distributions to those obtained with BrdU incorporation, confirming that this endogenous molecule is a useful S-phase marker during early embryogenesis. Mid-levels and caudal levels of the primitive streak showed the highest numbers of positive nuclei, and the highest proportion of labelling after cell density was accounted for. As with BrdU incorporation, the highest proportions of PCNA-positive nuclei were found towards the caudal regions of the epiblast and mesoderm. These results suggest that the differential growth of the caudal region of the embryo at this time is a direct consequence of elevated levels of cell proliferation in this region.(ABSTRACT TRUNCATED AT 400 WORDS)