GFP-PCNA as an S-phase marker in embryos during the first and subsequent cell cycles

Abstract

Proliferating cell nuclear antigen (PCNA) is a key component of the DNA replication machinery involved in the process of DNA elongation, recombination, methylation and repair. We have used PCNA fused with green fluorescent protein (GFP-PCNA) as a convenient tool to show the progress of S-phase in single embryos in vivo. Here we make a comparison between Hoechst 33342 and GFP-PCNA as in vivo event markers for DNA synthesis. Hoechst 33342 and DAPI (4,6-diamidino-2-phenylindole) have been used as a simple and rapid method for assessing membrane permeability and staining DNA in mammalian cells. However, it is difficult to use these dyes in living embryos during cell cycle progression studies over long periods of time as they are phototoxic. Moreover, though Hoechst staining reveals nuclear morphology, it gives no information about the progress of S-phase. We have microinjected or expressed a GFP-PCNA chimera to develop a method which enables visualization of S-phase in sea urchin and Caenorhabditis elegans embryos during the first and subsequent embryonic cell cycles and in Drosophila stage 4 embryos during syncytial nuclear divisions. We find that nuclear accumulation of GFP-PCNA correlates with S-phase onset. Loss of the chimera from the nucleus occurs when the nuclear envelope breaks down at mitosis. GFP-PCNA is a accurate and non-toxic marker of S-phase in embryos during early development.