INTRODUCTION Eukaryotic cells duplicate their DNA during a well defined period of the cell cycle, the S-phase, and the extent, timing and accuracy of DNA replication are carefully controlled to assure fidelity of genome propagation (reviewed by Kornberg and Baker, 1992). In vertebrates, DNA replication occurs at 100- 300 discrete intranuclear foci, each of which contains numerous replication forks (Hand, 1978; Nakamura et al., 1986; Nakayasu and Berezney, 1989; Mills et al., 1989). In yeast, origins of replication are specified by narrowly defined DNA regions, but in higher eukaryotic cells it appears that certain DNA sequences may direct initiation anywhere within a broad initiation zone (for a recent review see Hamlin and Dijkwel, 1995). It has been recently proposed that each initiation zone in the mammalian chromosome contains many sites where replica- tion can originate, but chromatin structure and/or the nuclear matrix may repress some sites while activating others (DePam- philis, 1993). The concept that nuclear organisation plays a major role in replication is further supported by the finding that early replicating DNA sequences are spatially segregated from late replicating DNA sequences in the nucleus, suggesting that replication of the mammalian genome follows a dynamic 4- dimensional order: specific regions of DNA occupying defined locations in the nucleus replicate at precise times during S- phase (reviewed by Spector, 1993). Contrasting with these observations, in cell-free extracts from Xenopus eggs the foci of replication of either Xenopus sperm nuclei, chicken ery- throcyte nuclei, Drosophila polytene nuclei, or bacteriophage λ are monotonously maintained during S-phase (Mills et al., 1989; Cox and Laskey 1991; Leno and Laskey 1991; Sleeman et al., 1992), suggesting that initiation sites fire more or less simultaneously. In addition, it has been shown that initiation of replication occurs at random sites on SV40 DNA injected into Xenopus eggs (Harland and Laskey, 1980; McTiernan and Stambrook, 1984) and apparently there are no specific sequence requirements for replication of DNA in these cells (Méchali and Kearsey, 1984; Hyrien and Méchali, 1993). Thus, in contrast with the data obtained in mammalian somatic cells, it appears that chromatin assembled in frog eggs does not organise into early and late replicating domains. A striking characteristic of fertilised eggs from Xenopus laevis is their ability to undergo many divisions producing approximately 20,000 cells in the same time as a mammalian egg divides only once (see Newport and Kirschner, 1982; Leno and Laskey, 1991). In the fast dividing cells from amphibian embryos, DNA replication is completed within 15 minutes, whereas in the mouse egg the duration of S-phase is 6-7 hours (Callan, 1973; Howlett and Bolton, 1985). Since S-phase lasts 6-10 hours in adult somatic cells from both amphibia and mammals, it is likely that at least some aspects of replication may be distinct in amphibian embryos. We therefore decided to compare the replication pattern previously described in Xenopus eggs with that of mammalian embryos. Following fertilisation, the mouse developmental program proceeds in a series of easily identifiable and well characterised steps. At approximately 4-9 hours after fertilisation the egg 889 Journal of Cell Science 110, 889-897 (1997) Printed in Great Britain © The Company of Biologists Limited 1997 JCS3496 The spatial and temporal organisation of replication sites during early mouse embryogenesis was analysed using high resolution confocal and video fluorescence microscopy. The results show that distinct replication patterns occur in the transcriptionally inactive pronuclei of 1-cell embryos as well as in the transcriptionally active nuclei from 2- and 16/32-cell embryos. This indicates that specific chromatin regions are replicated at different times during S-phase and provides the first evidence that mechanisms controlling the temporal and spatial replication of DNA are already present in the haploid pronuclei of the mammalian zygote. Furthermore the data demonstrate that the male and female pronuclei in one-cell embryos replicate their genomes asynchronously. Finally, we observe changes in the dynamics of embryonic genome replication during early development which correlate with gross chromatin structure transitions detected at the electron microscope level. Taken together these results indicate that DNA synthesis in the mouse zygote follows a defined four-dimen- sional order which may evolve during development and differentiation. Key words: DNA replication, Chromatin structure, Mouse embryo SUMMARY Genome replication in early mouse embryos follows a defined temporal and spatial order João Ferreira and Maria Carmo-Fonseca* Institute of Histology and Embryology, Faculty of Medicine, University of Lisbon, 1699 Lisboa codex, Portugal *Author for correspondence