Pluripotent cells of embryonic origin proliferate at unusually rapid rates and have a characteristic cell cycle structure with truncated gap phases. To define the molecular basis for this we have characterized the cell cycle control of murine embryonic stem cells and early primitive ectoderm-like cells. These cells display precocious Cdk2, cyclin A and cyclin E kinase activities that are conspicuously cell cycle independent. Suppression of Cdk2 activity significantly decreased cycling times of pluripotent cells, indicating it to be rate-limiting for rapid cell division, although this had no impact on cell cycle structure and the establishment of extended gap phases. Cdc2-cyclin B was the only Cdk activity that was identified to be cell cycle regulated in pluripotent cells. Cell cycle regulation of cyclin B levels and Y 15 regulation of Cdc2 contribute to the temporal changes in Cdc2-cyclin B activity. E2F target genes are constitutively active throughout the cell cycle, reflecting the low activity of pocket proteins such as p107 and pRb and constitutive activity of pRb-kinases. These results show that rapid cell division cycles in primitive cells of embryonic origin are driven by extreme levels of Cdk activity that lack normal cell cycle periodicity.

To understand cell cycle control mechanisms in early development and how they change during differentiation, we used embryonic stem cells to model embryonic events. Our results demonstrate that as pluripotent cells differentiate, the length of G 1 phase increases substantially. At the molecular level, this is associated with a significant change in the size of active cyclin-dependent kinase (Cdk) complexes, the establishment of cell cycle-regulated Cdk2 activity and the activation of a functional Rb-E2F pathway. The switch from constitutive to cell cycle-dependent Cdk2 activity coincides with temporal changes in cyclin A2 and E1 protein levels during the cell cycle. Transcriptional mechanisms underpin the downregulation of cyclin levels and the establishment of their periodicity during differentiation. As pluripotent cells differentiate and pRb/p107 kinase activities become cell cycle dependent, the E2F-pRb pathway is activated and imposes cell cycle-regulated transcriptional control on E2F target genes, such as cyclin E1. These results suggest the existence of a feedback loop where Cdk2 controls its own activity through regulation of cyclin E1 transcription. Changes in rates of cell division, cell cycle structure and the establishment of cell cycle-regulated Cdk2 activity can therefore be explained by activation of the E2F-pRb pathway. INTRODUCTIONCell proliferation is coordinated by the activity of cyclindependent kinase (Cdk) activities (Nigg, 1995). This family of kinases functions by regulating the activity of proteins required for progression through the different cell cycle phases and hence must themselves be tightly cell cycle regulated. At one level, this is achieved through the cell cycleregulated synthesis of cyclin regulatory subunits, which bind and activate their catalytic Cdk partner. Inactivation of Cdk activity from one cell cycle phase is required for transition into the next by a mechanism involving cyclin destruction (Tyers and Jorgensen, 2000;Breeden, 2003). Hence, the ordered synthesis and destruction of phase-specific cyclin regulatory subunits are essential elements of normal cell cycle progression. Cyclin expression levels are controlled, in part at least, through transcriptional regulation. Cell cycleregulated cyclin E1 and A2 transcription is controlled by E2F transcription factors that are subject to repression through recruitment of "pocket proteins," such as pRb and p107, to the promoter regions of these genes. Repression of E2F-dependent transcription is lifted through phosphorylation of pocket proteins by Cdk activities that become active during G 1 phase (Harbour et al., 1999;Zhang et al., 2000), allowing for the transcriptional activation of genes required for the G 1 -S transition, such as cyclin E1.Pluripotent cells of late preimplantation and early postimplantation embryos exhibit two remarkable features. First, their cell division rates are very short, and their division is controlled through unusual mechanisms of Cdk regulation. Second, they have extraordinary developmental...