Genome endoreduplication during mammalian development is a rare event for which the mechanism is unknown. It first appears when fibroblast growth factor 4 (FGF4) deprivation induces differentiation of trophoblast stem (TS) cells into the nonproliferating trophoblast giant (TG) cells required for embryo implantation. Here we show that RO3306 inhibition of cyclin-dependent protein kinase 1 (CDK1), the enzyme required to enter mitosis, induced differentiation of TS cells into TG cells. In contrast, RO3306 induced abortive endoreduplication and apoptosis in embryonic stem cells, revealing that inactivation of CDK1 triggers endoreduplication only in cells programmed to differentiate into polyploid cells. Similarly, FGF4 deprivation resulted in CDK1 inhibition by overexpressing two CDK-specific inhibitors, p57/KIP2 and p21/CIP1. TS cell mutants revealed that p57 was required to trigger endoreduplication by inhibiting CDK1, while p21 suppressed expression of the checkpoint protein kinase CHK1, thereby preventing induction of apoptosis. Furthermore, Cdk2 −/− TS cells revealed that CDK2 is required for endoreduplication when CDK1 is inhibited. Expression of p57 in TG cells was restricted to G-phase nuclei to allow CDK activation of S phase. Thus, endoreduplication in TS cells is triggered by p57 inhibition of CDK1 with concomitant suppression of the DNA damage response by p21.[Keywords: Endoreduplication; placentomegaly; trophoblast giant cell; p57; CDK1; DNA replication] Supplemental material is available at http://www.genesdev.org.

Cyclin-dependent kinases (CDKs) play a central role in the orderly transition from one phase of the eukaryotic mitotic cell division cycle to the next. In this context, p27 Kip1 (one of the CIP/KIP family of CDK specific inhibitors in mammals) or its functional analogue in other eukarya prevents a premature transition from G1 to S-phase. Recent studies have revealed that expression of a second member of this family, p57 Kip2 , is induced as trophoblast stem (TS) cells differentiate into trophoblast giant (TG) cells. p57 then inhibits CDK1 activity, an enzyme essential for initiating mitosis, thereby triggering genome endoreduplication (multiple S-phases without an intervening mitosis). Expression of p21 Cip1 , the third member of this family, is also induced in during differentiation of TS cells into TG cells where it appears to play a role in suppressing the DNA damage response pathway. Given the fact that p21 and p57 are unique to mammals, the question arises as to whether one or both of these proteins are responsible for the induction and maintenance of polyploidy during mammalian development. The road to polyploidyWhen metazoan cells proliferate, they employ the mitotic cell cycle in which separation of sibling chromosomes during mitosis (M-phase) and DNA synthesis during genome duplication (S-phase) are separated by two intervening gaps of time called the G1 and G2-phases to generate a repeating series of events: M→G1→S→G2→M. Cell division (cytokinesis) occurs immediately after mitosis. Cell growth occurs primarily during G1-phase. In addition, metazoan cells can exit their mitotic cell cycle and enter a quiescent state termed G0 in which the living state is maintained in the absence of either cell growth or proliferation. Mitotic cell cycles restrict genome duplication to once and only once per cell division. Therefore, G1-phase somatic cells contain two copies of their genome (2N or diploid), whereas somatic cells in G2 or M-phases are tetraploid (4N DNA). Cells with greater than 4N DNA content are referred to as polyploid.Polyploidy can result from aberrant DNA re-replication during S-phase. DNA re-replication occurs when newly assembled replication forks re-replicate parts of the genome that have already been replicated, resulting in replication bubbles within replication bubbles [1]. This occurs when one or more of the normal controls that prevent reutilization of replication origins during S-phase is circumvented. For example, DNA replication can be induced in some metazoan cells either by over-expression of Cdt1, a protein essential for loading the replicative MCM DNA helicase, or by suppression of the Cdt1 specific inhibitor geminin. Both changes promote loading of the MCM helicase at replication origins [2]. As DNA re-

Development of a fertilized egg into an adult human requires trillions of cell divisions, the vast majority of which duplicate their genome once and only once. Nevertheless, trophoblast giant cells and megakaryocytes in mammals circumvent this rule by duplicating their genome multiple times without undergoing cell division, a process generally referred to as 'endoreduplication'. In contrast, arthropods such as Drosophila endoreduplicate their genome in most larval tissues, as well as in many adult tissues. Endoreduplication requires that cells prevent entrance into or completion of mitosis and cytokinesis under conditions that permit assembly of prereplication complexes. In addition, cells must prevent induction of apoptosis in response to incomplete DNA replication or DNA damage that may occur during the ensuing sequence of 'endocycles'. Thus, developmentally regulated endoreduplication results in terminal cell differentiation. Recent progress has revealed both differences and similarities in the mechanisms employed by flies and mammals to change from mitotic cell cycles to 'endocycles'. The critical step, however, appears to be switching from a CDK-dependent form of the anaphase promoting complex (APC) to one that functions only in the absence of CDK activity.

Trophoblast stem (TS) cells proliferate in the presence of fibroblast growth factor 4, but in its absence, they differentiate into polyploid trophoblast giant (TG) cells that remain viable but nonproliferative. Differentiation is coincident with expression of the cyclin-dependent kinase (CDK)-specific inhibitors p21 and p57, of which p57 is essential for switching from mitotic cell cycles to endocycles. Here, we show that, in the absence of induced DNA damage, checkpoint kinase-1 (CHK1), an enzyme essential for preventing mitosis in response to DNA damage, functions as a mitogen-dependent protein kinase that prevents premature differentiation of TS cells into TG cells by suppressing expression of p21 and p57, but not p27, the CDK inhibitor that regulates mitotic cell cycles. CHK1 phosphorylates p21 and p57 proteins at specific sites, thereby targeting them for degradation by the 26S proteasome. TG cells lack CHK1, and restoring CHK1 activity in TG cells suppresses expression of p57 and restores mitosis. Thus, CHK1 is part of a “G2 restriction point” that prevents premature cell cycle exit in cells programmed for terminal differentiation, a role that CHK2 cannot play.