INTRODUCTION The development of a multicellular organism requires the careful coordination of cell division with growth and differentiation. In part, this coordination is achieved through integration of extracellular signals during the G1 phase, to which cells respond by either advancing into or withdrawing from another division cycle (Pardee, 1989). Ultimately, mitogenic and antiproliferative signals affect the cell-intrinsic cell-cycle machinery, of which the cyclin-dependent kinases (CDKs) are key components. The importance of the G1 control mechanisms is underscored by the finding that most, if not all, tumor cells have defects in one or more genes involved in G1 progression (Sherr, 1996). Despite extensive research, the in vivo functions of such genes have been poorly characterized. Significant insights have been gained from studying mammalian cells in tissue culture and gene alterations in human cancer. Members of the retinoblastoma (Rb) tumor- suppressor family (pRb, p107 and p130 in mammals) have been found to inhibit progression through the G1 phase (Sherr, 1996). This negative-regulatory function of the pRb protein is constrained by phosphorylation at multiple CDK phosphorylation sites. Sequential phosphorylations disrupt the binding between pRb and transcription factors such as E2F, thereby allowing these transcription factors to activate genes required for DNA synthesis and removing active transcriptional repression by pRb (Dyson, 1998). One of the critical targets of the E2F transcription factor is cyclin E (Dyson, 1998), which together with its partner CDK2 is required for the initiation of DNA replication (Duronio and O’Farrell, 1995; Knoblich et al., 1994; Ohtsubo et al., 1995; Tsai et al., 1993; van den Heuvel and Harlow, 1993). CDKs appear to act at multiple levels of this G1 control pathway. The first CDKs to become active in the cell cycle consist of a CDK4 or CDK6 catalytic subunit and a D-type cyclin regulatory subunit (Sherr, 1993). It is generally thought that cyclin D-dependent kinases initiate pRb phosphorylation in mid G1, while the subsequent activation of cyclin E-CDK2 kinases leads to completion of this process (Mittnacht, 1998). However, the presence of multiple D-type cyclins, CDK4/6 kinases and pRb-related proteins has hampered a direct demonstration of their functions in vivo. For example, it is not clear to what extent Cyclin D-CDK4/6 kinases are essential for pRb inactivation and cell-cycle progression in vivo. Moreover, it remains unknown whether Cyclin D has critical targets other than proteins of the pRb family. Potential targets include cyclin-dependent kinase inhibitors (CKIs) of the Cip/Kip family (Sherr and Roberts, 1999). p21 CIP1 and p27 KIP1 associate with cyclin E-CDK2 complexes and prevent their kinase activity. The same inhibitors also bind cyclin D and 4349 Development 128, 4349-4359 (2001) Printed in Great Britain © The Company of Biologists Limited 2001 DEV6543 We have investigated the regulation of cell-cycle entry in C. elegans, taking advantage of its largely invariant and completely described pattern of somatic cell divisions. In a genetic screen, we identified mutations in cyd-1 cyclin D and cdk-4 Cdk4/6. Recent results indicated that during Drosophila development, cyclin D-dependent kinases regulate cell growth rather than cell division. However, our data indicate that C. elegans cyd-1 primarily controls G1 progression. To investigate whether cyd-1 and cdk-4 solely act to overcome G1 inhibition by retinoblastoma family members, we constructed double mutants that completely eliminate the function of the retinoblastoma family and cyclin D-Cdk4/6 kinases. Inactivation of lin-35 Rb, the single Rb-related gene in C. elegans, substantially reduced the DNA replication and cell-division defects in cyd-1 and cdk-4 mutant animals. These results demonstrate that lin-35 Rb is an important negative regulator of G1/S progression and probably a downstream target for cyd-1 and cdk-4. However, as the suppression by lin-35 Rb is not complete, cyd-1 and cdk-4 probably have additional targets. An additional level of control over G1 progression is provided by Cip/Kip kinase inhibitors. We demonstrate that lin-35 Rb and cki-1 Cip/Kip contribute non- overlapping levels of G1/S inhibition in C. elegans. Surprisingly, loss of cki-1, but not lin-35, results in precocious entry into S phase. We suggest that a rate limiting role for cki-1 Cip/Kip rather than lin-35 Rb explains the lack of cell-cycle phenotype of lin-35 mutant animals. Key words: C. elegans, Cyclin, CDK, pRb, Cip/Kip, Cell cycle SUMMARY lin-35 Rb and cki-1 Cip/Kip cooperate in developmental regulation of G1 progression in C. elegans Mike Boxem and Sander van den Heuvel* Massachusetts General Hospital Cancer Center, Building 149, 13th Street, Charlestown, MA 02129, USA *Author for correspondence (e-mail: heuvel@helix.mgh.harvard.edu) Accepted 1 August 2001