Cell cycle entry in C. elegans development

Abstract

Cell division is controlled by a mechanism that uses Cyclins, in association with their Cyclin-dependent kinase partners (Cdk’s), to regulate the transitions in the cell cycle.Studies in mammalian cell culture and single cell eukaryotes such as budding and fission yeast have uncovered much about how Cyclin/Cdk complexes control processes such as mitosis and DNA replication during cell cycle progression. However, much less is known about how cell cycle regulators function in the context of animal development. This thesis uses the small nematodeC. elegans as a model system to study the developmental control of the cell cycle. In particular, we focused on cell cycle withdrawal upon differentiation in the C. elegansbodywall muscle. We set out to test how cell cycle withdrawal is maintained in these cells and which controls act to restrict proliferation of differentiated cells. Muscle-specific expression of the C. elegans G1/S Cyclin/Cdk’s CYD-1/CDK-4 and CYE-1/CDK-2 was sufficient to trigger cell cycle re-entry in the terminally differentiated bodywall muscle. CYD-1/CDK-4 and CYE-1/CDK-2 induced S-phase entry, DNA replication, mitosis-specific phospho-H3 staining and nuclear division. Furthermore, tissue-specific microarray analysis revealed that these dividing body wall muscles induce a highly cell cycle-specific transcriptional program enriched for E2F target genes, whilst keeping their bodywall muscle fate. However, the extent of cell cycle re-entry was still limited compared to the effect of G1/S Cyclin/Cdk expression in the intestine. A genetic enhancer screen in CYD-1/CDK-4 animals identified 2 mutants that showed an increase in extra divisions in the bodywall muscle. These mutations might define genes that are specifically required to restrict proliferation of differentiated cells. In addition, we studied the role of G1/S regulators in the stem cell-like divisions of the C. elegansepidermal seam cell lineage. Cell lineage tracing showed that deregulation of G1/S progression in this tissue does not only lead to a change in cell division timing and frequency, it also causes changes in cell fate. Furthermore, we investigated the kinase-independent role of the CDK-4 G1/S Cdk. In mammalian systems it has been hypothesized that Cdk4 plays a kinase-independent role in G1/S progression by sequestration of p27. Rescue of the C. eleganscdk-4 null mutant with a kinase-dead version of CDK-4 did not result in rescue of cell division, arguing against a kinase-independent role for CDK-4 in G1/S progression. Lastly, this thesis describes the cloning and analysis of the C. eleganslin-6 mutant. This mutant is defective in post-embryonic cell division and DNA replication. We demonstrate that lin-6 encodes for the MCM-4 component of the MCM2-7 pre-RC and replicativehelicase complex. lin-6/MCM-4 mutantslack replication checkpoint function and continue mitosis in the absence of DNA replication. Furthermore, the absence of DNA replication in lin-6/MCM-4 animals does not interfere with cell differentiation or S-phase entry. Finally, expression of lin-6/MCM-4 solely in the epidermis is sufficient to rescue the growth retardation and lethality of lin-6 mutants.Thus, lin-6/mcm-4 has conserved functions in DNA replication and replication checkpoint control, but also shows surprising tissue specific requirements