Coordination of cell proliferation and differentiation during C. elegans development

Abstract

The formation of a complex multi-cellular organism from a single fertilized egg is an intriguing yet highly complex process. It requires the generation of large numbers of cells, which at the appropriate times need to obtain specialized functions and morphologies, while assembling into well-defined structures, tissues, and organs. Most cells follow a gradual process of specialization with a final step, terminal differentiation, characterized by acquisition of a fully differentiated post-mitotic state. The temporal coupling between cell cycle withdrawal and differentiation is crucial for normal growth and development, as well as tissue homeostasis and cell replacement throughout life. In contrast, a failure to arrest proliferation and loss of differentiation can lead to a variety of diseases and are hallmarks of cancer cells. While obviously of great importance, it remains only partially understood how proliferation and differentiation are coordinated during development. In order to better understand these mechanisms, we use the small nematode Caenorhabditis elegans (C. elegans). C. elegans provides a very attractive model system for cell cycle studies because of its highly invariant pattern of cell division, the possibility of efficient genetics and its transparency which allows life imaging of all cells and cell divisions. Using C. elegans as a model system, we addressed what mechanisms control cell cycle exit, coordinate cell cycle arrest with differentiation and contribute to the maintenance of a non-proliferative state in terminally differentiated cells. We identified the negative cell cycle regulators Rb and FZR1 as the two critical downstream targets of CDK4-cyclin D in C. elegans and demonstrated that Rb and FZR1 act redundantly to control cell cycle exit both in C. elegans and human breast cancer cells. To further study the mechanisms required for cell cycle exit during differentiation, we developed a CRE-loxP-based conditional knockout and lineage tracing system for C. elegans. By combing cell-type-specific knockouts with genetic screening, we found that previously identified G1 regulators such as the above mentioned Rb and FZR1, act together with members of the SWI/SNF chromatin remodeling complex and lineage-specific transcription factors to ensure cell cycle exit during muscle differentiation in C. elegans. Combined inactivation of these regulators caused a failure in cell cycle exit and tumor growth. In addition, we examined the irreversibility of the post-mitotic state of terminally differentiated muscles and neurons. By overexpression of the G1 CDK-cyclins and inactivation of negative regulators of cell division we were able to induce partial cell cycle re-entry in both muscles and neurons. Moreover, micro-array analysis in these muscles provided insights in the transcriptional-program activated upon cell cycle re-entry in terminally differentiated cells. Experiments in rat-hippocampal neurons suggested that the mechanisms involved in maintenance of a non-proliferative state in terminally differentiated neurons may be conserved between C. elegans and mammals. Taken together, the work described in this thesis contributes to our understanding of the mechanisms that control cell cycle exit during differentiation which may be of great fundamental and clinical importance, both in terms of cancer biology and regenerative therapies.