dmFUCCI - a novel tool for studying cell proliferation in complex tissues. Norman Zielke, Jerome Korzelius, Monique van Straaten, Hanna Reuter, Katharina Bender, Gregor Schuhknecht, Juliette Pouch, Bruce Edgar. DKFZ/ZMBH Alliance, Im Neuenheimer Feld 282, 69120 Heidelberg, Germany.

   The development of complex tissues often involves strictly orchestrated lineages, in which only certain cell types proliferate at a time. The decision to proliferate is highly dependent on signals from surrounding cells and hence one of the future challenges is to study cell proliferation within its microenvironment. The recently introduced FUCCI system (Fluorescent Ubiquitination-based Cell Cycle Indicator) allows the monitoring of cell cycle phasing in living cells. In the original FUCCI system the sensor-constructs were ubiquitously expressed, limiting its usefulness because the analysis of cell proliferation in complex tissues requires specific labeling of small subpopulations of cells. To overcome these limitations we have generated a fly-specific FUCCI system (dmFUCCI), whose expression can be spatially and temporally controlled. The dmFUCCI system is based on E2F1 and Cyclin B, which are sequentially degraded by the ubiquitin ligases CRL4^Cdt2 and APC/C. Simultaneous expression of both dmFUCCI probes allows a distinction of all categories of interphase cells; the E2F1-based probe will mark cells in G1 phase; cells in S phase will be labeled by the Cyclin B-based sensor and cells residing in G2 phase will be positive for both markers. To allow maximum flexibility we have generated a series of fly lines expressing GFP/RFP or CFP/YFP versions of the dmFUCCI probes under control of UASt, UASp and QUAS promoters. We demonstrate that the dmFUCCI system is capable of recapitulating the developmentally programmed cell cycle patterns in developing eye and wing discs. Furthermore, we have applied the dmFUCCI method to the stem cell lineage of the adult midgut, which revealed that intestinal stem cells (ISCs) reside either in G1 or G2 phase instead of being halted at a specific cell cycle stage. Altogether, our work demonstrates that the dmFUCCI system is a valuable tool for visualizing cell cycle phasing during development and tissue homeostasis.