The cell-cycle transition from G1 to S phase has been difficult to visualize. We have harnessed antiphase oscillating proteins that mark cell-cycle transitions in order to develop genetically encoded fluorescent probes for this purpose. These probes effectively label individual G1 phase nuclei red and those in S/G2/M phases green. We were able to generate cultured cells and transgenic mice constitutively expressing the cell-cycle probes, in which every cell nucleus exhibits either red or green fluorescence. We performed time-lapse imaging to explore the spatiotemporal patterns of cell-cycle dynamics during the epithelial-mesenchymal transition of cultured cells, the migration and differentiation of neural progenitors in brain slices, and the development of tumors across blood vessels in live mice. These mice and cell lines will serve as model systems permitting unprecedented spatial and temporal resolution to help us better understand how the cell cycle is coordinated with various biological events.

Optical methods for viewing neuronal populations and projections in the intact mammalian brain are needed, but light scattering prevents imaging deep into brain structures. We imaged fixed brain tissue using Scale, an aqueous reagent that renders biological samples optically transparent but completely preserves fluorescent signals in the clarified structures. In Scale-treated mouse brain, neurons labeled with genetically encoded fluorescent proteins were visualized at an unprecedented depth in millimeter-scale networks and at subcellular resolution. The improved depth and scale of imaging permitted comprehensive three-dimensional reconstructions of cortical, callosal and hippocampal projections whose extent was limited only by the working distance of the objective lenses. In the intact neurogenic niche of the dentate gyrus, Scale allowed the quantitation of distances of neural stem cells to blood vessels. Our findings suggest that the Scale method will be useful for light microscopy-based connectomics of cellular networks in brain and other tissues.

By exploiting the cell-cycle-dependent proteolysis of two ubiquitination oscillators, human Cdt1 and geminin, which are the direct substrates of SCF Skp2 and APC Cdh1 complexes, respectively, Fucci technique labels mammalian cell nuclei in G 1 and S/G2/M phases with different colors. Transgenic mice expressing these G 1 and S/G2/M markers offer a powerful means to investigate the coordination of the cell cycle with morphogenetic processes. We attempted to introduce these markers into zebrafish embryos to take advantage of their favorable optical properties. However, although the fundamental mechanisms for cell-cycle control appear to be well conserved among species, the G 1 marker based on the SCF Skp2 -mediated degradation of human Cdt1 did not work in fish cells, probably because the marker was not ubiquitinated properly by a fish E3 ligase complex. We describe here the generation of a Fucci derivative using zebrafish homologs of Cdt1 and geminin, which provides sweeping views of cell proliferation in whole fish embryos. Remarkably, we discovered two anterior-toposterior waves of cell-cycle transitions, G 1/S and M/G1, in the differentiating notochord. Our study demonstrates the effectiveness of using the Cul4 Ddb1 -mediated Cdt1 degradation pathway common to all metazoans for the development of a G 1 marker that works in the nonmammalian animal model. cell cycle ͉ fluorescent protein ͉ imaging ͉ ubiquitination E ukaryotic cells ensure tight regulation of cell division by maintaining close control over the levels of cell-cycle proteins. For example, Cdt1 and geminin have opposite effects on DNA replication during S phase, and their levels fluctuate accordingly throughout the cell cycle (1, 2). Cdt1 levels are highest in G 1 phase just before DNA replication and decrease as cells transition into S phase, whereas geminin levels rise during S phase and fall during G 1 phase. Cells control Cdt1 and geminin activity at the protein level by ubiquitination, which precisely targets unwanted proteins for destruction.We harnessed the regulation of cell-cycle-dependent ubiquitination to develop a genetically encoded indicator for cell-cycle progression: Fucci ( fluorescent ubiquitination-based cell cycle indicator) (3). The original Fucci probe was generated by fusing mKO2 (monomeric Kusabira Orange2) and mAG (monomeric Azami Green) to the ubiquitination domains of human Cdt1 (hCdt1) and human geminin (hGem): hCdt1(30/120) and hGem(1/110), respectively. These two chimeric proteins, mKO2-hCdt1(30/120) and mAG-hGem(1/110) ( Fig. 1 A and B), accumulate reciprocally in the nuclei of transfected mammalian cells during the cell cycle, labeling nuclei of G 1 phase cells orange and those in S/G 2 /M phase green. Thus, these proteins function as effective G 1 and S/G 2 /M markers. We also developed a S/G 2 /M marker, mAG-hGem(1/60), which accumulates in both the nucleus and cytoplasm (4) and reveals the morphology of individual cells that have undergone DNA replication. This permits cell proliferation to be monitored along with t...

The precise pathways of memory T-cell differentiation are incompletely understood. Here we exploit transgenic mice expressing fluorescent cell cycle indicators to longitudinally track the division dynamics of individual CD8+ T cells. During influenza virus infection in vivo, naive T cells enter a CD62Lintermediate state of fast proliferation, which continues for at least nine generations. At the peak of the anti-viral immune response, a subpopulation of these cells markedly reduces their cycling speed and acquires a CD62Lhi central memory cell phenotype. Construction of T-cell family division trees in vitro reveals two patterns of proliferation dynamics. While cells initially divide rapidly with moderate stochastic variations of cycling times after each generation, a slow-cycling subpopulation displaying a CD62Lhi memory phenotype appears after eight divisions. Phenotype and cell cycle duration are inherited by the progeny of slow cyclers. We propose that memory precursors cell-intrinsically modulate their proliferative activity to diversify differentiation pathways.

The quiescent (G0) phase of the cell cycle is the reversible phase from which the cells exit from the cell cycle. Due to the difficulty of defining the G0 phase, quiescent cells have not been well characterized. In this study, a fusion protein consisting of mVenus and a defective mutant of CDK inhibitor, p27 (p27K−) was shown to be able to identify and isolate a population of quiescent cells and to effectively visualize the G0 to G1 transition. By comparing the expression profiles of the G0 and G1 cells defined by mVenus-p27K−, we have identified molecular features of quiescent cells. Quiescence is also an important feature of many types of stem cells, and mVenus-p27K−-transgenic mice enabled the detection of the quiescent cells with muscle stem cell markers in muscle in vivo. The mVenus-p27K− probe could be useful in investigating stem cells as well as quiescent cells.

Markers of cell cycle stage allow estimation of cell cycle dynamics in cell culture and during embryonic development. The Fucci system incorporates genetically encoded probes that highlight G1 and S/G2/M phases of the cell cycle allowing live imaging. However the available mouse models that incorporate Fucci are beset by problems with transgene inactivation, varying expression level, lack of conditional potential and/or the need to maintain separate transgenes-there is no transgenic mouse model that solves all these problems. To address these shortfalls we reengineered the Fucci system to create 2 bicistronic Fucci variants incorporating both probes fused using the Thosea asigna virus 2A (T2A) self cleaving peptide. We characterize these variants in stable 3T3 cell lines. One of the variants (termed Fucci2a) faithfully recapitulated the nuclear localization and cell cycle stage specific florescence of the original Fucci system. We go on to develop a conditional mouse allele (R26Fucci2aR) carefully designed for high, inducible, ubiquitous expression allowing investigation of cell cycle status in single cell lineages within the developing embryo. We demonstrate the utility of R26Fucci2aR for live imaging by using high resolution confocal microscopy of ex vivo lung, kidney and neural crest development. Using our 3T3 system we describe and validate a method to estimate cell cycle times from relatively short time-lapse sequences that we then apply to our neural crest data. The Fucci2a system and the R26Fucci2aR mouse model are compelling new tools for the investigation of cell cycle dynamics in cell culture and during mouse embryonic development.

SUMMARYFucci technology makes possible the distinction between live cells in the G 1 and S/G 2 /M phases by dual-color imaging. This technology relies upon ubiquitylation-mediated proteolysis, and transgenic mice expressing Fucci provide a powerful model system with which to study the coordination of the cell cycle and development. The mice were initially generated using the CAG promoter; lines expressing the G 1 and S/G 2 /M phase probes that emitted orange (mKO2) and green (mAG) fluorescence, respectively, were separately constructed. Owing to cell type-biased strength of the CAG promoter as well as the positional effects of random transgenesis, however, we noticed some variability in Fucci expression levels. To control more reliably the expression of cell cycle probes, we used different genetic approaches to create two types of reporter mouse lines with Fucci2 and Rosa26 transcriptional machinery. Fucci2 is a recently developed Fucci derivative, which emits red (mCherry) and green (mVenus) fluorescence and provides better color contrast than Fucci. A new transgenic line, R26p-Fucci2, utilizes the Rosa26 promoter and harbors the G 1 and S/G 2 /M phase probes in a single transgene to preserve their co-inheritance. In the other R26R-Fucci2 approach, the two probes are incorporated into Rosa26 locus conditionally. The Cre-mediated loxP recombination technique thus allows researchers to design cell-type-specific Fucci2 expression. By performing time-lapse imaging experiments using R26p-Fucci2 and R26-Fucci2 in which R26R-Fucci2 had undergone germline loxP recombination, we demonstrated the great promise of these mouse reporters for studying cell cycle behavior in vivo.

Eukaryotic cells spend most of their life in interphase of the cell cycle. Understanding the rich diversity of metabolic and genomic regulation that occurs in interphase requires the demarcation of precise phase boundaries in situ. Here, we report the properties of two genetically encoded fluorescence sensors, Fucci(CA) and Fucci(SCA), which enable real-time monitoring of interphase and cell-cycle biology. We re-engineered the Cdt1-based sensor from the original Fucci system to respond to S phase-specific CUL4-mediated ubiquitylation alone or in combination with SCF-mediated ubiquitylation. In cultured cells, Fucci(CA) produced a sharp triple color-distinct separation of G1, S, and G2, while Fucci(SCA) permitted a two-color readout of G1 and S/G2. Fucci(CA) applications included tracking the transient G1 phase of rapidly dividing mouse embryonic stem cells and identifying a window for UV-irradiation damage in S phase. These results show that Fucci(CA) is an essential tool for quantitative studies of interphase cell-cycle regulation.