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...