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. Doxorubicin treatment, and the resultant g2 arrest, leads to a significant increase in the number of cells where yb1 is not found in the cytoplasm, suggesting that its cellular localisation is variable during the cell cycle. Paw production of haploid and diploid androgenetic zebrafish including methodology for delayed in vitro fertilization john h. In zebrafish, the ntr system enables studies of cell function and cellular regeneration. To determine how cell cycle progression is affected, we imagedtcf21. Illuminating cell cycle progression in the developing zebrafish embryo mayu sugiyamaa,b, asako sakauesawanoa,c, tadahiro iimuraa,c,d, kiyoko fukamib, tetsuya kitaguchic, koichi kawakamie, hitoshi okamotof, shinichi higashijimag, and atsushi miyawakia,c,1 alaboratory for cell function and dynamics, advanced technology development group, brain science institute, riken, 21 hirosawa. The fucci indicator system is a powerful tool for spatiotemporal analysis of the cell cycle, but its utility has been restricted so far to a limited range of applications. Predicting stem cell fate changes by differential cell. Chapter 11 comprehensive approaches using luminescence. Illuminating cellcycle progression in the developing.
We examined cell proliferation and cell division in zebrafish lens epithelium by. A new transgenic line for studying the cell cycle from embryos to adults. Cell cycle regulatory molecules cyclins, cyclindependent kinases and cyclindependent kinase inhibitors and the cardiovascular system. Migrating neural crest cells nccs progress through the cell cycle with. Tracking neural crest cell cycle progression in vivo.
Cell cycle chromobody signal during the cell cycle. Remarkably, we discovered two anteriortoposterior waves of cell cycle transitions, g1s and mg1, in. Recently, a novel methodology was introduced that allows monitoring cell cycle. We examined cell proliferation and cell division in zebrafish lens epithelium by visualizing cell cycle phases and nuclear positions, using fluorescentlabeled geminin and histone proteins.
Thus, we devised a novel system allowing the cellcycle progression of ecs to be visualized in vivo. The present version substantially revises, corrects, and expands the earlier ones. Since zebrafish embryos are transparent, transgenic fish embryos. Cell proliferation is a key regulator of tissue morphogenesis. To enhance the dissemination of generally useful technological advances and transgenic lines, zebrafish is introducing technofish, which will highlight these. A novel hoxb cluster protein expressed in the hindbrain and pharyngeal. Various cell cycle sensors have been developed, starting with fucci in 2008. Accelerated cell division could increase the probability of ips cell generation andor cell cycle specific events such as dna replication might be the prerequisite for permitting the epigenetic changes to occur. Tracking neural crest cell cycle progression in vivo caltech authors. Metronidazole mtz has become the most commonly used prodrug substrate for eliciting cell loss in ntrexpressing transgenic zebrafish due to the cell specific nature of its cytotoxic. Live cell imaging of cell cycle and division premo fucci cell cycle sensor developed by miyawaki and colleagues, the fluorescence ubiquitination cell cycle indicator fucci, is a genetically encoded, twocolor red and green, indicator that lets you follow cell division within a cell population. In addition, we also generated a fucci derivative zfucci allowing visualization of cell cycle progression in zebrafish sugiyama et al. Expanding the utility of fucci reporters using facsbased.
Zebrafish are able to efficiently regenerate lost cardiac muscle, and is used as a model to understand why natural heart regeneration is blocked in mammals. Illuminating cellcycle progression in the developing zebrafish embryo. In g1, the chromobody signal is homogeneously distributed throughout the nucleus and cytoplasm. As fluorescent, ubiquitinationbased cell cycle indicator fucci lines to label different stages of cell cycle are available in zebrafish, one can cross nr1d1. Zebrafish, therefore, represent a novel model system with which to study this aspect of intestinal function and physiology. Here, we describe how to establish and validate the fucci system in murine pluripotent stem cells pscs and describe the utility of transgenic fucci mice. Zebrafish embryonic tissue differentiation is marked by concurrent.
Analysis of cell cycle by flow cytometry 303 cytometers are becton dickinson immunocytometry systems, beckmancoulter inc. A new transgenic line for studying the cell cycle from. It is an artificial activation of naturally occurring cell cycle checkpoints, induced by exogenous stimuli controlled by an experimenter. Hardcopies of the 4th edition of the zebrafish book can be obtained for a nominal fee from the zebrafish international resource center, 5274. The analysis of reprogramming events using the fucci cell cycle reporter may help to elucidate this issue. Thus, we devised a novel system allowing the cell cycle progression of ecs to be visualized in vivo. Visualizing spatiotemporal dynamics of multicellular cell. This technology relies upon ubiquitylationmediated proteolysis, and transgenic mice expressing fucci provide a powerful model system with which to study the coordination of the cell cycle and development. Muscle stem cells undergo extensive clonal drift during tissue growth via meox1mediated induction of g2 cell cycle arrest. A guide for the laboratory use of zebrafish danio rerio. This makes them particularly complimentary to the green asap1, if there is already another florescent protein in the same cell. We describe here the generation of a fucci derivative using zebrafish homologs of cdt1 and geminin, which provides sweeping views of cell. In g2, the foci disappear and the cell divides mitosis. Postlethwait analysis of protein and gene expression trevor jowett analysis of zebrafish development.
Enhanced cellspecific ablation in zebrafish using a. Pdf tracking neural crest cell cycle progression in vivo. These events include the duplication of its dna dna replication and some of its organelles, and subsequently the partitioning of its cytoplasm and other components into two daughter cells in a process called cell division. Remarkably, we discovered two anteriortoposterior waves of cell cycle transitions, g1s and mg1, in the differentiating notochord. Visualization of cellular electrical activity in zebrafish. Proliferation was low in the anterior region of lens epithelium and higher in the marginal zone anterior to the equator, suggesting that the. Chapter 15 assays for cell viability, proliferation and function molecular probes handbook a guide to fluorescent probes and labeling technologies 11th edition 2010. The cell cycle consists of two specific and distinct phases. It employs an s phasespecific cul4ddb1mediated ubiquitylation to sharply distinguish major cell cycle transitions and phases, particularly g1, s, and g2.
The technology analyzes living cells in a spatiotemporal manner using a dual color scheme of orange and green. A new transgenic line for studying the cell cycle from embryos to adults article pdf available in zebrafish 112 march 2014 with 515 reads how we measure reads. A new transgenic line for studying the cell cycle from embryos to adults cortney m. In this study we demonstrate in a developing in vivo model, how core promoter variations play a key role in defining transcriptional output in cells transitioning from a proliferative to cell lineage specifying phenotype. Fucci cells were mixed with nonfluorescent pac2 cells at a. The most commonly used fucci systems are ubiquitously expressed, making tracking of a specific cell. The cell cycle is the process by which eukaryotic cells duplicate and divide. Circadian clock synchronization of the cell cycle in zebrafish.
In the zebrafish version of fucci, which is called cellcycle illuminated cecyil, the. The cell cycle, or cell division cycle, is the series of events that take place in a cell that cause it to divide into two daughter cells. Here i describe several novel probes for cell functions that we developed in the last decade, including fluoppi, fucci, gepra, and unag. Visualization of cell cycle in mouse embryos with fucci2. Vnp fish with cell cycle reporter lines to examine the relationship between circadian clock and cell cycle in vivo.
Circadian clock regulation of the cell cycle in the zebrafish intestine. William detrich iii cell cycles and development in the embryonic zebrafish donald a. The circadian pacemaker can be set to this wide range of periods, and so by imaging cell cycle progression in individual cells, using the fucci cell cycle reporter system, it is possible to directly test two hypotheses. A combination of s and mphase markers, termed fucci, was developed for human and zebrafish cells 1, 2, while in plants. Visualizing the metazoan proliferationdifferentiation. Fission yeast in fission yeast the g 1 and sphases are relatively short each accounting for 10% of the time it takes to complete the cell cycle, whereas g 2 is. Visualizing the cell cycle behavior of individual cells within living organisms. Illuminating cell cycle progression in the developing zebrafish embryo mayu sugiyamaa,b, asako sakauesawanoa,c, tadahiro iimuraa,c,d, kiyoko fukamib, tetsuya kitaguchic, koichi kawakamie, hitoshi okamotof, shinichi higashijimag, and atsushi miyawakia,c,1 alaboratory for cell function and dynamics, advanced technology development group, brain science institute, riken, 21 hirosawa, wakocity.
Live cell imaging of cell cycle and divisionpremo fucci. Fucci technology makes possible the distinction between live cells in the g 1 and sg 2 m phases by dualcolor imaging. One experiment involves timelapse imaging of cells stably expressing a fucci derivative fucci2, which allows for the exploration of the spatiotemporal patterns of cell cycle dynamics during structural and behavioral changes of cultured cells. The high proliferation rate of these cells was evidenced by the large fraction of cells with green nuclei figure 2a, 2549 hr. Cell cycle regulatory molecules cyclins, cyclindependent. After cells were plated on a glass coverslip, they proliferated as clusters maintaining cell cell adhesion with their neighbors figure 2a, 1 hr. Cellular and developmental biology, part b, volume 4 3rd edition. Visualizing the cellcycle progression of endothelial. Since zebrafish embryos are transparent, transgenic fish embryos expressing zfucci probes provide a powerful tool for visualizing cell cycle dynamics in living animals. These indicators enable researchers to explore the cell cycle expansively. This investigation identified that the fucci system can successfully be used to segregate cells on the basis of cell cycle stage and cycling. For example, asap1 and quasar can be combined with fucci zebrafish 30 for studying the relationship between cell cycle and electric potential changes.
The zebrafish book a guide for the laboratory use of zebrafish danio. Spatial pattern of cell geometry and celldivision orientation in. Part of the methods in molecular biology book series mimb, volume 70. Abstract transgenic expression of bacterial nitroreductase ntr facilitates chemicallyinducible targeted cell ablation. Fucci fluorescent ubiquitinationbased cell cycle indicator sakauesawano et al. By exploiting the cellcycle 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 sg 2 m phases with different colors. To examine cell cycle progression during emt, we examined fucci expressing, stably transformed nmumg cells. Cell growth occurs through an ordered sequence of events the cell cycle, which consists of four distinct phases, g1, s, g2 and m phase. Visualizing the cellcycle progression of endothelial cells in zebrafish. The cell cycle, mitosis and meiosis university of leicester. Genetically encoded tools for optical dissection of the. Biology free fulltext circadian clocks in fishwhat. In february 2008 atsushi miyawaki from the riken basic science institute reported that his group has developed a method, fucci that will allow cell cycle researchers to visualize cell cycle progression.
The software used to deconvolute the dna content frequency histograms, to estimate the proportions of cells in the respective phases of the cycle, is available. Zebrafish embryonic tissue differentiation is marked by. This chapter focuses on visualization of cell cycle progression in plant tissues using the. In the repertoire of fucci probes, fucci ca is ideal as an allpurpose, highresolution interphase sensor. Induced cell cycle arrest is the use of a chemicals or genetic manipulation to artificially halt progression through the cell cycle. Live cell imaging reveals that the location of yb1 is linked to progression through the cell cycle. Fluorescent ubiquitinationbased cell cycle indicator fucci is a sophisticated technology which can easily determine g 1 andor sg 2 m phases of the cell cycle. Cellular processes like genome duplication and cell division stop. The cell cycle, mitosis and meiosis learning objective this learning material is about the life cycle of a cell and the series of stages by which genetic materials are duplicated and partitioned to produce two daughter cells with the same genetic component as the parent cell. Cytrap marker systems for in vivo visualization of cell cycle. The zebrafish fucci cell line used in this study has been previously described downey et al.