Previous studies have applied the Brainbow technology to zebrafish and have shown that Cre induction can generate many distinct colors from microinjected Brainbow plasmid DNA ( Pan et al., 2011) or β-actin-2:Brainbow transgenes ( Gupta and Poss, 2012). The stochastic recombination events in individual progenitor cells are inherited by their progeny, resulting in clones marked by different colors ( Gupta and Poss, 2012 Snippert et al., 2010 Tabansky et al., 2012). In addition to enhancing visual resolution, Brainbow can also be used as a multi-lineage marker ( Buckingham and Meilhac, 2011 Kretzschmar and Watt, 2012). The unique combination provides each cell a distinct color, allowing resolution of individual cell boundaries. Remarkably, transgenic mice that carried multiple reporter insertions showed a large variety of colors owing to stochastic recombination and combinatorial expression of fluorescent proteins in each cell ( Fig. Expression of one and only one of these three proteins (per one copy of the construct) is achieved by the use of Lox sites, the recognition sites for Cre recombinase. The Brainbow construct contains a promoter followed by three fluorescent proteins: RFP, CFP and YFP ( Fig. One potential solution to this problem involves labeling adjacent cells with many different colors, which was achieved by the development of Brainbow ( Lichtman et al., 2008 Livet et al., 2007). The Zebrabow tool set presented here provides a resource for next-generation color-based anatomical and lineage analyses in zebrafish. Using the cornea as a model system, we provide evidence that embryonic corneal epithelial clones are replaced by large, wedge-shaped clones formed by centripetal expansion of cells from the peripheral cornea. Fourth, we demonstrate that Zebrabow can be used for long-term lineage analysis. Third, we show that UAS:Zebrabow lines can be used in combination with Gal4 to generate broad or tissue-specific expression patterns and facilitate tracing of axonal processes. Second, we find that colors are inherited equally among daughter cells and remain stable throughout embryonic and larval stages. First, we show that the broadly expressed ubi:Zebrabow line provides diverse color profiles that can be optimized by modulating Cre activity. Here, we describe Zebrabow (Zebrafish Brainbow) tools for in vivo multicolor imaging in zebrafish. The random combination of fluorescent proteins provides a way to distinguish adjacent cells, visualize cellular interactions and perform lineage analyses. Previous studies have demonstrated that Cre recombinase-mediated recombination can lead to combinatorial expression of spectrally distinct fluorescent proteins (RFP, YFP and CFP) in neighboring cells, creating a ‘Brainbow’ of colors. However, distinguishing and following cells over extended time periods remains difficult. Among vertebrates, zebrafish is uniquely suited for in vivo imaging owing to its small size and optical translucency. Advances in imaging and cell-labeling techniques have greatly enhanced our understanding of developmental and neurobiological processes.
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