Zebrabow: multispectral cell labeling for cell tracing and lineage analysis in zebrafish

Y. Albert Pan ( 0 Tom Freundlich Tamily A. Weissman David Schoppik X. Cindy Wang Steve Zimmerman Brian Ciruna Joshua R. Sanes Jeff W. Lichtman Alexander F. Schier ) 0 Present address: Institute of Molecular Medicine and Genetics, Medical College of Georgia, Georgia Regents University , Augusta, GA 30912, USA SUMMARY Advances in imaging and cell-labeling techniques have greatly enhanced our understanding of developmental and neurobiological processes. Among vertebrates, zebrafish is uniquely suited for in vivo imaging owing to its small size and optical translucency. However, distinguishing and following cells over extended time periods remains difficult. 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. The random combination of fluorescent proteins provides a way to distinguish adjacent cells, visualize cellular interactions and perform lineage analyses. Here, we describe Zebrabow (Zebrafish Brainbow) tools for in vivo multicolor imaging in zebrafish. First, we show that the broadly expressed ubi:Zebrabow line provides diverse color profiles that can be optimized by modulating Cre activity. Second, we find that colors are inherited equally among daughter cells and remain stable throughout embryonic and larval stages. 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. Fourth, we demonstrate that Zebrabow can be used for long-term lineage analysis. 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. The Zebrabow tool set presented here provides a resource for next-generation color-based anatomical and lineage analyses in zebrafish. INTRODUCTION A deeper understanding of developmental and neurobiological processes requires high-resolution visualization of cell lineages and assemblies. The accessibility and translucency of zebrafish make it an ideal system for dissecting the cellular basis of vertebrate development. Indeed, substantial progress has been made in labeling zebrafish cells and following their trajectory through development. Initial advances based on cell labeling with organic dyes resulted in the establishment of fate maps, lineage diagrams and neural circuits (Kimmel and Law, 1985). The discovery of genetically encoded fluorescent proteins in conjunction with novel transgenic and microscopy technologies allowed for visualization of different cell types and subcellular compartments (Distel et al., 2006; Kawakami, 2004; Keller et al., 2008; Megason, 2009). Despite these significant developments, current technologies still have several limitations. When a large number of cells are labeled, individual cells are often difficult to distinguish. In the nervous system, overlapping axons and dendrites cannot be resolved with conventional fluorescence microscopy, rendering it difficult to trace the precise connectivity of individual neurons. Similar problems arise during time-lapse visualization owing to the low speed and resolution of current confocal and multiphoton technologies. To circumvent this challenge, two or three fluorescent colors (Distel et al., 2006; Hatta et al., 2006; Megason, 2009; Teddy et al., 2005) or faster imaging techniques (Dunsby, 2008; Huisken and Stainier, 2007; Keller et al., 2008) have been used. In these studies, however, each cell is labeled with the same set of colors (for example membrane in red and nuclei in green), which provides no distinction between adjacent cells. 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 Brainbow construct contains a promoter followed by three fluorescent proteins: RFP, CFP and YFP (Fig. 1A). 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. 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. 1B). The unique combination provides each cell a distinct color, allowing resolution of individual cell boundaries. 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 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). 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 -actin-2:Brainbow transgenes (Gupta and Poss, 2012). These studies were restricted to early embryogenesis and heart tissue, respectively, and have not addressed several key questions: can Brainbow be used in a wide variety of tissues? What are the optimal strategies to achieve broad or tissue-specific labeling? How can color diversity be maximized? How stable are colors over time and through cell divisions? Can Brainbow be used for clonal analysis in multiple organs? Here, we address these issues and describe tools and methods that will allow for the broad application of the Brainbow technology in zebrafish (Zebrabow): we describe new transgenic lines for ubiquitous or tissue-specific multicolor labeling; we show that multicolor labeling facilitates axonal tracing; we maximize color diversity by optimizing Cre activity; we show that colors are stable over time and after cell divisions; and we demonstrate that Zebrabow can be used for long-term clonal analysis in a wide variety of tissues. Long-term time-lapse imaging shows that the zebrafish corneal epithelium undergoes dramatic changes in clonal structures during development and suggests that late-born corneal clones are formed by centripetal expansion from the periphery. The Zebrabow tools and methods reported here provide an important resource to facilitate multicolor fluorescent labeling in a wide variety of applications. MATERIALS AND METHODS Zebrafish husbandry and strains Zebrafish from the TL/AB strain were maintained using standard procedures (Westerfield, 2000). Embryos were raised at 28.5C in embryo water containing 0.1% Methylene Blue hydrate (Sigma, St Louis, MO, USA) and 0.03% Instant Ocean sea salt (United Pet Group, Cincinnati, OH, USA). At 24 hours post-fertilization (hpf), embryos were transferr (...truncated)