To trace cell lineages in a developing vertebrate and to observe, in vivo, how behaviors of individual cells are affected by the genes they express, we created a zebrafish line containing a transgene called mosaic analysis in zebrafish (MAZe), built around a self-excising hsp70:Cre cassette. Heat shock triggers Cre recombinase–mediated recombination in a random subset of cells, bringing the transcriptional activator Gal4:VP16 under control of the EF1α promoter. Gal4-VP16 then activates expression of a fluorescent protein from an upstream activating sequence (UAS) promoter. Marked clones of cells expressing any desired gene product can be generated by crossing MAZe fish with other lines containing UAS-driven transgenes. The number of clones induced, and their time of origin, could be varied by adjusting heat-shock timing and duration. As an alternative to heat shock, we introduced Cre under a tissue-specific promoter in MAZe fish to generate clones in a designated tissue.
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We thank M. Tada (University College London) for the UAS:gfp transgenic line; R. Kelsh (University of Bath) for the Sox10:gfp transgenic line and the Sox10 promoter sequence; J. Clarke (Kings College London) for the Gap42-GFP plasmid; M. Capecchi (University of Utah School of Medicine) for the pACN plasmid; H. Gerhardt (London Research Institute, Cancer Research UK) for the Cre antibody; L. Zimmerman (National Institute for Medical Research, London) for the γ-crystallin promoter; P. Taylor and D. Martin for fish husbandry; J. Clarke, S. Wilson and D. Ish-Horowicz for comments on the manuscript; and Cancer Research UK for funding.
The authors declare no competing financial interests.
Supplementary Figures 1–2 (PDF 7981 kb)
Myoblast fusion (MAZe + membrane GFP). A MAZe-labeled myoblast (left red arrow) fuses with neighboring unlabeled myoblast in the dorsal part of a trunk somite. The unlabeled myoblast nucleus becomes fluorescent after cytoplasm continuity is observed (yellow arrow). Thereafter, both nuclei are fluorescent (red arrows) and the intensity of their fluorescence increases over time. MAZe embryos were injected with 100 pg of mRNA encoding membrane-targeted GFP at the 1-2 cell stage and heat-shocked at 39°C for 45 min at 8 h.p.f.; confocal images were obtained from ~18 h.p.f. every 14 min. Each time point corresponds to a single optical section of 3 μm depth. Anterior to the left, dorsal up. (MOV 1667 kb)
Myoblast fusion (MAZe + UAS:gfp). A myoblast labeled with MAZe and GFP (red arrow) in the medial part of a trunk somite fuses with a neighboring unlabeled myoblast. The myoblast cytoplasm (observed with GFP fluorescence) elongates to span the entire length of the somite, and subsequently a second fluorescent nucleus (second red arrow) is observed in the same cell. Embryos obtained from crossing MAZe to UAS:gfp fish were heat shocked at 39°C for 45 min at 8 h.p.f. and confocal images were obtained from ~18 h.p.f. every 14 min. Each time point corresponds to a single optical section of 3 μm depth. (MOV 1031 kb)
Dividing MAZe-labeled cell. A MAZe-labeled cell situated just dorsal to the notochord (white arrow) divides. As the nuclear membrane breaks down the fluorescent protein diffuses to fill the cytoplasm of the cell. After cytokinesis the nlsRFP becomes re-localized to the nuclei of the daughter cells, labeling each nucleus with equal intensity. MAZe embryos were heat shocked at 39°C for 45 min at 8 h.p.f. and confocal and bright-field images were obtained from ~30 h.p.f. every 8 min. Each time point corresponds to a projection of confocal sections merged with the corresponding bright field image. Anterior to the left, dorsal up. (MOV 909 kb)
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Collins, R., Linker, C. & Lewis, J. MAZe: a tool for mosaic analysis of gene function in zebrafish. Nat Methods 7, 219–223 (2010). https://doi.org/10.1038/nmeth.1423
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