Abstract
BackgroundThe use of genetically-encoded fluorescent proteins has revolutionized the fields of cell and developmental biology and in doing so redefined our understanding of the dynamic morphogenetic processes that shape the embryo. With the advent of more accessible and sophisticated imaging technologies as well as an abundance of fluorescent proteins with different spectral characteristics, the dynamic processes taking place in situ in living cells and tissues can now be probed. Photomodulatable fluorescent proteins are one of the emerging classes of genetically-encoded fluorescent proteins.ResultsWe have compared PA-GFP, PS-CFP2, Kaede and KikGR four readily available and commonly used photomodulatable fluorescent proteins for use in ES cells and mice. Our results suggest that the green-to-red photoconvertible fluorescent protein, Kikume Green-Red (KikGR), is most suitable for cell labeling and lineage studies in ES cells and mice because it is developmentally neutral, bright and undergoes rapid and complete photoconversion. We have generated transgenic ES cell lines and strains of mice exhibiting robust widespread expression of KikGR. By efficient photoconversion of KikGR we labeled subpopulations of ES cells in culture, and groups of cells within ex utero cultured mouse embryos. Red fluorescent photoconverted cells and their progeny could be followed for extended periods of time.ConclusionTransgenic ES cells and mice exhibiting widespread readily detectable expression of KikGR are indistinguishable from their wild type counterparts and are amenable to efficient photoconversion. They represent novel tools for non-invasive selective labeling specific cell populations and live imaging cell dynamics and cell fate. Genetically-encoded photomodulatable proteins such as KikGR represent emergent attractive alternatives to commonly used vital dyes, tissue grafts and genetic methods for investigating dynamic behaviors of individual cells, collective cell dynamics and fate mapping applications.
Highlights
The use of genetically-encoded fluorescent proteins has revolutionized the fields of cell and developmental biology and in doing so redefined our understanding of the dynamic morphogenetic processes that shape the embryo
We found the green-to-red photoconvertible protein KikGR, engineered from the coral Favia favus [25], to be the best suited for cell labeling and fate mapping due to its specific and efficient photoconversion and outstanding brightness compared to the other photomodulatable fluorescent proteins tested in this study
Photoconversion of cells and ex utero culture of embryos to visualize cell dynamics in situ Since their spectral properties can be modulated in defined regions of interest, photoconvertible fluorescent proteins provide greater control of labeling specificity over conventional fluorescent proteins
Summary
The use of genetically-encoded fluorescent proteins has revolutionized the fields of cell and developmental biology and in doing so redefined our understanding of the dynamic morphogenetic processes that shape the embryo. Several approaches have been established for labeling cells in developing mouse embryos, the mammalian genetically-tractable model of choice, including dye injections, electroporation of nucleic acids or proteins into single or groups of cells [1,2] as well as grafting of genetically-distinct tissues [3,4] or using chimeras [57]. Most of these techniques are invasive, and only effective when the tissue or cells of interest are easy accessible for manipulation. In combination with increasingly sophisticated imaging technology, for example laser scanning confocal or multiphoton microscopy, and on-stage cultures, geneticallyencoded fluorescent proteins can provide high resolution information on dynamic cell behaviors
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