A bright single-cell resolution live imaging reporter of Notch signaling in the mouse.

BACKGROUND

Live imaging provides an essential methodology for understanding complex and dynamic cell behaviors and their underlying molecular mechanisms. Genetically-encoded reporter expressing mouse strains are an important tool for use in live imaging experiments. Such reporter strains can be engineered by placing cis-regulatory elements of interest to direct the expression of desired reporter genes. If these cis-regulatory elements are downstream targets, and thus activated as a consequence of signaling pathway activation, such reporters can provide read-outs of the signaling status of a cell. The Notch signaling pathway is an evolutionary conserved pathway operating in multiple developmental processes as well as being the basis for several congenital diseases. The transcription factor CBF1 is a central evolutionarily conserved component of the Notch signaling pathway. It binds the active form of the Notch receptor (NICD) and subsequently binds to cis-regulatory regions (CBF1 binding sites) in the promoters of Notch responsive genes. In this way, CBF1 binding sites represent a good target for the design of a Notch signaling reporter.

RESULTS

To generate a single-cell resolution Notch signaling reporter, we used a CBF responsive element to direct the expression of a nuclear-localized fluorescent protein. To do this, we linked 4 copies of a consensus CBF1 binding site to the basal simian virus 40 (SV40) promoter, placed this cassette in front of a fluorescent protein fusion comprising human histone H2B linked to the yellow fluorescent protein (YFP) Venus, one of the brightest available YFPs. We used the CBF:H2B-Venus construct to generate both transgenic embryonic mouse stem (ES) cell lines and a strain of transgenic mice that would report Notch signaling activity.

CONCLUSION

By using multiple CBF1 binding sites together with a subcellular-localized, genetically-encoded fluorescent protein, H2B-Venus, we have generated a transgenic strain of mice that faithfully recapitulates Notch signaling at single-cell resolution. This is the first mouse reporter strain in which individual cells transducing a Notch signal can be visualized. The improved resolution of this reporter makes it ideal for live imaging developmental processes regulated by the Notch signaling pathway as well as a short-term lineage tracer of Notch expressing cells due to the perdurance of the fluorescent reporter. Taken together, the CBF:H2B-Venus mouse strain is a unique tool to study and understand the morphogenetic events regulated by the Notch signaling pathway.