Abstract
BackgroundLive 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.ResultsTo 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.ConclusionBy 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.
Highlights
Live imaging provides an essential methodology for understanding complex and dynamic cell behaviors and their underlying molecular mechanisms
Generation of CBF:H2B-Venus transgenic embryonic mouse stem (ES) cells Since previous studies have reported that mouse ES cells express Notch receptors and ligands [11], we investigated whether the CBF:H2B-Venus transgene was expressed in pluripotent mouse embryonic stem (ES) cells
We have generated a novel single-cell resolution transgenic strain of mice that serves as a transcriptional read-out of Notch signaling
Summary
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. 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. Cell-cell contact and the interaction of the NECD with one of the ligands expressed by a neighboring cell leads to a cascade of proteolytic events involving an S2 cleavage of the Notch receptor, and a cleavage at the S3 site by γ-secretase This series of proteolytic cleavages results in the release of the NICD from the membrane. The binding of NICD to CBF1 replaces co-factors of the Groucho family that function to repress target genes and in doing so allows the recruitment of the co-activator mastermind-like (MAML) promoting the formation of a CBF1-NICD-MAML complex that leads to transcriptional activation of target genes including members of the Hes and Hey families of bHLH transcription factors [1]
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