title: A bright single-cell resolution live imaging reporter of Notch signaling in the mouse.
abstract: 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Developmental Biology Program, Sloan-Kettering Institute, New York, NY, USA.
authors.forename: Sonja
authors.initials: S
authors.lastname: Nowotschin
authors.forename: Panagiotis
authors.initials: P
authors.lastname: Xenopoulos
authors.forename: Nadine
authors.initials: N
authors.lastname: Schrode
authors.forename: Anna-Katerina
authors.initials: AK
authors.lastname: Hadjantonakis
mesh_terms: D000818:Animals; D001921:Brain / Q000378:metabolism; D054262:Gastrulation; D015870:Gene Expression; D017930:Genes, Reporter; D006657:Histones / Q000235:genetics; D051819:Immunoglobulin J Recombination Signal Sequence-Binding Protein / Q000235:genetics / Q000378:metabolism; D007668:Kidney / Q000378:metabolism; D051379:Mice; D011401:Promoter Regions, Genetic; D051880:Receptors, Notch / Q000378:metabolism*; D015398:Signal Transduction
chemical_list: D006657:Histones; D051819:Immunoglobulin J Recombination Signal Sequence-Binding Protein; C497571:Rbpj protein, mouse; D051880:Receptors, Notch