A bright single-cell resolution live imaging reporter of Notch signaling in the mouse Journal Article
| Authors: | Nowotschin, S.; Xenopoulos, P.; Schrode, N.; Hadjantonakis, A. K. |
| Article 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. 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. © 2013 Nowotschin et al.; licensee BioMed Central Ltd. |
| Keywords: | signal transduction; promoter region; genetics; mouse; animal; metabolism; animals; mice; mus; gene expression; gfp; notch receptor; mus musculus; kidney; brain; histone; promoter regions, genetic; receptors, notch; reporter gene; simian virus 40; genes, reporter; histones; cell tracking; live imaging; mouse embryo; gastrulation; immunoglobulin j recombination signal sequence binding protein; immunoglobulin j recombination signal sequence-binding protein; reporter strain; notch signaling; venus; h2b-venus; cbf/rbpj; rbpj protein, mouse |
| Journal Title: | BMC Developmental Biology |
| Volume: | 13 |
| Issue: | 1 |
| ISSN: | 1471-213X |
| Publisher: | Biomed Central Ltd |
| Date Published: | 2013-04-25 |
| Start Page: | 15 |
| Language: | English |
| DOI: | 10.1186/1471-213x-13-15 |
| PROVIDER: | scopus |
| PMCID: | PMC3663770 |
| PUBMED: | 23617465 |
| DOI/URL: | |
| Notes: | --- - "Export Date: 25 September 2013" - "Source: Scopus" |
Altmetric
Citation Impact
BMJ Impact Analytics
MSK Authors
-
37Nowotschin -
191Hadjantonakis -
11Xenopoulos -
10Schrode
Related MSK Work