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A sensitive and bright single-cell resolution live imaging reporter of Wnt/ß-catenin signaling in the mouse.

BMC developmental biology | Dec 21, 2010

BACKGROUND: Understanding the dynamic cellular behaviors and underlying molecular mechanisms that drive morphogenesis is an ongoing challenge in biology. Live imaging provides the necessary methodology to unravel the synergistic and stereotypical cell and molecular events that shape the embryo. Genetically-encoded reporters represent an essential tool for live imaging. Reporter strains can be engineered by placing cis-regulatory elements of interest to direct the expression of a desired reporter gene. In the case of canonical Wnt signaling, also referred to as Wnt/β-catenin signaling, since the downstream transcriptional response is well understood, reporters can be designed that reflect sites of active Wnt signaling, as opposed to sites of gene transcription, as is the case with many fluorescent reporters. However, even though several transgenic Wnt/β-catenin reporter strains have been generated, to date, none provides the single-cell resolution favored for live imaging studies. RESULTS: We have placed six copies of a TCF/Lef responsive element and an hsp68 minimal promoter in front of a fluorescent protein fusion comprising human histone H2B to GFP and used it to generate a strain of mice that would report Wnt/β-catenin signaling activity. Characterization of developmental and adult stages of the resulting TCF/Lef:H2B-GFP strain revealed discrete and specific expression of the transgene at previously characterized sites of Wnt/β-catenin signaling. In support of the increased sensitivity of the TCF/Lef:H2B-GFP reporter, additional sites of Wnt/β-catenin signaling not documented with other reporters but identified through genetic and embryological analysis were observed. Furthermore, the sub-cellular localization of the reporter minimized reporter perdurance, and allowed visualization and tracking of individual cells within a cohort, so facilitating the detailed analysis of cell behaviors and signaling activity during morphogenesis. CONCLUSION: By combining the Wnt activity read-out efficiency of multimerized TCF/Lef DNA binding sites, together with the high-resolution imaging afforded by subcellularly-localized fluorescent fusion proteins such as H2B-GFP, we have created a mouse transgenic line that faithfully recapitulates Wnt signaling activity at single-cell resolution. The TCF/Lef:H2B-GFP reporter represents a unique tool for live imaging the in vivo processes triggered by Wnt/β-catenin signaling, and thus should help the formulation of a high-resolution understanding of the serial events that define the morphogenetic process regulated by this signaling pathway.

Pubmed ID: 21176145 RIS Download

Mesh terms: Animals | Endoderm | Gastrulation | Green Fluorescent Proteins | Mice | Mice, Transgenic | Primitive Streak | Signal Transduction | Single-Cell Analysis | TCF Transcription Factors | Wnt Proteins | beta Catenin

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Cellular Imaging and Analysis

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Induced Mutant Resource

THIS RESOURCE IS NO LONGER IN SERVICE, documented on June 08, 2012. The function of the IMR is to select, import, cryopreserve, maintain, and distribute these important strains of mice to the research community. To improve their value for research, the IMR also undertakes genetic development of stocks, such as transferring mutant genes or transgenes to defined genetic backgrounds and combining transgenes and/or targeted mutations to create new mouse models for research. The function of the IMR is to: * select biomedically important stocks of transgenic, chemically induced, and targeted mutant mice * import these stocks into the Jackson Laboratory by rederivation procedures that rid them of any pathogens they might carry * cryopreserve embryos from these stocks to protect them against accidental loss and genetic contamination * backcross the mutation onto an inbred strain, if necessary * distribute them to the scientific community More than 1000 mutant stocks have been accepted by the IMR from 1992 through December 2006. Current holdings include models for research on cancer; breast cancer; immunological and inflammatory diseases; neurological diseases; behavioral, cardiovascular and heart diseases; developmental, metabolic and other diseases; reporter (e.g., GFP) and recombinase (e.g., cre/loxP) strains. About eight strains a month are being added to the IMR holdings. Research is being conducted on improved methods for assisted reproduction and speed congenic production. Most of the targeted mutants arrive on a mixed 129xC57BL/6 genetic background, and as many of these as possible are backcrossed onto an inbred strain (usually C57BL/6J). In addition, new mouse models are being created by intercrossing carriers of specific transgenes and/or targeted mutations. Simple sequence length polymorphism DNA markers are being used to characterize and evaluate differences between inbred strains, substrains, and embryonic stem cell lines.

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