Wnt signaling is a key, evolutionarily conserved, cellular signal transduction pathway required and reiteratively used for diverse biological functions. Precise regulation of pathway activity is required for proper embryonic development, and in adulthood, for tissue homeostasis. By contrast, impaired Wnt signaling activity can lead to embryonic defects and disease progression. Wnt proteins encompass a large family of secreted glycoproteins that trigger their outcome through different downstream cascades, among them the canonical Wnt/ß-catenin pathway, which activates transcription of target genes by the stabilization and nuclear localization of ß-catenin, a transcriptional co-activator protein.
In the absence of ligand, cytoplasmatic ß-catenin is phosphorylated and targeted for degradation by a protein complex consisting of the scaffolding proteins Axin, APC and the kinase GSK3ß. Once phosphorylated, ß-catenin is recognized by the ubiquitin ligase Trcp, which targets it for proteasomal degradation. Upon binding of the Wnt ligand to the receptor complex formed by Frizzled (Fz) and LRP5/6, Dishevelled (Dvl) is recruited by Fz leading to LRP5/6 phosphorylation and Axin recruitment. Loss of Axin from the degradation complex dismantles the complex and releases ß-catenin. Once stabilized, ß-catenin translocates to the nucleus. As a transcriptional coactivator, ß-catenin together with the T c
ell-specific transcription f
ymphoid enhancer-binding factor 1 (TCF/Lef) family of transcription factors induces the transcription of downstream genes (reviewed in [1
Over the past fifteen years several transgenic mouse strains have been established to monitor Wnt/ß-catenin pathway activity during development, homeostasis and disease progression (reviewed in [4
]). These reporter constructs are generally derived from the TOPFLASH
]. They consist of a series of multimerized DNA binding sites for TCF/Lef (TCF/Lefn
), which together with a minimal promoter (promotermin)
, drive expression of a reporter gene. Thus, in principle, such TCF/Lefn-promotermin:reporter
constructs label cells that are actively transducing a Wnt signal.
As a validation of their utility, several variant Wnt/ß-catenin reporter mouse strains have been characterized, are readily available and have been used to determine Wnt signaling status in a broad spectrum of applications. First generation constructs usually comprised a LacZ reporter (e.g. [6
]), whereas later generation versions provided a quantifiable readout and promoted live imaging applications by incorporating fluorescent protein reporters such as GFP [7
]. Even so, because of their robust expression and resistance to fixation, LacZ reporters have often been preferable for higher resolution analysis of sectioned tissues. By contrast, native fluorescent proteins, even though desirable for live imaging applications, usually cannot be visualized at single-cell resolution, and often do not withstand fixation and post-processing. Thus, none of the existing TCF/Lefn-promotermin:reporter
constructs, or derivative mouse strains, facilitate single-cell resolution imaging of Wnt/ß-catenin pathway activity that can be quantified in live as well as in fixed tissues.
We therefore sought to generate an improved, third generation, Wnt/ß-catenin reporter, that would incorporate a bright fluorescent reporter which could be live imaged at single-cell resolution and also quantified, but which would withstand fixation and therefore could also be visualized in tissue sections. To do so, we designed a reporter construct that combined the Wnt/ß-catenin signaling read-out efficiency of multimerized TCF/Lef DNA binding sites with the single-cell resolution and quantifiable reporter expression afforded by fluorescent histone fusions. Fluorescent proteins fused to histones, for example human histone H2B, are localized to the nucleus because they remain bound to chromatin, and as such allow the visualization and tracking of individual cells. H2B fusions also provide details of cell divisions, including the plane of division and identification of daughter cells. They also reveal nuclear fragmentation which is often associated with cell death [9
]. We placed the H2B-GFP cassette under the control of six TCF/Lef
response elements and the hsp68
minimal promoter in a configuration identical to previously reported TCF/Lef-LacZ
reporter mice [11
]. We then used this construct to generate a derivative TCF/Lef:H2B-GFP
We recovered several founder transgenic lines which exhibited equivalent expression, demonstrating that TCF/Lef:H2B-GFP reporter expression was independent of integration site. Characterization of the TCF/Lef:H2B-GFP strain of mice revealed bright single-cell resolution reporter expression that spatio-temporally recapitulated TCF/Lef-LacZ reporter expression during mouse embryonic development. Moreover, given its improved sensitivity, the TCF/Lef:H2B-GFP strain revealed additional sites of reporter expression, in the visceral endoderm and epiblast of the pre-gastrula stage mouse embryo, tissues suggested through genetic and expression analyses to possess active Wnt/ß-catenin signaling, that is not reflected by existing Wnt/ß-catenin signaling reporters.
In summary, we have generated a transgenic mouse strain that serves as a quantitative, non-invasive single-cell resolution read-out of Wnt/ß-catenin signaling in the mouse. The TCF/Lef:H2B-GFP reporter currently represents an improved tool for imaging the in vivo processes triggered by canonical Wnt signaling pathway activation.