Abstract
BackgroundUnderstanding 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.ResultsWe 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.ConclusionBy 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.
Highlights
Understanding the dynamic cellular behaviors and underlying molecular mechanisms that drive morphogenesis is an ongoing challenge in biology
Since the transcription factor/lymphoid enhancer-binding factor 1 (TCF/Lef):H2B-GFP transgenic is the first Wnt reporter line to reveal the dynamics of Wnt signaling activity in cells of the visceral endoderm of E5.5 embryos, we focused on this stage to study the behavior of cells expressing the reporter
Even thought Wnt reporter lines based on the multimerization of Lef/ TCF binding sites which act as transcriptional reporters, might not be ideal readouts of active Wnt signaling, they are at present some of the best tools available to accurately identify the major sites of Wnt activity
Summary
Understanding the dynamic cellular behaviors and underlying molecular mechanisms that drive morphogenesis is an ongoing challenge in biology. Even though several transgenic Wnt/b-catenin reporter strains have been generated, to date, none provides the single-cell resolution favored for live imaging studies. 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. 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. SS-catenin together with the T cell-specific transcription factor/lymphoid enhancer-binding factor 1 (TCF/Lef) family of transcription factors induces the transcription of downstream genes (reviewed in [1,2,3])
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