Visualization and exploration of Tcf/Lef function using a highly responsive Wnt/Β-catenin signaling-reporter transgenic zebrafish

Division of Cell Regulation Systems, Department of Immunobiology and Neuroscience, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
Developmental Biology (Impact Factor: 3.55). 07/2012; 370(1):71-85. DOI: 10.1016/j.ydbio.2012.07.016
Source: PubMed


Evolutionarily conserved Tcf/Lef transcription factors (Lef1, Tcf7, Tcf7l1, and Tcf7l2) mediate gene expression regulated by Wnt/β-catenin signaling, which has multiple roles in early embryogenesis, organogenesis, adult tissue homeostasis, and tissue regeneration. However, the spatiotemporal dynamics of Tcf/Lef activity during these events remain poorly understood. We generated stable transgenic zebrafish lines carrying a new Wnt/β-catenin signaling reporter, Tcf/Lef-miniP:dGFP. The reporter revealed the transcriptional activities of four Tcf/Lef members controlled by Wnt/β-catenin signaling, which were expressed in known Wnt/β-catenin signaling-active sites during embryogenesis, organ development and growth, and tissue regeneration. We used the transgenic lines to demonstrate the contribution of Tcf/Lef-mediated Wnt/β-catenin signaling to the development of the anterior lateral line, dorsal and secondary posterior lateral lines, and gill filaments. Thus, these reporter lines are highly useful tools for studying Tcf/Lef-mediated Wnt/β-catenin signaling-dependent processes.

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Available from: Koichi Kawakami, Jul 10, 2014
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    • "These results suggest that Hipk2 is essential for endogenous b-catenin stability in zebrafish embryos. To monitor the specific role of Hipk2 in the Wnt/b-catenin pathway, we used a transgenic zebrafish line carrying a Wnt/b-catenin signaling reporter construct (OTM:d2EGFP) comprising a destabilized GFP (d2EGFP) driven by a promoter containing multiple Tcf/Lef binding sites (Shimizu et al., 2012). d2EGFP expression was attenuated by hipk2 knockdown using MOs at 4, 8, and 10 hr postfertilization (hpf) (Figures 1B, S2B, and S2C), suggesting that Hipk2 is required for b-catenin pathway activation throughout early zebrafish embryogenesis. "
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    ABSTRACT: The phosphoprotein Dishevelled (Dvl) is a common essential component of Wnt/β-catenin and Wnt/planar cell polarity (PCP) signaling pathways. However, the regulation and significance of Dvl phosphorylation are not fully understood. Here, we show that homeodomain-interacting protein kinase 2 (Hipk2) facilitates protein phosphatase 1 catalytic subunit (PP1c)-mediated dephosphorylation of Dvl via its C-terminal domain and that this dephosphorylation blocks ubiquitination and consequent degradation mediated by the E3 ubiquitin ligase Itch, which targets the phosphorylated form of Dvl proteins. Inhibition of Hipk2 or PP1c function reduces Dvl protein levels and suppresses Wnt/β-catenin and Wnt/PCP pathway-dependent events in mammalian cells and zebrafish embryos, suggesting that Hipk2 and PP1c are essential for maintaining Dvl protein levels that are sufficient to activate Wnt signaling. We also show that Wnt-3a, a Wnt/β-catenin ligand, induces dissociation of the Dvl-Hipk2-PP1c complex and Dvl degradation under high-cell-density conditions. This regulation may be a negative feedback mechanism that fine-tunes Wnt/β-catenin signaling.
    Cell Reports 09/2014; 8(5). DOI:10.1016/j.celrep.2014.07.040 · 8.36 Impact Factor
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    • "Thus, we speculate that the accumulative process of í µí»½-catenin is not primarily mediated by í µí»½catenin mRNA but instead by the state of í µí»½-catenin, such as phosphorylation or dephosphorylation. The role of Tcf7/Lef1 as either a repressor or activator in Wnt/í µí»½-catenin signaling is controversial [41] [42]. We found that the treatment of BMSCs with SP led to an increased expression of Lef1 and Tcf7, which appeared to exert a positive effect on the activation of SPinduced Wnt/í µí»½-catenin signaling. "
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    ABSTRACT: Our previous work showed that implanting a sensory nerve or vascular bundle when constructing vascularized and neurotized bone could promote bone osteogenesis in tissue engineering. This phenomenon could be explained by the regulatory function of neuropeptides. Neuropeptide substance P (SP) has been demonstrated to contribute to bone growth by stimulating the proliferation and differentiation of bone marrow stem cells (BMSCs). However, there have been no prior studies on the association between Wnt signaling and the mechanism of SP in the context of BMSC differentiation. Our results have shown that SP could enhance the differentiation of BMSCs by activating gene and protein expression via the Wnt pathway and by translocating β-catenin, which can be inhibited by Wnt signaling blocker treatment or by the NK-1 antagonist. SP could also increase the growth factor level of bone morphogenetic protein-2 (BMP-2). Additionally, SP could enhance the migration ability of BMSCs, and the promotion of vascular endothelial growth factor (VEGF) expression by SP has been studied. In conclusion, SP could induce osteoblastic differentiation via the Wnt pathway and promote the angiogenic ability of BMSCs. These results indicate that a vascularized and neurotized tissue-engineered construct could be feasible for use in bone tissue engineering strategies.
    BioMed Research International 06/2014; 2014:596023. DOI:10.1155/2014/596023 · 3.17 Impact Factor
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    • "Both the TOPdGFP (Dorsky et al. 2002) and the TCFsiam lines (Moro et al. 2012), in which the minimal promoters used are cFos and siamois, respectively, show Wnt-dependent activation of the reporter transgene in several tissue contexts . On the other hand, however, very recent work by Shimizu et al. (2012) argued that in several Wnt reporter lines, including TOPdGFP and TCFSiam, the minimal promoters were derived from the promoters of natural genes, containing not only TATA, but also sequences derived from each gene promoter, which may be affected by non-specific signals. The authors suggested that the use of an artificial minimal promoter, such as the pGL4 vectorderived promoter called miniP, might increase the responsiveness and sensitivity of the reporter protein detection, reducing potentially unspecific signal. "
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    ABSTRACT: In the last years, we have seen the emergence of different tools that have changed the face of biology from a simple modeling level to a more systematic science. The transparent zebrafish embryo is one of the living models in which, after germline transformation with reporter protein-coding genes, specific fluorescent cell populations can be followed at single-cell resolution. The genetically modified embryos, larvae and adults, resulting from the transformation, are individuals in which time lapse analysis, digital imaging quantification, FACS sorting and next-generation sequencing can be performed in specific times and tissues. These multifaceted genetic and cellular approaches have permitted to dissect molecular interactions at the subcellular, intercellular, tissue and whole-animal level, thus allowing integration of cellular and developmental genetics with molecular imaging in the resulting frame of modern biology. In this review, we describe a new step in the zebrafish road to system biology, based on the use of transgenic biosensor animals expressing fluorescent proteins under the control of signaling pathway-responsive cis-elements. In particular, we provide here the rationale and details of this powerful tool, trying to focus on its huge potentialities in basic and applied research, while also discussing limits and potential technological evolutions of this approach.
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