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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    • "However, these reporter mice do not faithfully reproduce endogenous β-catenin transcriptional activity (Ahrens et al., 2011; Al Alam et al., 2011). In addition, several zebrafish reporter lines that express fluorescent proteins under the control of a β-catenin/Tcf-responsive promoter have been generated (Dorsky et al., 2002; Moro et al., 2012; Shimizu et al., 2012). However, they might not be suitable for studying β-catenin-dependent transcription in ECs, since the reporter gene expression is not restricted to the vasculature. "
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    ABSTRACT: β-catenin regulates the transcription of genes involved in diverse biological processes, including embryogenesis, tissue homeostasis and regeneration. Endothelial cell (EC)-specific gene-targeting analyses in mice have revealed that β-catenin is required for vascular development. However, the precise function of β-catenin-mediated gene regulation in vascular development is not well understood, since β-catenin regulates not only gene expression but also the formation of cell-cell junctions. To address this question, we have developed a novel transgenic zebrafish line that allows the visualization of β-catenin transcriptional activity specifically in ECs and discovered that β-catenin-dependent transcription is central to the bone morphogenetic protein (Bmp)-mediated formation of venous vessels. During caudal vein (CV) formation, Bmp induces the expression of aggf1, a putative causative gene for Klippel-Trenaunay syndrome, which is characterized by venous malformation and hypertrophy of bones and soft tissues. Subsequently, Aggf1 potentiates β-catenin transcriptional activity by acting as a transcriptional co-factor, suggesting that Bmp evokes β-catenin-mediated gene expression through Aggf1 expression. Bmp-mediated activation of β-catenin induces the expression of Nr2f2 (also known as Coup-TFII), a member of the nuclear receptor superfamily, to promote the differentiation of venous ECs, thereby contributing to CV formation. Furthermore, β-catenin stimulated by Bmp promotes the survival of venous ECs, but not that of arterial ECs. Collectively, these results indicate that Bmp-induced activation of β-catenin through Aggf1 regulates CV development by promoting the Nr2f2-dependent differentiation of venous ECs and their survival. This study demonstrates, for the first time, a crucial role of β-catenin-mediated gene expression in the development of venous vessels. © 2015. Published by The Company of Biologists Ltd.
    Development 01/2015; 142(3). DOI:10.1242/dev.115576 · 6.46 Impact Factor
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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 · 1.58 Impact Factor
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