Wnt/β-catenin signaling has an essential role in the initiation of limb regeneration

University of Washington Seattle, Seattle, Washington, United States
Developmental Biology (Impact Factor: 3.55). 07/2007; 306(1):170-8. DOI: 10.1016/j.ydbio.2007.03.014
Source: PubMed


Anuran (frog) tadpoles and urodeles (newts and salamanders) are the only vertebrates capable of fully regenerating amputated limbs. During the early stages of regeneration these amphibians form a "blastema", a group of mesenchymal progenitor cells that specifically directs the regrowth of the limb. We report that wnt-3a is expressed in the apical epithelium of regenerating Xenopus laevis limb buds, at the appropriate time and place to play a role during blastema formation. To test whether Wnt/beta-catenin signaling is required for limb regeneration, we created transgenic X. laevis tadpoles that express Dickkopf-1 (Dkk1), a specific inhibitor of Wnt/beta-catenin signaling, under the control of a heat-shock promoter. Heat-shock immediately before limb amputation or during early blastema formation blocked limb regeneration but did not affect the development of contralateral, un-amputated limb buds. When the transgenic tadpoles were heat-shocked following the formation of a blastema, however, they retained the ability to regenerate partial hindlimb structures. Furthermore, heat-shock induced Dkk1 blocked fgf-8 but not fgf-10 expression in the blastema. We conclude that Wnt/beta-catenin signaling has an essential role during the early stages of limb regeneration, but is not absolutely required after blastema formation.

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    • "Wild-type (WT) Xenopus laevis adults were purchased from domestic animal vendors. Heatshock-inducible Dkk1GFP F0 Tg Xenopus was prepared as previously reported (Yokoyama et al. 2007, 2011). The tdTomato under control of the gamma-crystallin promoter was inserted downstream of hsp70-Dkk1GFP to select Tg individuals (Fig. 1A; Yokoyama et al. 2011). "
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    ABSTRACT: Urodele amphibians (newts and salamanders) and anuran amphibians (frogs) are excellent research models to reveal mechanisms of three-dimensional organ regeneration since they have exceptionally high regenerative capacity among tetrapods. However, the difficulty in manipulating gene expression in cells in a spatially restricted manner has so far hindered elucidation of the molecular mechanisms of organ regeneration in amphibians. Recently, local heat shock by laser irradiation has enabled local gene induction even at the single-cell level in teleost fishes, nematodes, fruit flies and plants. In this study, local heat shock was made with infrared laser irradiation (IR-LEGO) by using a gene expression inducible system in transgenic animals containing a heat shock promoter, and gene expression was successfully induced only in the target region of two amphibian species, Xenopus laevis and Pleurodeles waltl (a newt), at postembryonic stages. Furthermore, we induced spatially restricted but wider gene expression in Xenopus laevis tadpoles and froglets by applying local heat shock by a temperature-controlled metal probe (temperature stimulator). The local gene manipulation systems, the IR-LEGO and the temperature stimulator, enable us to do a rigorous cell lineage trace with the combination of the Cre-LoxP system as well as to analyze gene function in a target region or cells with less off-target effects in the study of amphibian regeneration.
    Full-text · Article · Oct 2015 · Development Growth and Regeneration
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    • "To detect transcripts on sectioned samples, in situ hybridization on frozen sections was carried out as previously described (Ohgo et al., 2010) with slight modification. To synthesize an antisense RNA probe for gfp, CMV-GFP5 plasmid (Yokoyama et al., 2007) was linearized with BamHI and transcribed with T7 RNA polymerase (Roche). Probes for prrx1 (Suzuki et al., 2005) and dusp6 (Gomez et al., 2005) were synthesized as previously described. "
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    ABSTRACT: Many amphibians can regenerate limbs, even in adulthood. If a limb is amputated, the stump generates a blastema that makes a complete, new limb in a process similar to developmental morphogenesis. The blastema is thought to inherit its limb-patterning properties from cells in the stump, and it retains the information despite changes in morphology, gene expression, and differentiation states required by limb regeneration. We hypothesized that these cellular properties are maintained as epigenetic memory through histone modifications. To test this hypothesis, we analyzed genome-wide histone modifications in Xenopus limb bud regeneration. The trimethylation of histone H3 at lysine 4 (H3K4me3) is closely related to an open chromatin structure that allows transcription factors access to genes, whereas the trimethylation of histone H3 at lysine 27 (H3K27me3) is related to a closed chromatin state that blocks the access of transcription factors. We compared these two modification profiles by high-throughput sequencing of samples prepared from the intact limb bud and the regenerative blastema by chromatin immunoprecipitation. For many developmental genes, histone modifications at the transcription start site were the same in the limb bud and the blastema, were stable during regeneration, and corresponded well to limb properties. These results support our hypothesis that histone modifications function as a heritable cellular memory to maintain limb cell properties, despite dynamic changes in gene expression during limb bud regeneration in Xenopus. Copyright © 2015. Published by Elsevier Inc.
    Full-text · Article · Aug 2015 · Developmental Biology
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    • "Inhibition of Wnt/ß-catenin signaling in the regenerating tail fin or limb bud blocks regeneration (Wehner et al., 2014; Yokoyama et al., 2007), suggesting that the pathway may be Fig. 3. Representative images of gfap:Cre ERT2 -labeled progeny in the regenerating blastema. (A–C) At 5 dpi one mCherry þ cell is GFAP þ (arrow), while a neighboring cell is GFAP À (arrowhead). "
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    ABSTRACT: Spinal cord injury results in permanent sensorimotor loss in mammals, in part due to a lack of injury-induced neurogenesis. The regeneration of neurons depends upon resident neural progenitors, which in zebrafish persist throughout the central nervous system as radial glia. However the molecular mechanisms regulating spinal cord progenitors remain uncharacterized. Wnt/ß-catenin signaling is necessary for the regenerative response of multiple tissues in zebrafish as well as other vertebrates, but it is not known whether the pathway has a role in spinal cord regeneration. Here we show that spinal radial glia exhibit Wnt/ß-catenin activity as they undergo neurogenesis following transection. We then use Cre-mediated lineage tracing to label the progeny of radial glia and show that Wnt/ß-catenin signaling is required for progenitors to differentiate into neurons. Finally, we show that axonal regrowth after injury also requires Wnt/ß-catenin signaling, suggesting coordinated roles for the pathway in functional recovery. Our data thus establish Wnt/ß-catenin pathway activation as a necessary step in spinal cord regeneration. Copyright © 2015. Published by Elsevier Inc.
    Full-text · Article · Apr 2015 · Developmental Biology
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