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Wnt/ß-Catenin Signaling Regulates Vertebrate Limb Regeneration

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Wnt/ß-Catenin Signaling Regulates Vertebrate Limb Regeneration Downloaded from genesdev.cshlp.org on September 26, 2021 - Published by Cold Spring Harbor Laboratory Press RESEARCH COMMUNICATION ␤ epithelia that, like the regenerating AEC, is required for Wnt/ -catenin signaling the proliferation of mesenchymal cells, and therefore for regulates vertebrate limb normal limb development. Here we show that reduction in Wnt and BMP signaling during limb regeneration in regeneration axolotls, Xenopus laevis, and zebrafish induce alter- ations in the formation of the AEC that prevent normal 1 Yasuhiko Kawakami, Concepción Rodriguez fin/limb regeneration. More importantly, by performing Esteban,1 Marina Raya,2 Hiroko Kawakami,1 gain of function experiments of the Wnt/␤-catenin path- Merce`Martı´,2 Ilir Dubova,1,2 and way during appendage regeneration, we demonstrate Juan Carlos Izpisúa Belmonte1,2,3 that this pathway promotes Xenopus and zebrafish limb/ fin regeneration. The ability of this pathway to promote 1Gene Expression Laboratory, The Salk Institute for Biological regeneration is not only restricted to normally regener- Studies, La Jolla, California 92037, USA; 2Center for ating organisms, since activation of Wnt signaling during Regenerative Medicine of Barcelona, 08003 Barcelona, Spain limb development in the chick embryo enables regenera- tion of the AER. While obviously not identical processes, The cellular and molecular bases allowing tissue regen- the similarities encountered in the molecular and cellu- eration are not well understood. By performing gain- and lar processes involved during limb embryogenesis and loss-of-function experiments of specific members of the limb regeneration suggest a mechanism whereby varia- Wnt pathway during appendage regeneration, we dem- tions in the concentration and/or spatiotemporal distri- onstrate that this pathway is not only necessary for re- bution of developmental regulators may allow regenera- tion to occur. generation to occur, but it is also able to promote regen- eration in axolotl, Xenopus, and zebrafish. Furthermore, Results and Discussion we show that changes in the spatiotemporal distribution of ␤-catenin in the developing chick embryo elicit apical Even though the Wnt signaling pathway has been shown ectodermal ridge and limb regeneration in an organism to have a role in the regenerating zebrafish caudal fin previously thought not to regenerate. Our studies may (Poss et al. 2000a), the urodele tail (Caubit et al. 1997), and hydra head (Bode 2003), a direct demonstration for provide valuable insights toward a better understanding any of the pathway components in altering or eliciting of adult tissue regeneration. vertebrate tissue regeneration is still lacking. To test a Supplemental material is available at http://www.genesdev.org. direct participation of this pathway in vertebrate regen- eration, we developed an adenovirus-mediated protocol Received July 26, 2006; revised version accepted October 4, (Supplementary Fig. S1; Materials and Methods) that al- 2006. lowed us to perform gain- and loss-of-function experi- ments of specific members of the Wnt pathway during axolotl, Xenopus, and zebrafish regeneration. Regeneration is a complex biological process by which After amputation, axolotls have the ability to regener- animals restore shape, structure, and function of body ate their entire appendages, thus making them one of the parts lost to injury or experimentally amputated. In this most studied model systems in tissue regeneration (Bry- regard, regeneration of vertebrate appendages has been ant et al. 2002; Tanaka 2003; Brockes and Kumar 2005). one of the most extensively studied model systems (Tso- To determine the involvement of the Wnt pathway in nis 2000; Brockes and Kumar 2002). Urodeles and ze- this process, we microinjected an adenoviral vector en- brafish are among the vertebrate species that retain a gineered to express Axin1 (an intracellular inhibitor of significant limb regenerative ability during adulthood. In the Wnt pathway), Ad-Axin1 (Kawakami et al. 2001) dur- these species, limb regeneration proceeds by the forma- ing forelimb regeneration. This treatment led to a de- tion of a growth zone or blastema at the end of the crease in the regeneration capacity of the infected ani- stump. For regeneration to occur, this event needs to be mals and resulted in spike-like regenerated limbs lacking preceded by the formation of the apical ectodermal cap all of the digits (n = 5, 60%) (Fig. 1A,B). The alterations in (AEC), the multilayered epithelia that covers the wound the regeneration capacity after Wnt down-regulation surface after amputation (Bryant et al. 2002; Poss et al. were not restricted to the adult limb but were also ob- 2003). During the last decade, and in part with the help served in the embryo (data not shown) and larvae (Fig. of the knowledge gathered during the embryogenesis of 1C,D), since infection of amputated, developing limbs the vertebrate limb, some of the molecular and cellular with an adenovirus, Ad-Dkk1, harboring another inhibi- processes involved in AEC and blastema formation have tor of the Wnt pathway, Dkk1 (Glinka et al. 1998), been unveiled (Capdevila and Izpisua Belmonte 2001; showed similar regeneration defects as those observed in Poss et al. 2003). Members of the Wnt and bone morpho- adult infected regenerating limbs (Supplementary Table genetic protein (BMP) signaling pathways have been S1). shown to be required in vertebrates for the formation of The fact that two different Wnt antagonists acting ei- the apical ectodermal ridge (AER), a pseudostratified ther extracellularly or intracellularly are able to perturb limb regeneration indicates the necessity but does not [Keywords: Wnt; regeneration; limb; apical ectodermal cap; p63] address the sufficiency of Wnt signaling for inducing re- 3 Corresponding author. generation in axolotls, animals that exhibit this capabil- E-MAIL [email protected]; FAX (858) 453-2573. Article published online ahead of print. Article and publication date are ity throughout their life cycle. On the other hand, the online at http://www.genesdev.org/cgi/doi/10.1101/gad.1475106. amphibian Xenopus laevis, while an equally efficient re- GENES & DEVELOPMENT 20:000–000 © 2006 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/06; www.genesdev.org 1 Downloaded from genesdev.cshlp.org on September 26, 2021 - Published by Cold Spring Harbor Laboratory Press Kawakami et al. generator as a tadpole, loses its regenerative capacity af- Supplementary Table S1). Overall, these results not only ter metamorphosis. Complete limb regenerates are ob- demonstrate the necessity of Wnt signaling in the early tained only up to stages 50–51, after which the capacity stages of axolotl and Xenopus limb regeneration, but also to regenerate declines progressively until stage 58, when reflect on its ability to promote limb regeneration at spe- it is completely abolished (Dent 1962; Muneoka et al. cific stages. They also hint at the stage-dependent re- 1986). With the objective of assessing the ability of Wnt quirement of other factors, besides induction of the Wnt signaling to promote regeneration, we microinjected an canonical pathway, to elicit regeneration in Xenopus. adenovirus Ad-CA-␤-catenin, containing a constitu- While the axolotl and Xenopus have been at the fore- tively activated, form of ␤-catenin (Funayama et al. 1995) front of regeneration research (Bryant et al. 2002; Maden in amputated limbs at stages 53–54, when their regen- and Hind 2003; Tanaka 2003; Slack et al. 2004; Brockes eration potential is lost or greatly diminished. In most and Kumar 2005; Suzuki et al. 2005), the lack of molecu- cases only partially regenerated limbs displaying large lar and genetic resources has hindered a rapid progres- deviations from normal patterning were observed. In a sion of this field. The zebrafish has proved to be an ex- few others, complete and well-patterned limbs or limbs cellent model system to further enhance the cellular and with incomplete distal structures, although slightly de- molecular basis of tissue regeneration (Akimenko et al. layed in their development when compared with their 2003; Poss et al. 2003). contralateral, nonamputated limbs, were obtained (Fig. Induction of lef1 expression, a target of Wnt signaling, 1H–K; Supplementary Table S2). On the other hand, Wnt during zebrafish caudal fin regeneration was the first im- signaling could not induce regeneration after the limbs plication of Wnt signaling in zebrafish regeneration (Poss have completely lost the ability to regenerate at stage 58 et al. 2000a). After amputation of the zebrafish caudal (Fig. 1L,M). As was the case for axolotls, down-regulation fin, microinjection of the fin rays with the Ad-Dkk1 vi- of Wnt activity in Xenopus also impaired regeneration at rus resulted in an abrupt halt in the regeneration that stages where the potential was still present (Fig. 1E–G; normally would take place in an amputated, but not in- fected or Ad-eGFP-infected, caudal fin (Fig. 2A,B; data not shown). Similarly, down-regulation of the Wnt path- way in amputated pectoral fins led to a decrease in their regeneration capability (Fig. 2E,F; Supplementary Table S1). Thus, as in axolotls and Xenopus, blockade of the Wnt signaling pathway impairs the ability of different zebrafish appendages to regenerate. The variations in the ability to block regeneration by Ad-Dkk1 among the dif- ferent species may reflect differences in adenovirus in- fectivity and/or differences in promoter efficiency to drive transgene expression in the animal model systems Figure 1. Wnt/␤-catenin signaling is required for limb regeneration in axolotls and Xenopus. (A–D) Gross morphology and scanning electron microscope (SEM) images of adult (A,B) and larvae (C,D) axolotls. (A) A fully regenerated wild-type adult axolotl forelimb develops 2 mo after amputation at the elbow level. (B) Microinjec- tion of Ad-Axin1 after forelimb amputation prevented regeneration of the distal elements (red arrowhead). (C) SEM image of an axolotl larvae limb at the stage at which amputation was performed (stage 45). (D) Ventral view of axolotl larvae limbs 1 mo after amputation and virus injection.
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