The Hippo signaling pathway is conserved from insects to mammals and is important for multiple processes, including cell proliferation, apoptosis and tissue homeostasis. Hippo signaling is also crucial for regeneration, including intercalary regeneration, of the whole body in the flatworm and of the leg in the cricket. However, its role in vertebrate epimorphic regeneration is unknown. Therefore, to identify principles of regeneration that are conserved among bilaterians, we investigated the role of Hippo signaling in the limb bud regeneration of an anuran amphibian, Xenopus laevis. We found that a transcription factor, Yap1, an important downstream effector of Hippo signaling, is upregulated in the regenerating limb bud. To evaluate Yap1's function in limb bud regeneration, we made transgenic animals that expressed a dominant-negative form of Yap under a heat-shock promoter. Overexpression of a dominant-negative form of Yap in tadpoles reduced cell proliferation, induced ectopic apoptosis, perturbed the expression domains of limb-patterning genes including hoxa13, hoxa11, and shh in the regenerating limb bud. Transient expression of a dominant-negative Yap in transgenic tadpoles also caused limb bud regeneration defects, and reduced intercalary regeneration.These results indicate that Yap1 has a crucial role in controlling the limb regenerative capacity in Xenopus, and suggest that the involvement of Hippo signaling in regeneration is conserved between vertebrates and invertebrates. This finding provides molecular evidence that common principles underlie regeneration across phyla, and may contribute to the development of new therapies in regenerative medicine.
Bibliographical noteFunding Information:
We thank Drs. Hajime Ogino and Haruki Ochi for technical advice on I-SceI meganuclease-mediated transgenesis, Drs. Hiroshi Sasaki and Yoshikazu Hirate for providing expression constructs of Hippo components, and Dr. Jose F. de Celis for the mkp3 construct. We thank Drs. Stefan Hoppler, Tim Mohun, Masahiko Hibi, and Roger Tsien for their respective gifts of the Xenopus hsp70 promoter, Xenopus γ-crystalline promoter, 2A peptide, and tdTomato. We thank Yoshiko Yoshizawa-Ohuchi for frog care and Natsume Sagawa for maintaining the frog facility. This work was supported by MEXT and JSPS KAKENHI Grant number 22124005 to HY, JSPS KAKENHI Grant number 25870058 to HY, “Funding Program for Next Generation World-Leading Researchers” [LS007] from the Cabinet Office, Government of Japan to KT, the Kurata Memorial Hitachi Science and Technology Foundation to HY, and the Asahi Glass Foundation to HY.
- Hippo pathway
- Limb bud regeneration