The size and shape of tissues are tightly controlled by synchronized processes among cells and tissues to produce an integrated organ. The Hippo signaling pathway controls both cell proliferation and apoptosis by dual signal-transduction states regulated through a repressive kinase cascade. Yap1 and Tead, transcriptional regulators that act downstream of the Hippo signaling kinase cascade, have essential roles in regulating cell proliferation. In amphibian limb or tail regeneration, the local tissue outgrowth terminates when the correct size is reached, suggesting that organ size is strictly controlled during epimorphic organ-level regeneration. We recently demonstrated that Yap1 is required for the regeneration of Xenopus tadpole limb buds (Hayashi et al., 2014, Dev. Biol. 388, 57-67), but the molecular link between the Hippo pathway and organ size control in vertebrate epimorphic regeneration is not fully understood. To examine the requirement of Hippo pathway transcriptional regulators in epimorphic regeneration, including organ size control, we inhibited these regulators during Xenopus tadpole tail regeneration by overexpressing a dominant-negative form of Yap (dnYap) or Tead4 (dnTead4) under a heat-shock promoter in transgenic animal lines. Each inhibition resulted in regeneration defects accompanied by reduced cell mitosis and increased apoptosis. Single-cell gene manipulation experiments indicated that Tead4 cell-autonomously regulates the survival of neural progenitor cells in the regenerating tail. In amphibians, amputation at the proximal level of the tail (deep amputation) results in faster regeneration than that at the distal level (shallow amputation), to restore the original-sized tail with similar timing. However, dnTead4 overexpression abolished the position-dependent differential growth rate of tail regeneration. These results suggest that the transcriptional regulators in the Hippo pathway, Tead4 and Yap1, are required for general vertebrate epimorphic regeneration as well as for organ size control in appendage regeneration. In regenerative medicine, these findings should contribute to the development of three-dimensional organs with the correct size for a patient's body.
Bibliographical noteFunding Information:
We thank Drs. Hiroshi Sasaki and Yoshikazu Hirate for providing the cDNA encoding the dominant-negative form of Tead4 (dnTead4). We thank Drs. Stefan Hoppler, Tim Mohun, Masahiko Hibi, and Roger Tsien for the Xenopus hsp70 promoter, Xenopus γ-crystallin promoter, 2A peptide, and tdTomato, respectively. We thank Dr. Yumi Izutsu for providing the hsp70-GFP Tg Xenopus frogs. We thank Yoshiko Yoshizawa-Ohuchi and Natsume Sagawa for excellent animal care. We thank all the staff members of the Spectrography and Bioimaging Facility and the NIBB Core Research Facilities for kind support in setting up the IR-LEGO experiments in Xenopus. This work was supported by MEXT and JSPS KAKENHI Grant number 22124005 to HY, 25124704 to H. Ochi, 25124705 to H. Ogino, JSPS KAKENHI Grant number 25870058 to HY, “Funding Program for Next Generation World-Leading Researchers ” (Grant no. LS007 ) from the Cabinet Office, Government of Japan to KT, CREST (JST) to H. Ogino, the Kurata Memorial Hitachi Science and Technology Foundation to HY, and the Asahi Glass Foundation to HY. This work was also supported by the NIBB Collaborative Research Program (Grant no. 12-367 , 13-347 , 14-330 ) to HY. This work was also supported by MEXT and JSPS KAKENHI Grant number 22124001 .
© 2014 Elsevier Inc.
- Hippo pathway
- Size control
- Tail regeneration