Yap1 and Hippo signaling pathway are essential for Xenopus limb bud regeneration

 This article is based on the paper entitled “Yap1, transcription regulator in the Hippo signaling pathway is required for Xenopus limb bud regeneration” written by Shinichi Hayashi, Koji Tamura, and Hitoshi Yokoyama and was published in Developmental Biology Volume 388 on April 2014.


Regeneration is commonly found among animals in order to maintain form and function throughout life. Regeneration restores the normal structure and function of organs and tissues, essential for survival. All multicellular organisms have some form of regeneration, but the ability and performance of the process differ among species. For example, worms, planarians, and members in the phylum of echinoderms, can regenerate whole animals from tiny fragments of almost any part of the body after amputation (Bonfanti, 2011). In contrast, some fish have the ability to regenerate complex body structures such as the limb, tail, eye, lens, and even the heart.  In amphibians, only anuran tadpoles and urodeles can fully regenerate amputated limbs (Staube and Tanaka, 2006).

photo of Xenopus laevis obtained from http://hermes.mbl.edu/news/press_releases/2010/2010_pr_08_17.html

Model organisms are essential in providing knowledge on regenerative properties. They help provide an understanding of the modes and mechanism involved in the regenerative process in order to better understand how to use these properties for future biomedicine. Though the ability of regeneration is different among species, the species may have common mechanisms such as amphibian limb bud regeneration which include: signal transduction and signaling molecules, tissue interaction among wound epithelium, mesenchyme and neural axons, and positional information and intercalation (Alvarado and Tsonis, 2006).

In the amphibian regeneration process, an epithelial sheet, the wound epithelium covers the amputation plane to protect stump cells from the external environment which thickens and becomes the AEC (apical epithelial cap). The AEC acts as a signaling center for regeneration of the limb. A protein encoded by fgf8 is expressed in AEC covering the limb blastema, a cluster of highly proliferative and undifferentiated mesenchymal cells derived from the stump. The blastema is reconstituted to the same architecture as the original limb guided by AP (anterior-posterior) position information and determined by Shh (Sonic Hedgehog) (Torok et al., 1999).

Hippo Signaling Pathway & Yap 1 expression

The Hippo Signaling Pathway is used for proper regulation of organ growth. The pathway transduces signals from the plasma membrane to the nucleus where it regulates gene expression. It is also important for cell proliferation, apoptosis and tissue homeostasis. Yap 1 is a key transcriptional regulator of the Hippo Signaling. It is inactivated when phosphorylated and trapped in the cytoplasm but when dephosphorylated it is translocated in the nucleus to activate target genes through DNA-binding proteins (Halder and Johnson, 2011).

Experimental Strategy

The experimental strategy is to analyze the contribution and importance of the Hippo pathway with a focus on yap1 and how it effects limb bud regeneration. Xenopus tadpoles were used in the experiment. Some methods used in this experiment are immunohistochemistry and RT-PCR (Reverse transcriptase-polymerase chain reaction).


The Hippo Signaling pathway is expressed in the blastema

Gene expression of the Hippo pathway components in the blastema of limb bud regeneration were examined by amputating a stage 52 tadpole limb bud at knee level and extracting RNA from blastema at 5 days post amputation. At stage 52, the limb bud has the ability to regenerate completely from the blastema. RT-PCR was performed for the core components of the Hippo pathway and as shown in figure 1A the core Hippo components were detected in the regenerative blastema.


Figure 1. A) RT-PCR of Hippo Signaling Pathway main components expressed during limb bud regeneration. (B-D) yap1 mRNA expression. B) yap1 mRNA is strongly expressed in the stump. C) yap1 mRNA is expressed througout the intact limb bud. D) The black line indicates where the limb was amputated. yap1 mRNA is strongly expressed in blastema but is also present in the stump (Hayashi, Tamura, and Yokoyama, 2014).

Yap 1 is upregulated in the blastema

Next, a study for the presence of yap1 in the blastema was performed due to the fact that yap1 regulates the on/off state for transcription of the Hippo target genes. Spatial patterning of yap1 mRNA expression in the limb bud blastema was examined and detected throughout the unoperated stage 51 limb bud (Figure 1B). However at 3 days post amputation, the yap1 expression was found to be greater in the blastema which indicates transcription of yap1 is upregulated in blastema cells (Figure 1D).

Yap 1 is in the nucleus during regeneration

Immunostaining using the antibody, anti-Yap1, showed that the protein was located in the developing limb bud. From figure 2, it can be seen that at 5 days post amputation, yap1 was present in both the intact limb bud and the regenerative blastema.  A confocal microcopy shows that yap1 protein  is in the blastema cells (Figure J-L) but shows that the protein is only present within the cytoplasm of the cells of the intact bud (Figure G-L). These findings show that yap1 is activated and translocated into the nucleus of blastema cells during regeneration.


Figure 2. Yap1 protein in limb bud regeneration in comparison to intact limb bud. White line indicates where amputated limb started. (A-C) No signal of antibody was detected in the intact limb and the 5 dpa (days post amputation) blastema. (D-F) yap1 protein detected in both intact and 5dpa. (G-L) Confocal microscopy, high magnification images of areas D-F. (G-I) yap1 protein is present in the cytoplasm of intact limb bud. (J-L) yap1 protein is localized in the nuclei of blastema cells (Hayashi, Tamura, and Yokoyama, 2014).


Yap 1 is necessary for limb bud regeneration

A dominant-negative form of Yap (dnYap) was used under control heat-shock promoter to create transgenic Xenopus tadpoles allowing overexpression of dnYap to inhibit endogenous yap1 function. As shown in figure 3, the transgene induction was diminished until 14 days post amputation, where the dnYap transgenic (Tg) froglets showed clear defects and reduction in size of skeletal elements in contrast to the wildtype (WT)  froglets which regenerated a complete limb. This indicates that yap1 expression is essential for Xenopus limb bud regeneration.


Figure 3. Loss of Yap1 function causing limp bud regeneration defects. (F-I) Regeneration in wildtype. (J-M) Regeneration in dnYap Tg (Hayashi, Tamura and Yokoyama, 2014).


The loss of Yap1 results in regeneration defects

Investigation whether the loss of yap1 function affected re-patterning of regenerating limb bud was performed by examining expressed patterns of region specific genes. From figure 4, it is apparent that the Tg tadpoles showed slowed growth of the gene or no gene expression whatsoever suggesting that yap1 expression is correlated to the outgrowth of the blastema and provision of cells from the stump.


Figure 4. Comparison of gene expression pattern between WT and Tg. (A,C, E, G, I) Normal expression of genes shown in wildtype. (B,D,F,H, J) Reduced or disrupted patterns of genes are expressed in dnYap transgenic blastemas (Hayashi, Tamura, Yokoyama, 2014).



From the results, it can be concluded that yap1 activity is upregulated in the blastema and plays an essential role to the Xenopus limb bud regeneration. The study also showed that yap1 gene expression translocates from the cytoplasm to the nucleus during activation in the blastema cells.  It is also important to note that the loss of yap1 function causeS regeneration defects in the limb bud and that yap1 is necessary for the Hippo Signaling Pathway.


Overall, the article was well-written. The heading and subheading provided an easy read. The conclusions after each paragraph in the results portion of the article gave a better understanding of what the main goal in each study of the experiment was exactly aiming for. The discussion, however, could have been written better in the sense that the authors would mention Wnt signaling and BMP without explaining the importance of these signals and how it affected the study. But, the figures and supplemental section helped in proper explanation of the experiment performed although the figure captions could have used more detail.


Bonfanti, L., 2011. From hydra regeneration to human brain structural plasticity: a long trip through narrowing roads. Sci. World J. 11, 1270–1299. http://www.hindawi.com/journals/tswj/2011/936817/abs/

Halder, G., Johnson, R.L., 2011. Hippo signaling: growth control and beyond. Development. 138, 9–22. http://dev.biologists.org/content/138/1/9.full

Sanchez Alvarado, A., Tsonis, P.A., 2006. Bridging the regeneration gap: genetic insights from diverse animal models. Nat. Rev. Genet. 7, 873–884. http://planaria.stowers.org/publications/NRG2006.pdf

Straube, W.L., Tanaka, E.M., 2006. Reversibility of the differentiated state: regeneration in amphibians. Artif. Org. 30, 743–755. http://onlinelibrary.wiley.com/doi/10.1111/j.1525-1594.2006.00296.x/pdf

Torok, M.A., Gardiner, D.M., Izpisua-Belmonte, J.C., Bryant, S.V., 1999. Sonic hedgehog (shh) expression in developing and regenerating axolotl limbs. J. Exp. Zool. 284, 197–206. http://onlinelibrary.wiley.com/doi/10.1002/(SICI)1097-010X(19990701)284:2%3C197::AID-JEZ9%3E3.0.CO;2-F/pdf


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