As I have discussed before in this blog, the Hippo signalling pathway has a conserved function in controlling organ size and patterning through the regulation of the balance between cell proliferation and cell death. During regeneration these processes must be tightly regulated so the regenerated organs and structures attain proper sizes. However, not much is known yet about the role of Hippo signalling during regeneration. Previous reports have shown that this pathway is required for proper insect leg as well as Macrostomum (flatworm) regeneration. In fact, for Macrostomum, I discussed those results in this blog (https://regenerationinnature.wordpress.com/2013/06/27/the-hippo-pathway-in-macrostomum-homeostasis-and-regeneration/).
Now a recent paper from the laboratory of Hitoshi Yokoyama reports a role for Yap1 during regeneration of the Xenopus limb bud (http://www.ncbi.nlm.nih.gov/pubmed/24491818). The transcriptional co-factor Yap1 (Yorkie in invertebrates) is the canonical effector of the Hippo signalling pathway. When the Hippo pathway is activated, Yap1 gets phosphorylated and becomes inactive as it gets trapped in the cytoplasm. On the other side, active dephosphorylated Yap1 can enter the nucleus where it activates the expression of its target genes. In other systems it has been shown how the over activation of Yap1 (or Yorkie) leads to an increase of proliferation and a decrease of apoptosis. Conversely, the inactivation of Yap1 results in decreased cell proliferation and higher levels of apoptosis.
In this study, the authors analysed the regeneration of amputated limb buds of Xenopus tadpoles at stage 52. In a first set of experiments they found that all the main core components of the Hippo pathway, including some regulators and target genes, were expressed in 5-day blastemas. As Yap1 is the effector of the pathway they focussed on this gene. In situ hybridizations showed that Yap1 was upregulated within the blastema. Taking advantage of the cross-reaction of an antibody against human Yap1 they could see how in intact limb buds Yap1 was in the cytoplasm whereas, upon amputation, Yap1 was localized in the nuclei of blastema cells. These results suggested that Yap1 was translocated to the nuclei during regeneration where it could activate its target genes. Next, the authors characterized the function of Yap1 during regeneration by using a dominant-negative form of this gene (dnYap) under control of a heat shock promoter. Overexpression of dnYap resulted in impaired limb regeneration characterized by a reduction in the size and number of skeletal elements, as well as a reduction in the number of digits in those regenerated limbs. Interestingly, the overexpression of dnYap did not apparently affect the normal development of the uncut contralateral limb bud.
In addition to these external defects, the authors checked the expression of several patterning genes with well-defined and region-specific patterns. The overexpression of dnYap impaired the expression of hoxa13 and hoxa11 so both genes partially overlapped in contrast to their expression in well-separated domains in controls. Also, the expression of shh, fgf8 and mkp3 was clearly downregulated. Moreover, the small blastemas of dnYap animals contained much less differentiated muscle and innervation. As the Hippo pathway controls cell proliferation and apoptosis the authors checked how these events were affected after overexpressing dnYap. The number of mitotic cells significantly decreased within the blastema as well as in the stump. On the other side, ectopic apoptotic cells were found the stump. These results suggested that the impaired regeneration phenotype was associated to missregulation of cell proliferation and apoptosis. Again, the overexpression of dnYap did not affect cell proliferation, apoptosis or differentiation in uncut limb buds and they developed into normal limbs.
In summary, this paper shows how Yap1 is required for limb bud regeneration in Xenopus. In this context Yap1 appears to have a conserved role in the regulation of cell proliferation and apoptosis. Future experiments should determine whether other elements of this pathway as well as its upstream regulators are equally conserved between vertebrates and invertebrates and are also required for regeneration.