The Hippo signalling pathway has been evolutionary conserved and shown to play a key role in controlling organ size through regulating the balance between cell proliferation and cell death. Also, in mammals the Hippo pathway acts as a tumor suppressor. When the Hippo pathway gets activated the transcriptional coactivator Yorkie (known as Yap in mammals) is repressed. On the other side, when the Hippo pathway is inactive, Yorkie is active. Yorkie induces cell proliferation and inhibits apoptosis and, therefore, its silencing results in a decrease in cell proliferation and increase in cell death.
A recent paper from the laboratory of Eugene Berezikov reports on the function of the Hippo pathway in the flatworm Macrostomum lignano (http://www.ncbi.nlm.nih.gov/pubmed/23495768). Similarly to freshwater planarians, M. lignano is a growing model to study stem cell-based regeneration. All these different flatworms are an excellent model in which to study the role of signalling pathways on stem cell regulation in vivo. In this study, the authors first isolated the homologues in M. lignano of the core components of the Hippo pathway including Hippo (Hpo), Salvador (Sav), Warts (Wts), Mats and Yorkie (Yki/Yap). Whole mount in situ hybridizations showed that in adult animals Hpo, Sav, Wts and Mats show similar expression patterns, being enriched in gonads and also found in neoblasts (pluripotent stem cells) and differentiated tissues. On the other hand, Yap is expressed specifically in the gonads and neoblasts as its expression is completely abrogated after irradiation (a way to eliminate all the neoblasts and dividing cells in flatworms).
Then the authors moved forward to functionally characterize all these genes first in adult homeostatic animals and then during regeneration. In adults, 2 weeks after RNAi treatment to silence Hpo, Sav, Wts and Mats the animals start to develop small outgrowths along the body. After 4-6 weeks there is an increase in the size of these outgrowths and epidermal bulges form throughout the body. Finally, all animals die within 8 weeks of treatment. In contrast, the silencing of Yap results in head regression followed by ventral curling and lysis of the whole animal, a phenotype that resembles those obtained in freshwater planarians when they are depleted of neoblasts. Compared to freshwater planarians M. lignano shows more limited regenerative abilities; however, when amputated behind the head they can regenerate a new posterior part. After Hpo(RNAi) animals regenerate a new posterior part but, remarkably, this new part appears to be larger than in controls. Moreover, the animals also develop bulges around the cutting site and show other morphological aberrations and disrupted allometric scaling. However, cell differentiation does not seem to be impaired in them. All these morphological phenotypes correlate with an increased cell proliferation detected in the blastemas of these animals after silencing Hpo. In contrast the silencing of Yap results in a significant decrease in the number of proliferating cells within the blastema, which blocks the regeneration of a new posterior region. In few days all the animals die.
Finally, the authors used BrdU to label neoblasts after silencing Hpo and Yap in adult homeostatic animals. By day 10 of treatment the number of S-phase cells is significantly higher in Hpo(RNAi) and significantly lower in Yap(RNAi), compared to controls. By day 20 not only the number of S-phase cells was much higher after Hpo(RNAi) but also were found all throughout the body, compared to the more restricted spatial distribution found in controls.
In summary, the results reported in this paper clearly show that the Hippo pathway is conserved in the flatworm M. lignano and, more importantly, that plays a key role in regulating neoblast biology during homeostasis and regeneration. Similarly to mammals the Hippo pathway acts as a tumor suppressor in these flatworms as Hpo(RNAi) animals develop outgrowths and bulges all throughout the body. Also, Yap appears to be required to maintain neoblasts self-renewal, which agrees with the role of Yap in maintaining the pluripotency of mammalian embryonic and induced stem cells. Further studies in M. lignano could help to characterize upstream and downstream elements of this pathway to better understand how the Hippo pathway regulates the size of the regenerating parts by controlling stem cell proliferation, cell death and differentiation.