Yorkie is required to restrict the injury responses in planarians

Abstract

Regeneration requires the precise integration of cues that initiate proliferation, direct differentiation, and ultimately re-pattern tissues to the proper size and scale. Yet how these processes are integrated with wounding responses remains relatively unknown. The freshwater planarian, Schmidtea mediterranea, is an ideal model to study the stereotyped proliferative and transcriptional responses to injury due to its high capacity for regeneration. Here, we characterize the effector of the Hippo signalling cascade, yorkie, during planarian regeneration and its role in restricting early injury responses. In yki(RNAi) regenerating animals, wound responses are hyper-activated such that both stem cell proliferation and the transcriptional wound response program are heighted and prolonged. Using this observation, we also uncovered novel woundinduced genes by RNAseq that were de-repressed in yki(RNAi) animals compared with controls. Additionally, we show that yki(RNAi) animals have expanded epidermal and muscle cell populations, which we hypothesize are the increased sources of wound-induced genes. Finally, we show that in yki(RNAi) animals, the sensing of the size of an injury by eyes or the pharynx is not appropriate, and the brain, gut, and midline cannot remodel or scale correctly to the size of the regenerating fragment. Taken together, our results suggest that yki functions as a key molecule that can integrate multiple aspects of the injury response including proliferation, apoptosis, injury-induced transcription, and patterning. Author summaryThe planarian displays a remarkable ability to regenerate any tissue from mere fragments of its original size. This high capacity to regenerate is attributed to the abundant population of pluripotent adult stem cells. In response to an injury, such as an amputation, stem cells proliferate and replace the lost tissues de novo (epimorphosis), whereas existing tissue must rescale to the correct proportions in relation to the new fragment size (morphallaxis). Currently, the molecules that control either the responses to injury or the ones that mediate size and scaling are not well understood. For instance, how are the injury responses precisely activated and shut down to ensure regenerating tissues are not underor overgrown? Here, we find that Yki, the effector of the Hippo signalling cascade, is a critical molecule that influences several injury processes during regeneration. Loss of Yki function in regenerating animals resulted in increased and temporally dysregulated expression of wound-induced genes, proliferation, and apoptosis. Genes that are injury induced were mis-expressed in yki(RNAi) animals, which also showed increases in the epidermal and muscle cell populations. Taken together, our findings suggest that the injury responses must be restricted to ensure proper regenerative outcomes of correct scale, and that Yki is a key regulator in these processes.

Hippo signaling controls cell cycle and restricts cell plasticity in planarians

“…Inhibition of yki in regenerating animals led to an increase in the numbers of differentiated neurons and photoreceptors ( Fig 4C and S6D Fig ), in direct opposition to the phenotype observed following hippo inhibition. In agreement with our results, a recent study [ 43 ] reported a general increase in the numbers of several types of differentiated cells in yki (RNAi) planarians. Taken together, these results indicate that a conserved Hippo-Yki signal regulates cell differentiation during planarian regeneration.…”

“…Moreover, the observed defects in differentiation may be caused by the dedifferentiation of postmitotic cells in pre-existing tissue after hippo RNAi, as previously described in intact animals, leading to misexpression by differentiated muscle cells of signaling molecules required for proper differentiation [ 60 ]. This possibility is consistent with the phenotype generated after yki inhibition in planarians, in which an increase in signaling molecules and differentiated cells is observed [ 43 ] ( Fig 4C and S6 Fig ).…”

The DEAD-box helicase DDX56 is a conserved stemness regulator in normal and cancer stem cells

“…TUNEL was performed as previously described (Lin and Pearson, 2017;Pellettieri et al, 2010). Briefly, worms were labeled with DIG-11-dUTP using terminal deoxynucleotidyl transferase for 4 h at 37 C, and then detected with anti-DIG-POD (1:1000) and tyramide amplification.…”

“…qPCR was performed as previously described (Lin and Pearson, 2017). Briefly, RNA was isolated using Trizol reagent and cDNA was prepared using SuperScript III Reverse Transcriptase (Invitrogen).…”

Rebuilding a planarian: from early signaling to final shape

“…Those results show the relevance of the insulin-TOR pathway in the control of growth in planarians, although further studies are required for a deeper understanding.The Hippo pathway is a conserved signaling pathway that integrates signals controlling proliferation, cell death, and differentiation during development (Pfleger, 2017). While inhibition of yki results in blotted planarians due to altered homeostasis of the excretory system (Lin and Pearson 2014), in-depth molecular analyses have shown that yki RNAi animals display a hyper-activated wound response (Lin and Pearson 2017). Thus, inhibition of yki induces blastema regeneration, with increases both in cell number and in the number of differentiated cells.…”