We also identified the two main components of mTORC2, RICTOR and SIN1, which we named Smed-rictor and Smed-sin1, respectively (Figure S13A; GenBank Accession Numbers “type”:”entrez-nucleotide”,”attrs”:”text”:”JN815259″,”term_id”:”383386066″,”term_text”:”JN815259″JN815259 selleckbio and “type”:”entrez-nucleotide”,”attrs”:”text”:”JN815260″,”term_id”:”383386068″,”term_text”:”JN815260″JN815260). Figure 4 Smed-tor and Smed-raptor are necessary for the response to injury and blastema growth. Consistent with their broad organismal role we observed that, in addition to being expressed broadly in the planarian body, Smed-tor and Smed-raptor were expressed in most neoblasts (Figure 4B and 4C). Abnormal neoblast proliferation in Smed-tor RNAi animals has been already described [8].

Although Smed-tor(RNAi) animals were able to close wounds after amputation, they were not able to form blastemas, even after more than 25 dR (Figure 4D and 4E). These animals lacked the first mitotic regeneration peak (P<0.01) and mitotic levels during regeneration were lower than controls (P<0.05) (Figure 4F). This difference was not due to a decrease in the number of neoblasts present before amputation (Figure S5), suggesting that the effects we observe are related to the control of mitotic responses to wounding and subsequent regeneration. Consistent with RAPTOR phenocopying TOR in other organisms [34], Smed-raptor(RNAi) induced the same phenotype as Smed-tor(RNAi), albeit with a weaker penetrance (Figure 4D).

Although qPCR experiments showed that RNAi experiments downregulated Smed-lst8 expression in a similar way to Smed-raptor after RNAi (Figure S12C), we observed a relatively weak phenotype that was nonetheless in agreement with the phenotypes described for Smed-tor and Smed-raptor (Figure S12B). RNAi for TORC2 components Smed-rictor and Smed-sin1 did not show any apparent phenotype after more than 40 days of regeneration, even after three rounds of RNAi injections or combining RNAi of both genes (Figure S13B). The Anacetrapib mRNA levels were however downregulated at similar values as in Smed-raptor RNAi experiments or Smed-smg-1 RNAi experiments (Figure S13C). Interestingly, it has already been shown that mTOR signalling is important for the proper balance of stem cell self-renewal and differentiation. For instance, mTOR downregulation has been shown to suppress embryonic stem cell self-renewal while enhancing endodermal and mesodermal differentiation [35]. We wanted to investigate if down-regulation of Smed-tor and Smed-raptor, in addition to decreasing, proliferation would enhance differentiation.