Different requirements for conserved post-transcriptional regulators in planarian regeneration and stem cell maintenance

Abstract

Planarian regeneration depends on the presence and precise regulation of pluripotent adult somatic stem cells named neoblasts, which differentiate to replace cells of any missing tissue. A characteristic feature of neoblasts is the presence of large perinuclear nonmembranous organelles named "chromatoid bodies", which are comparable to ribonucleoprotein structures found in germ cells of organisms across different phyla. In order to better understand regulation of gene expression in neoblasts, and potentially the function and composition of chromatoid bodies, we characterized homologues to known germ and soma ribonucleoprotein granule components from other organisms and analyzed their function during regeneration of the planarian Dugesia japonica. Expression in neoblasts was detected for 49 of 55 analyzed genes, highlighting the prevalence of post-transcriptional regulation in planarian stem cells. RNAi-mediated knockdown of two factors [ago-2 and bruli] lead to loss of neoblasts, and consequently loss of regeneration, corroborating with results previously reported for a bruli ortholog in the planarian Schmidtea mediterranea (Guo et al., 2006). Conversely, depletion mRNA turnover factors [edc-4 or upf-1], exoribonucleases [xrn-1 or xrn-2], or DEAD box RNA helicases [Djcbc-1 or vas-1] inhibited planarian regeneration, but did not reduce neoblast proliferation or abundance. We also found that depletion of cap-dependent translation initiation factors eIF-3A or eIF-2A interrupted cell cycle progression outside the M-phase of mitosis. Our results show that a set of post-transcriptional regulators is required to maintain the stem cell identity in neoblasts, while another facilitates proper differentiation. We propose that planarian neoblasts maintain pluripotency by employing mechanisms of post-transcriptional regulation exhibited in germ cells and early development of most metazoans.

Comprehensive gene expression analyses in pluripotent stem cells of a planarian, Dugesia japonica

“…Cells expressing #008 (piwiA) completely disappeared in the X-ray-irradiated animals ( Fig. 3D), indicating that this gene was specifically expressed in the neoblasts, as previously described (Yoshida-Kashikawa et al, 2007;Rouhana et al, 2010;Shibata et al, 2010). Genes such as #012 (P2X-A) and #032 (unknown) showed the same expression patterns as #008 (piwiA) in intact and X-ray-irradiated animals (data not shown).…”

“…#008 (piwiA), showed a typical pattern of neoblast-specific expression, in which the distribution of positive cells was observed in the mesenchymal space from behind the eyes to the tail ( Fig. 3C; Yoshida-Kashikawa et al, 2007;Rouhana et al, 2010;. Cells expressing #008 (piwiA) completely disappeared in the X-ray-irradiated animals ( Fig.…”

Stem cells and neural signalling: the case of neoblast recruitment and plasticity in low dose X-ray treated planarians

“…CBs are electron dense perinuclear granules composed of RNA and proteins that decrease in number and size during cell differentiation, and, for this reason, are thought to be required for neoblast self-renewal and proliferation . In support of this hypothesis, a set of RNA-binding factors has been found to be necessary to maintain neoblast pluripotency (YoshidaKashikawa et al, 2007;Rouhana et al, 2010), and silencing of Smed-SmB, a homolog of a member of the LSm protein family, results in loss of CBs organization and severe neoblasts proliferation failure (Fernandéz-Taboada et al, 2010). Moreover, Solana and colleagues (2009) demonstrated that a tudor homolog, Spoltud-1, is a component of CBs and is required for neoblast maintenance.…”

Small RNA pathways in Schmidtea mediterranea

“…Recent studies in planarians identified several genes that encode RNA binding proteins such as TIA-1, LSm, eIF-4A, eIF-4G, TRDR-1, SMN, CBC-1, TUD-1, PIWI, DICER-1, FMRP-1 and Ago-2. These genes are expressed in neoblasts and some of them were shown to be essential for neoblast function and regeneration (Rouhana et al, 2010). In C. elegans, Drosophila and mammals, most of these proteins are associated with various RNP granules such as P-bodies, stress granules and chromatoid bodies (Kotaja et al, 2007).…”