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A mex3 homolog is required for differentiation during planarian stem cell lineage development.

Zhu SJ, Hallows SE, Currie KW, Xu C, Pearson BJ - Elife (2015)

Bottom Line: In this study, we used transcriptional profiling of irradiation-sensitive and irradiation-insensitive cell populations and RNA interference (RNAi) functional screening to uncover markers and regulators of postmitotic progeny.We also demonstrated the utility of using mex3-1(RNAi) animals to identify additional progenitor markers.These results identified mex3-1 as a cell fate regulator, broadly required for differentiation, and suggest that mex3-1 helps to mediate the balance between ASC self-renewal and commitment.

View Article: PubMed Central - PubMed

Affiliation: Program in Developmental and Stem Cell Biology, The Hospital for Sick Children, Toronto, Canada.

ABSTRACT
Neoblasts are adult stem cells (ASCs) in planarians that sustain cell replacement during homeostasis and regeneration of any missing tissue. While numerous studies have examined genes underlying neoblast pluripotency, molecular pathways driving postmitotic fates remain poorly defined. In this study, we used transcriptional profiling of irradiation-sensitive and irradiation-insensitive cell populations and RNA interference (RNAi) functional screening to uncover markers and regulators of postmitotic progeny. We identified 32 new markers distinguishing two main epithelial progenitor populations and a planarian homolog to the MEX3 RNA-binding protein (Smed-mex3-1) as a key regulator of lineage progression. mex3-1 was required for generating differentiated cells of multiple lineages, while restricting the size of the stem cell compartment. We also demonstrated the utility of using mex3-1(RNAi) animals to identify additional progenitor markers. These results identified mex3-1 as a cell fate regulator, broadly required for differentiation, and suggest that mex3-1 helps to mediate the balance between ASC self-renewal and commitment.

No MeSH data available.


Related in: MedlinePlus

Identification and analysis of MEX3 homologs in S. mediterranea.(A) Phylogenetic analysis of the mex3 gene family in planarians shows that they are likely the results of planarian-specific duplications. A Bayesian phylogeny was run as outlined in the ‘Materials and methods’. Only posterior probabilities 50% and above are shown. S. mediterranea sequences are in red. Multiple mex3 homologs could not be found in individual species of other flatworms. (B) Expression levels of mex3-2 and mex3-3 in RNAseq of FACS-isolated populations and control intact worms. Expression of mex3 homologs by WISH after lethal irradiation (60 Gy) is shown below. (C) Phenotypes of mex3-2 and mex3-3 RNAi animals during homeostasis (upper panel) and during regeneration after amputation (lower panel). Species sequences used in the phylogeny: Smed = Schmidtea mediterranea; S. mansoni = Schistosoma mansoni; Echinococcus = Echinococcus multilocularis; Aplysia = Aplysia californica; Ci = Ciona intestinalis; Lg = Lottia gigantea; Dm = Drosophila melanogaster; Tc = Tribolium castaneum; Nvit = Nasonia vitripennis; Mm = Mus musculus; Xt = Xenopus tropicalis; Nvect = Nematostella vectensis; Sp = Strongylocentrotus purpuratus.DOI:http://dx.doi.org/10.7554/eLife.07025.012
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fig3s1: Identification and analysis of MEX3 homologs in S. mediterranea.(A) Phylogenetic analysis of the mex3 gene family in planarians shows that they are likely the results of planarian-specific duplications. A Bayesian phylogeny was run as outlined in the ‘Materials and methods’. Only posterior probabilities 50% and above are shown. S. mediterranea sequences are in red. Multiple mex3 homologs could not be found in individual species of other flatworms. (B) Expression levels of mex3-2 and mex3-3 in RNAseq of FACS-isolated populations and control intact worms. Expression of mex3 homologs by WISH after lethal irradiation (60 Gy) is shown below. (C) Phenotypes of mex3-2 and mex3-3 RNAi animals during homeostasis (upper panel) and during regeneration after amputation (lower panel). Species sequences used in the phylogeny: Smed = Schmidtea mediterranea; S. mansoni = Schistosoma mansoni; Echinococcus = Echinococcus multilocularis; Aplysia = Aplysia californica; Ci = Ciona intestinalis; Lg = Lottia gigantea; Dm = Drosophila melanogaster; Tc = Tribolium castaneum; Nvit = Nasonia vitripennis; Mm = Mus musculus; Xt = Xenopus tropicalis; Nvect = Nematostella vectensis; Sp = Strongylocentrotus purpuratus.DOI:http://dx.doi.org/10.7554/eLife.07025.012

Mentions: The self-renewal of ASCs and the appropriate differentiation of postmitotic progeny are the driving force behind homeostatic cell turnover and regeneration of all tissues in planarians (Newmark and Sánchez Alvarado, 2000; Rossi et al., 2008; Baguna, 2012; van Wolfswinkel et al., 2014). RNAi against genes required for differentiation, such as p53, CHD4, zfp-1, and vasa-1, results in the decline of postmitotic progeny without depletion of ASCs, and subsequent defects in tissue homeostasis and regeneration (Pearson and SánchezAlvarado, 2010; Scimone et al., 2010; Wagner et al., 2012). To determine whether progeny-enriched genes were regulators of postmitotic fates, we used RNAi knockdown against our set of 100 X2-enriched and 20 WThighXlow genes and screened for the above phenotypes. In agreement with previous data, RNAi against prog-1 and prog-2 separately or together did not yield any detectable phenotype (Eisenhoffer et al., 2008). Unexpectedly, none of the new epithelial progenitor markers yielded phenotypes upon knockdown either, suggestive of considerable functional redundancy among these genes. In contrast, knockdown of a gene encoding a homolog to the RNA-binding protein MEX3, Smed-mex3-1 (mex3-1) produced phenotypes highly suggestive of defective stem cell lineage progression. mex3-1(RNAi) was completely penetrant and lethal, resulting in ventral curling, head regression, and dorsal lesioning during homeostasis, as well as loss of regenerative ability after amputation (Figure 3A–C), indicative of epithelial homeostasis defects as well as overall stem cell impairment (Bardeen and Baetjer, 1904; Reddien et al., 2005a). Using reciprocal BLAST, we identified two other MEX3 homologs in S. mediterranea (mex3-2 and mex3-3, transcripts SmedASXL_000637 and SmedASXL_01505, respectively), which both contained two KH RNA-binding domains and had top reciprocal BLAST hits in mouse, fly, and C. elegans. These additional MEX3 homologs were neither irradiation-sensitive nor produced observable phenotypes after knockdown and thus not investigated further (Figure 3—figure supplement 1).10.7554/eLife.07025.011Figure 3.mex3-1 is required for tissue homeostasis and regeneration.


A mex3 homolog is required for differentiation during planarian stem cell lineage development.

Zhu SJ, Hallows SE, Currie KW, Xu C, Pearson BJ - Elife (2015)

Identification and analysis of MEX3 homologs in S. mediterranea.(A) Phylogenetic analysis of the mex3 gene family in planarians shows that they are likely the results of planarian-specific duplications. A Bayesian phylogeny was run as outlined in the ‘Materials and methods’. Only posterior probabilities 50% and above are shown. S. mediterranea sequences are in red. Multiple mex3 homologs could not be found in individual species of other flatworms. (B) Expression levels of mex3-2 and mex3-3 in RNAseq of FACS-isolated populations and control intact worms. Expression of mex3 homologs by WISH after lethal irradiation (60 Gy) is shown below. (C) Phenotypes of mex3-2 and mex3-3 RNAi animals during homeostasis (upper panel) and during regeneration after amputation (lower panel). Species sequences used in the phylogeny: Smed = Schmidtea mediterranea; S. mansoni = Schistosoma mansoni; Echinococcus = Echinococcus multilocularis; Aplysia = Aplysia californica; Ci = Ciona intestinalis; Lg = Lottia gigantea; Dm = Drosophila melanogaster; Tc = Tribolium castaneum; Nvit = Nasonia vitripennis; Mm = Mus musculus; Xt = Xenopus tropicalis; Nvect = Nematostella vectensis; Sp = Strongylocentrotus purpuratus.DOI:http://dx.doi.org/10.7554/eLife.07025.012
© Copyright Policy
Related In: Results  -  Collection

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fig3s1: Identification and analysis of MEX3 homologs in S. mediterranea.(A) Phylogenetic analysis of the mex3 gene family in planarians shows that they are likely the results of planarian-specific duplications. A Bayesian phylogeny was run as outlined in the ‘Materials and methods’. Only posterior probabilities 50% and above are shown. S. mediterranea sequences are in red. Multiple mex3 homologs could not be found in individual species of other flatworms. (B) Expression levels of mex3-2 and mex3-3 in RNAseq of FACS-isolated populations and control intact worms. Expression of mex3 homologs by WISH after lethal irradiation (60 Gy) is shown below. (C) Phenotypes of mex3-2 and mex3-3 RNAi animals during homeostasis (upper panel) and during regeneration after amputation (lower panel). Species sequences used in the phylogeny: Smed = Schmidtea mediterranea; S. mansoni = Schistosoma mansoni; Echinococcus = Echinococcus multilocularis; Aplysia = Aplysia californica; Ci = Ciona intestinalis; Lg = Lottia gigantea; Dm = Drosophila melanogaster; Tc = Tribolium castaneum; Nvit = Nasonia vitripennis; Mm = Mus musculus; Xt = Xenopus tropicalis; Nvect = Nematostella vectensis; Sp = Strongylocentrotus purpuratus.DOI:http://dx.doi.org/10.7554/eLife.07025.012
Mentions: The self-renewal of ASCs and the appropriate differentiation of postmitotic progeny are the driving force behind homeostatic cell turnover and regeneration of all tissues in planarians (Newmark and Sánchez Alvarado, 2000; Rossi et al., 2008; Baguna, 2012; van Wolfswinkel et al., 2014). RNAi against genes required for differentiation, such as p53, CHD4, zfp-1, and vasa-1, results in the decline of postmitotic progeny without depletion of ASCs, and subsequent defects in tissue homeostasis and regeneration (Pearson and SánchezAlvarado, 2010; Scimone et al., 2010; Wagner et al., 2012). To determine whether progeny-enriched genes were regulators of postmitotic fates, we used RNAi knockdown against our set of 100 X2-enriched and 20 WThighXlow genes and screened for the above phenotypes. In agreement with previous data, RNAi against prog-1 and prog-2 separately or together did not yield any detectable phenotype (Eisenhoffer et al., 2008). Unexpectedly, none of the new epithelial progenitor markers yielded phenotypes upon knockdown either, suggestive of considerable functional redundancy among these genes. In contrast, knockdown of a gene encoding a homolog to the RNA-binding protein MEX3, Smed-mex3-1 (mex3-1) produced phenotypes highly suggestive of defective stem cell lineage progression. mex3-1(RNAi) was completely penetrant and lethal, resulting in ventral curling, head regression, and dorsal lesioning during homeostasis, as well as loss of regenerative ability after amputation (Figure 3A–C), indicative of epithelial homeostasis defects as well as overall stem cell impairment (Bardeen and Baetjer, 1904; Reddien et al., 2005a). Using reciprocal BLAST, we identified two other MEX3 homologs in S. mediterranea (mex3-2 and mex3-3, transcripts SmedASXL_000637 and SmedASXL_01505, respectively), which both contained two KH RNA-binding domains and had top reciprocal BLAST hits in mouse, fly, and C. elegans. These additional MEX3 homologs were neither irradiation-sensitive nor produced observable phenotypes after knockdown and thus not investigated further (Figure 3—figure supplement 1).10.7554/eLife.07025.011Figure 3.mex3-1 is required for tissue homeostasis and regeneration.

Bottom Line: In this study, we used transcriptional profiling of irradiation-sensitive and irradiation-insensitive cell populations and RNA interference (RNAi) functional screening to uncover markers and regulators of postmitotic progeny.We also demonstrated the utility of using mex3-1(RNAi) animals to identify additional progenitor markers.These results identified mex3-1 as a cell fate regulator, broadly required for differentiation, and suggest that mex3-1 helps to mediate the balance between ASC self-renewal and commitment.

View Article: PubMed Central - PubMed

Affiliation: Program in Developmental and Stem Cell Biology, The Hospital for Sick Children, Toronto, Canada.

ABSTRACT
Neoblasts are adult stem cells (ASCs) in planarians that sustain cell replacement during homeostasis and regeneration of any missing tissue. While numerous studies have examined genes underlying neoblast pluripotency, molecular pathways driving postmitotic fates remain poorly defined. In this study, we used transcriptional profiling of irradiation-sensitive and irradiation-insensitive cell populations and RNA interference (RNAi) functional screening to uncover markers and regulators of postmitotic progeny. We identified 32 new markers distinguishing two main epithelial progenitor populations and a planarian homolog to the MEX3 RNA-binding protein (Smed-mex3-1) as a key regulator of lineage progression. mex3-1 was required for generating differentiated cells of multiple lineages, while restricting the size of the stem cell compartment. We also demonstrated the utility of using mex3-1(RNAi) animals to identify additional progenitor markers. These results identified mex3-1 as a cell fate regulator, broadly required for differentiation, and suggest that mex3-1 helps to mediate the balance between ASC self-renewal and commitment.

No MeSH data available.


Related in: MedlinePlus