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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

RNAseq analysis of mex3-1(RNAi) animals.(A) RNAseq was performed on mex3-1(RNAi) animals 12 days after RNAi, and X1 enrichment is compared to fold change after mex3-1 knockdown. Each gray dot represents one transcript. Established stem cell genes (from Figure 1B), which were found to be significantly upregulated in mex3-1(RNAi), are highlighted in blue (p < 0.01). The fold changes of a subset of known stem cell genes (involved in proliferation, pan-stem cell gene, or subclass identity) are shown on the right. (B) Upregulation of the cell cycle genes PCNA and H2B in RNAseq analysis was confirmed by WISH of mex3-1(RNAi) worms 6 days after RNAi. (C) From the top down-regulated genes after mex3-1 knockdown, 21 uncharacterized genes were chosen for cloning and expression analysis by WISH (11/21 genes are shown). mex3-1(RNAi) animals were stained 12 days after RNAi to confirm that target genes were down-regulated. Genes were named based on the best BLASTx result to mouse when the Expect value <1 × e−5, or based on transcript number if no homology was found. Scale bars, 200 μm.DOI:http://dx.doi.org/10.7554/eLife.07025.021
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fig7s1: RNAseq analysis of mex3-1(RNAi) animals.(A) RNAseq was performed on mex3-1(RNAi) animals 12 days after RNAi, and X1 enrichment is compared to fold change after mex3-1 knockdown. Each gray dot represents one transcript. Established stem cell genes (from Figure 1B), which were found to be significantly upregulated in mex3-1(RNAi), are highlighted in blue (p < 0.01). The fold changes of a subset of known stem cell genes (involved in proliferation, pan-stem cell gene, or subclass identity) are shown on the right. (B) Upregulation of the cell cycle genes PCNA and H2B in RNAseq analysis was confirmed by WISH of mex3-1(RNAi) worms 6 days after RNAi. (C) From the top down-regulated genes after mex3-1 knockdown, 21 uncharacterized genes were chosen for cloning and expression analysis by WISH (11/21 genes are shown). mex3-1(RNAi) animals were stained 12 days after RNAi to confirm that target genes were down-regulated. Genes were named based on the best BLASTx result to mouse when the Expect value <1 × e−5, or based on transcript number if no homology was found. Scale bars, 200 μm.DOI:http://dx.doi.org/10.7554/eLife.07025.021

Mentions: RNAseq of mex3-1(RNAi) whole animals 12 days after RNAi was performed to provide a broad and comprehensive overview of gene expression changes. Given that mex3-1 RNAi specifically eliminates postmitotic progeny fates, we reasoned that this approach may offer a more selective method than X2-FACS enrichment in order to identify additional progenitor-specific transcripts both within and outside the epithelial lineage. In agreement with our data demonstrating hyper-proliferation and expansion of the entire stem cell compartment after mex3-1 knockdown, 13/59 known stem cell-specific transcripts were significantly upregulated upon knockdown of mex3-1 (p < 0.01, Figure 7—figure supplement 1A, Supplementary file 4). These included cell cycle genes, two of which were further confirmed by WISH (PCNA and H2B; Figure 7—figure supplement 1B). Importantly, we also observed an approximately 1.5-fold increase in soxP-1, zfp-1, and piwi-1 transcripts in mex3-1(RNAi) animals (Figure 7—figure supplement 1A; Supplementary file 4), concordant with the increase in stem cell subclasses quantified by cell counting (Figure 5C,D).


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)

RNAseq analysis of mex3-1(RNAi) animals.(A) RNAseq was performed on mex3-1(RNAi) animals 12 days after RNAi, and X1 enrichment is compared to fold change after mex3-1 knockdown. Each gray dot represents one transcript. Established stem cell genes (from Figure 1B), which were found to be significantly upregulated in mex3-1(RNAi), are highlighted in blue (p < 0.01). The fold changes of a subset of known stem cell genes (involved in proliferation, pan-stem cell gene, or subclass identity) are shown on the right. (B) Upregulation of the cell cycle genes PCNA and H2B in RNAseq analysis was confirmed by WISH of mex3-1(RNAi) worms 6 days after RNAi. (C) From the top down-regulated genes after mex3-1 knockdown, 21 uncharacterized genes were chosen for cloning and expression analysis by WISH (11/21 genes are shown). mex3-1(RNAi) animals were stained 12 days after RNAi to confirm that target genes were down-regulated. Genes were named based on the best BLASTx result to mouse when the Expect value <1 × e−5, or based on transcript number if no homology was found. Scale bars, 200 μm.DOI:http://dx.doi.org/10.7554/eLife.07025.021
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fig7s1: RNAseq analysis of mex3-1(RNAi) animals.(A) RNAseq was performed on mex3-1(RNAi) animals 12 days after RNAi, and X1 enrichment is compared to fold change after mex3-1 knockdown. Each gray dot represents one transcript. Established stem cell genes (from Figure 1B), which were found to be significantly upregulated in mex3-1(RNAi), are highlighted in blue (p < 0.01). The fold changes of a subset of known stem cell genes (involved in proliferation, pan-stem cell gene, or subclass identity) are shown on the right. (B) Upregulation of the cell cycle genes PCNA and H2B in RNAseq analysis was confirmed by WISH of mex3-1(RNAi) worms 6 days after RNAi. (C) From the top down-regulated genes after mex3-1 knockdown, 21 uncharacterized genes were chosen for cloning and expression analysis by WISH (11/21 genes are shown). mex3-1(RNAi) animals were stained 12 days after RNAi to confirm that target genes were down-regulated. Genes were named based on the best BLASTx result to mouse when the Expect value <1 × e−5, or based on transcript number if no homology was found. Scale bars, 200 μm.DOI:http://dx.doi.org/10.7554/eLife.07025.021
Mentions: RNAseq of mex3-1(RNAi) whole animals 12 days after RNAi was performed to provide a broad and comprehensive overview of gene expression changes. Given that mex3-1 RNAi specifically eliminates postmitotic progeny fates, we reasoned that this approach may offer a more selective method than X2-FACS enrichment in order to identify additional progenitor-specific transcripts both within and outside the epithelial lineage. In agreement with our data demonstrating hyper-proliferation and expansion of the entire stem cell compartment after mex3-1 knockdown, 13/59 known stem cell-specific transcripts were significantly upregulated upon knockdown of mex3-1 (p < 0.01, Figure 7—figure supplement 1A, Supplementary file 4). These included cell cycle genes, two of which were further confirmed by WISH (PCNA and H2B; Figure 7—figure supplement 1B). Importantly, we also observed an approximately 1.5-fold increase in soxP-1, zfp-1, and piwi-1 transcripts in mex3-1(RNAi) animals (Figure 7—figure supplement 1A; Supplementary file 4), concordant with the increase in stem cell subclasses quantified by cell counting (Figure 5C,D).

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