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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

Characterization of down-regulated genes in mex3-1(RNAi) animals.(A) Expression of three genes down-regulated after mex3-1 RNAi was assessed in lethally irradiated worms by WISH. Scale bar, 200 μm. (B) Expression of two mex3-1(RNAi)-down-regulated genes in late progeny was examined by dFISH with AGAT-1. Percentages of cells that express AGAT-1 are indicated in the top-right of each panel. Images shown are confocal projections spanning 4 μm in depth. Scale bar, 100 μm. (C) Functional analysis by RNAi knockdown of the new pharyngeal progenitor marker SmedASXL_059179. Expression of SmedASXL_059179 after knockdown was examined by WISH. Scale bar, 200 μm. (D) Diagram outlining schedule of RNAi feeds and amputation for assessment of pharynx regeneration. Shown is WISH of the pharynx marker laminin in regenerating tail fragments. Scale bar, 200 μm.DOI:http://dx.doi.org/10.7554/eLife.07025.022
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fig7s2: Characterization of down-regulated genes in mex3-1(RNAi) animals.(A) Expression of three genes down-regulated after mex3-1 RNAi was assessed in lethally irradiated worms by WISH. Scale bar, 200 μm. (B) Expression of two mex3-1(RNAi)-down-regulated genes in late progeny was examined by dFISH with AGAT-1. Percentages of cells that express AGAT-1 are indicated in the top-right of each panel. Images shown are confocal projections spanning 4 μm in depth. Scale bar, 100 μm. (C) Functional analysis by RNAi knockdown of the new pharyngeal progenitor marker SmedASXL_059179. Expression of SmedASXL_059179 after knockdown was examined by WISH. Scale bar, 200 μm. (D) Diagram outlining schedule of RNAi feeds and amputation for assessment of pharynx regeneration. Shown is WISH of the pharynx marker laminin in regenerating tail fragments. Scale bar, 200 μm.DOI:http://dx.doi.org/10.7554/eLife.07025.022

Mentions: We chose 21 down-regulated and uncharacterized transcripts for validation by WISH. By RNAseq, all were irradiation-sensitive but not X2-enriched (Figure 7A, Supplementary file 4) and thus could be classified as WThighXlow. We found that 20/21 transcripts produced a prog-like pattern in control worms and exhibited severely reduced expression in mex3-1(RNAi) worms, as anticipated from RNAseq (Figure 7B, Figure 7—figure supplement 1C). The remaining transcript, SmedASXL_059179, was highly expressed near the proximal end of the pharynx, throughout the pharynx proper, and was similarly dependent on mex3-1 for its expression (Figure 7B). Three transcripts were confirmed to be irradiation-sensitive by WISH (Figure 7—figure supplement 2A), and two of these were verified to be additional late progeny markers based on their high degree of co-localization with AGAT-1 expression (Figure 7—figure supplement 2B). We predicted that SmedASXL_059179+ cells may represent a pharyngeal progenitor cell type, and indeed, we observed PIWI-1+SmedASXL_059179+ cells near the proximal base of the pharynx (Figure 7C). However, no regulatory roles were uncovered for this gene, as RNAi against SmedASXL_059179 did not result in apparent perturbations to pharynx function during homeostasis or regeneration (Figure 7—figure supplement 2C,D). Overall, these results further support a role for mex3-1 as a critical regulator of differentiation toward multiple lineages and also demonstrate the utility of transcriptional analysis of mex3-1(RNAi) in identifying additional markers of tissue-specific progenitor populations, as an alternative to the criteria of irradiation-sensitivity and FACS localization.


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)

Characterization of down-regulated genes in mex3-1(RNAi) animals.(A) Expression of three genes down-regulated after mex3-1 RNAi was assessed in lethally irradiated worms by WISH. Scale bar, 200 μm. (B) Expression of two mex3-1(RNAi)-down-regulated genes in late progeny was examined by dFISH with AGAT-1. Percentages of cells that express AGAT-1 are indicated in the top-right of each panel. Images shown are confocal projections spanning 4 μm in depth. Scale bar, 100 μm. (C) Functional analysis by RNAi knockdown of the new pharyngeal progenitor marker SmedASXL_059179. Expression of SmedASXL_059179 after knockdown was examined by WISH. Scale bar, 200 μm. (D) Diagram outlining schedule of RNAi feeds and amputation for assessment of pharynx regeneration. Shown is WISH of the pharynx marker laminin in regenerating tail fragments. Scale bar, 200 μm.DOI:http://dx.doi.org/10.7554/eLife.07025.022
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4507787&req=5

fig7s2: Characterization of down-regulated genes in mex3-1(RNAi) animals.(A) Expression of three genes down-regulated after mex3-1 RNAi was assessed in lethally irradiated worms by WISH. Scale bar, 200 μm. (B) Expression of two mex3-1(RNAi)-down-regulated genes in late progeny was examined by dFISH with AGAT-1. Percentages of cells that express AGAT-1 are indicated in the top-right of each panel. Images shown are confocal projections spanning 4 μm in depth. Scale bar, 100 μm. (C) Functional analysis by RNAi knockdown of the new pharyngeal progenitor marker SmedASXL_059179. Expression of SmedASXL_059179 after knockdown was examined by WISH. Scale bar, 200 μm. (D) Diagram outlining schedule of RNAi feeds and amputation for assessment of pharynx regeneration. Shown is WISH of the pharynx marker laminin in regenerating tail fragments. Scale bar, 200 μm.DOI:http://dx.doi.org/10.7554/eLife.07025.022
Mentions: We chose 21 down-regulated and uncharacterized transcripts for validation by WISH. By RNAseq, all were irradiation-sensitive but not X2-enriched (Figure 7A, Supplementary file 4) and thus could be classified as WThighXlow. We found that 20/21 transcripts produced a prog-like pattern in control worms and exhibited severely reduced expression in mex3-1(RNAi) worms, as anticipated from RNAseq (Figure 7B, Figure 7—figure supplement 1C). The remaining transcript, SmedASXL_059179, was highly expressed near the proximal end of the pharynx, throughout the pharynx proper, and was similarly dependent on mex3-1 for its expression (Figure 7B). Three transcripts were confirmed to be irradiation-sensitive by WISH (Figure 7—figure supplement 2A), and two of these were verified to be additional late progeny markers based on their high degree of co-localization with AGAT-1 expression (Figure 7—figure supplement 2B). We predicted that SmedASXL_059179+ cells may represent a pharyngeal progenitor cell type, and indeed, we observed PIWI-1+SmedASXL_059179+ cells near the proximal base of the pharynx (Figure 7C). However, no regulatory roles were uncovered for this gene, as RNAi against SmedASXL_059179 did not result in apparent perturbations to pharynx function during homeostasis or regeneration (Figure 7—figure supplement 2C,D). Overall, these results further support a role for mex3-1 as a critical regulator of differentiation toward multiple lineages and also demonstrate the utility of transcriptional analysis of mex3-1(RNAi) in identifying additional markers of tissue-specific progenitor populations, as an alternative to the criteria of irradiation-sensitivity and FACS localization.

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