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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

Co-localization of progeny markers with dFISH.Co-expression of X2-enriched and WThighXlow early and late progeny markers with established lineage markers (prog-1, prog-2, and AGAT-1) or with each other was determined using dFISH. Confocal projections spanning 6–10 μm are shown. Smaller panels on the left show individual channels with expression of one gene; larger panels on the right show merged channels. Numbers indicate the percent of magenta-labeled cells, which co-express the green-labeled transcript. 200–2000 cells were counted for each dFISH combination. Location of eyespots is marked by asterisks; anterior, up.DOI:http://dx.doi.org/10.7554/eLife.07025.009
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fig2s3: Co-localization of progeny markers with dFISH.Co-expression of X2-enriched and WThighXlow early and late progeny markers with established lineage markers (prog-1, prog-2, and AGAT-1) or with each other was determined using dFISH. Confocal projections spanning 6–10 μm are shown. Smaller panels on the left show individual channels with expression of one gene; larger panels on the right show merged channels. Numbers indicate the percent of magenta-labeled cells, which co-express the green-labeled transcript. 200–2000 cells were counted for each dFISH combination. Location of eyespots is marked by asterisks; anterior, up.DOI:http://dx.doi.org/10.7554/eLife.07025.009

Mentions: Prior to determining how the new progeny markers fit into this putative lineage using double fluorescent WISH (dFISH), we first duplicated previous experiments with piwi-1, prog-1, and AGAT-1 in order to establish baseline values of overlap. Congruent with previous studies, we found a small amount of overlap between piwi-1 with prog-1, and minimal with AGAT-1: 7.2% ± 2.1 of prog-1+ cells were piwi-1+, while merely 0.29% ± 0.50 of AGAT-1+ cells were piwi-1+ (Supplementary file 3). In contrast to a previous study that found nearly 45% overlap between the progeny populations (Eisenhoffer et al., 2008), we found that only 5.4% ± 2.4 of prog-1+ cells co-expressed low levels of AGAT-1, while 5.2% ± 3.2 of AGAT-1+ cells had prog-1 expression. dFISH with new progeny markers also showed little overlap between early and late progeny, confirming that these progeny transcripts mark two mainly non-overlapping populations (Figure 2B, Figure 2—figure supplement 3), and that previous overlap was likely an dFISH artifact. It has been shown that PIWI-1 protein has a wider expression domain than piwi-1 mRNA, reflecting the perdurance of PIWI-1 into postmitotic progeny (Guo et al., 2006; Wenemoser and Reddien, 2010). It is unknown how differentiated these piwi-1−PIWI-1+ cells are, but they are clearly in a transition from a stem cell gene expression state to various postmitotic fates. In support of these data, we found that 17.8% ± 12.2 of prog-1+ cells to be PIWI-1+, while 1.6% ± 1.2 of AGAT-1+ cells had detectable PIWI-1 expression (Figure 2B, Supplementary file 3).


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)

Co-localization of progeny markers with dFISH.Co-expression of X2-enriched and WThighXlow early and late progeny markers with established lineage markers (prog-1, prog-2, and AGAT-1) or with each other was determined using dFISH. Confocal projections spanning 6–10 μm are shown. Smaller panels on the left show individual channels with expression of one gene; larger panels on the right show merged channels. Numbers indicate the percent of magenta-labeled cells, which co-express the green-labeled transcript. 200–2000 cells were counted for each dFISH combination. Location of eyespots is marked by asterisks; anterior, up.DOI:http://dx.doi.org/10.7554/eLife.07025.009
© Copyright Policy
Related In: Results  -  Collection

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

fig2s3: Co-localization of progeny markers with dFISH.Co-expression of X2-enriched and WThighXlow early and late progeny markers with established lineage markers (prog-1, prog-2, and AGAT-1) or with each other was determined using dFISH. Confocal projections spanning 6–10 μm are shown. Smaller panels on the left show individual channels with expression of one gene; larger panels on the right show merged channels. Numbers indicate the percent of magenta-labeled cells, which co-express the green-labeled transcript. 200–2000 cells were counted for each dFISH combination. Location of eyespots is marked by asterisks; anterior, up.DOI:http://dx.doi.org/10.7554/eLife.07025.009
Mentions: Prior to determining how the new progeny markers fit into this putative lineage using double fluorescent WISH (dFISH), we first duplicated previous experiments with piwi-1, prog-1, and AGAT-1 in order to establish baseline values of overlap. Congruent with previous studies, we found a small amount of overlap between piwi-1 with prog-1, and minimal with AGAT-1: 7.2% ± 2.1 of prog-1+ cells were piwi-1+, while merely 0.29% ± 0.50 of AGAT-1+ cells were piwi-1+ (Supplementary file 3). In contrast to a previous study that found nearly 45% overlap between the progeny populations (Eisenhoffer et al., 2008), we found that only 5.4% ± 2.4 of prog-1+ cells co-expressed low levels of AGAT-1, while 5.2% ± 3.2 of AGAT-1+ cells had prog-1 expression. dFISH with new progeny markers also showed little overlap between early and late progeny, confirming that these progeny transcripts mark two mainly non-overlapping populations (Figure 2B, Figure 2—figure supplement 3), and that previous overlap was likely an dFISH artifact. It has been shown that PIWI-1 protein has a wider expression domain than piwi-1 mRNA, reflecting the perdurance of PIWI-1 into postmitotic progeny (Guo et al., 2006; Wenemoser and Reddien, 2010). It is unknown how differentiated these piwi-1−PIWI-1+ cells are, but they are clearly in a transition from a stem cell gene expression state to various postmitotic fates. In support of these data, we found that 17.8% ± 12.2 of prog-1+ cells to be PIWI-1+, while 1.6% ± 1.2 of AGAT-1+ cells had detectable PIWI-1 expression (Figure 2B, Supplementary file 3).

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