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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

Statistical correlations between new and published data sets.Scatter plots of transcripts (gray circles) with reads per million values of 1000 or less, displayed as log-transformed values. The black diagonal line represents a Pearson correlation coefficient of 1. The corresponding Pearson correlation coefficients are shown at the top of each panel. All correlation-test p-values were found to be equal to 0 (Student's t-test). As expected, the replicates in each data are highly correlated, and the Pearson correlation coefficients between the replicates in X1 or X2 are greater than 0.95. Furthermore, sequencing from whole irradiated planarians is highly correlated.DOI:http://dx.doi.org/10.7554/eLife.07025.004
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fig1s1: Statistical correlations between new and published data sets.Scatter plots of transcripts (gray circles) with reads per million values of 1000 or less, displayed as log-transformed values. The black diagonal line represents a Pearson correlation coefficient of 1. The corresponding Pearson correlation coefficients are shown at the top of each panel. All correlation-test p-values were found to be equal to 0 (Student's t-test). As expected, the replicates in each data are highly correlated, and the Pearson correlation coefficients between the replicates in X1 or X2 are greater than 0.95. Furthermore, sequencing from whole irradiated planarians is highly correlated.DOI:http://dx.doi.org/10.7554/eLife.07025.004

Mentions: Previously, we published two replicates of Illumina RNAseq of the X2 cell fraction to a depth of 206 million reads (Labbe et al., 2012). Here, we sequenced a third replicate to 63 million reads. We found a very high correlation across all of our sequencing replicates, as well as with two irradiated samples from a previous study, which we subsequently analyzed along with our irradiated sequencing (Figure 1—figure supplement 1, Supplementary file 1) (Onal et al., 2012; Resch et al., 2012; Solana et al., 2012). To identify transcripts enriched in the X2 cell fraction, we used the program DESeq (Anders and Huber, 2010) to compare RNAseq from purified X1 and X2 cells vs whole irradiated animals at 7 days after exposure to 60–100 Gray (Gy) of γ-irradiation (Anders and Huber, 2010; Solana et al., 2012; Fernandes et al., 2014). This identified 2839 X1 and 1512 X2 transcripts with a p-value ≤ 0.01 (Figure 1A,B, Supplementary file 1). It is important to note that X1 and X2 cells shared the majority of their transcriptional profiles (Figure 1C), and bona fide progeny markers can be highly expressed in the X1 fraction, while bona fide stem cell markers can be highly expressed in the X2 fraction (Figure 1A,B). Therefore, to be considered X2-enriched, we imposed the additional criterion that the expression ratio of X2/X1 was >1, to exclude transcripts jointly expressed in both irradiation-sensitive populations. This eliminated previously known stem cell genes, such as piwi-1 and -2, PCNA (proliferating cell nuclear antigen), and cyclinB (Figure 1A; 8th, 45th, 57th, and 80th highest enriched X2 genes, respectively), and reduced the total number of enriched X2 genes to 735 (Figure 1D, Supplementary file 1) (Orii et al., 2005; Reddien et al., 2005b; Eisenhoffer et al., 2008). Finally, we observed 66 transcripts that were highly expressed in wild-type animals, yet exhibited low counts in irradiated worms and in both X1 and X2 cell fractions (WThighXlow, Figure 1D, Supplementary file 1). From the remaining 735 X2-specific genes as well as these 66 other irradiation-sensitive transcripts, we hypothesized that these represented multiple types of irradiation-sensitive progenitor cells. We next cloned the top 100 X2-specific and 20 WThighXlow transcripts for expression and functional analyses (Figure 1D, Supplementary file 2). Genes were annotated based on the top BLAST hit in mouse, when the Expect value passed the threshold of e−5.10.7554/eLife.07025.003Figure 1.Transcriptional analysis of irradiation-sensitive cell populations in Schmidtea mediterranea.


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)

Statistical correlations between new and published data sets.Scatter plots of transcripts (gray circles) with reads per million values of 1000 or less, displayed as log-transformed values. The black diagonal line represents a Pearson correlation coefficient of 1. The corresponding Pearson correlation coefficients are shown at the top of each panel. All correlation-test p-values were found to be equal to 0 (Student's t-test). As expected, the replicates in each data are highly correlated, and the Pearson correlation coefficients between the replicates in X1 or X2 are greater than 0.95. Furthermore, sequencing from whole irradiated planarians is highly correlated.DOI:http://dx.doi.org/10.7554/eLife.07025.004
© Copyright Policy
Related In: Results  -  Collection

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

fig1s1: Statistical correlations between new and published data sets.Scatter plots of transcripts (gray circles) with reads per million values of 1000 or less, displayed as log-transformed values. The black diagonal line represents a Pearson correlation coefficient of 1. The corresponding Pearson correlation coefficients are shown at the top of each panel. All correlation-test p-values were found to be equal to 0 (Student's t-test). As expected, the replicates in each data are highly correlated, and the Pearson correlation coefficients between the replicates in X1 or X2 are greater than 0.95. Furthermore, sequencing from whole irradiated planarians is highly correlated.DOI:http://dx.doi.org/10.7554/eLife.07025.004
Mentions: Previously, we published two replicates of Illumina RNAseq of the X2 cell fraction to a depth of 206 million reads (Labbe et al., 2012). Here, we sequenced a third replicate to 63 million reads. We found a very high correlation across all of our sequencing replicates, as well as with two irradiated samples from a previous study, which we subsequently analyzed along with our irradiated sequencing (Figure 1—figure supplement 1, Supplementary file 1) (Onal et al., 2012; Resch et al., 2012; Solana et al., 2012). To identify transcripts enriched in the X2 cell fraction, we used the program DESeq (Anders and Huber, 2010) to compare RNAseq from purified X1 and X2 cells vs whole irradiated animals at 7 days after exposure to 60–100 Gray (Gy) of γ-irradiation (Anders and Huber, 2010; Solana et al., 2012; Fernandes et al., 2014). This identified 2839 X1 and 1512 X2 transcripts with a p-value ≤ 0.01 (Figure 1A,B, Supplementary file 1). It is important to note that X1 and X2 cells shared the majority of their transcriptional profiles (Figure 1C), and bona fide progeny markers can be highly expressed in the X1 fraction, while bona fide stem cell markers can be highly expressed in the X2 fraction (Figure 1A,B). Therefore, to be considered X2-enriched, we imposed the additional criterion that the expression ratio of X2/X1 was >1, to exclude transcripts jointly expressed in both irradiation-sensitive populations. This eliminated previously known stem cell genes, such as piwi-1 and -2, PCNA (proliferating cell nuclear antigen), and cyclinB (Figure 1A; 8th, 45th, 57th, and 80th highest enriched X2 genes, respectively), and reduced the total number of enriched X2 genes to 735 (Figure 1D, Supplementary file 1) (Orii et al., 2005; Reddien et al., 2005b; Eisenhoffer et al., 2008). Finally, we observed 66 transcripts that were highly expressed in wild-type animals, yet exhibited low counts in irradiated worms and in both X1 and X2 cell fractions (WThighXlow, Figure 1D, Supplementary file 1). From the remaining 735 X2-specific genes as well as these 66 other irradiation-sensitive transcripts, we hypothesized that these represented multiple types of irradiation-sensitive progenitor cells. We next cloned the top 100 X2-specific and 20 WThighXlow transcripts for expression and functional analyses (Figure 1D, Supplementary file 2). Genes were annotated based on the top BLAST hit in mouse, when the Expect value passed the threshold of e−5.10.7554/eLife.07025.003Figure 1.Transcriptional analysis of irradiation-sensitive cell populations in Schmidtea mediterranea.

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