Increased level of transcripts and increased frequency of long poly(A) tails are restricted to neoblast-containing cell populations.

<p>(A–B) Quantification of the level of expression by qRT-PCR of the neoblast markers <i>Smedwi-1</i>, <i>Smedtud-1</i>, <i>Smed-vasa-1</i> and <i>Smed-pcna</i> (A) and <i>Smed-not1</i> (B) in FACS sorted populations X1, X2 and Xins in <i>control(RNAi)</i> (c) and <i>Smed-not1(RNAi)</i> (n) animals 10 and 15 days after RNAi, normalized expression and relative to respective X1 <i>control(RNAi)</i> samples. (A) <i>Smedwi-1</i>, <i>Smedtud-1</i>, <i>Smed-vasa-1</i> and <i>Smed-pcna</i> transcripts accumulate progressively after 10 and 15 days of RNAi in X1 and X2 cells, the two fractions that contain neoblasts to different extents, but this accumulation is not observed in Xins cells, which contain differentiated cells exclusively, including CNS cells. (B) <i>Smed-not1</i> is significantly depleted across all three cell fractions, showing that the absence of accumulation and increased frequency of long poly(A) tails of neoblast mRNAs that are expressed also in CNS is not due to absence of effective gene knock down in differentiated cells. Error bars represent standard deviation and asterisks represent statistical significance in A–B (C–E) PAT assays reflecting the distribution of mRNA poly(A) tail lengths for the neoblast and CNS expressed mRNAs <i>Smedtud-1</i> and <i>Smed-vasa-1</i> (C), the neoblast specific mRNAs <i>Smed-pcna</i> and <i>Smedwi-1</i> (D) and the housekeeping and tissue specific mRNAs <i>Smed-mhc</i> and <i>Smed-ef-2</i> (E) in FACS sorted populations X1, X2 and Xins from <i>control(RNAi)</i> (c) and <i>Smed-not1(RNAi)</i> (n) animals 10 and 15 days after RNAi. Size markers used are represented in blue, the theoretical length of the amplicon corresponding to the deadenylated mRNA species given the primers used in each assay is given in green. (C). The marked differences in poly(A) tail length distribution detected for the neoblast and CNS mRNAs <i>Smed-vasa-1</i> and <i>Smedtud-1</i> are only detected in X1 and X2 but not in Xins FACS sorted populations, showing that the fractions of these mRNA populations localised in the CNS show no differences after <i>Smed-not1</i> knock down. (D) The marked differences in poly(A) tail length distribution detected for the neoblast specific mRNAs <i>Smed-pcna</i> and <i>Smedwi-1</i> are only detected in X1 and X2 but the mRNAs are not detected in Xins FACS sorted populations. (E) No differences in poly(A) tail length distribution are detected for the tissue specific mRNA <i>Smed-mhc</i>, and only slight differences are detected in X1 and X2 but not in Xins fractions for the housekeeping mRNA <i>Smed-ef-2</i>.</p

<i>Smed-not1(RNAi)</i> animals maintain mitotic neoblasts.

<p>(A) Quantification of mitosis by counting of h3p-positive cells in whole mount immunohistochemistry on <i>control(RNAi)</i> and <i>Smed-not1(RNAi)</i> animals 5, 10, 15 and 20 days after RNAi (N = 7 per time point). No significant differences are detected. Representative <i>control(RNAi)</i> (B) and different <i>Smed-not1(RNAi)</i> worms (C–E) 20 days after RNAi, immunostained with the mitotic marker h3p (h3p, green) and counterstained with phospho-tyrosine (p-tyr, red) in order to show head regression defects. <i>Smed-not1(RNAi)</i> animals still display detectable mitotic cells, even as head regression defects occur. The number of mitotic cells detected is smaller in the animals with the most severe head regression defects (D–E). Anterior is to the left. Scale bars: 500 µm. (F) Quantification of FACS sorted X1 cells in <i>control(RNAi)</i> and <i>Smed-not1(RNAi)</i> animals 10 and 15 days after RNAi, and wild type irradiated animals. While no significant differences are observed 10 days after knock down, <i>Smed-not1(RNAi)</i> animals show a reduced but significant decrease in percentage of X1 cells. Error bars represent standard deviation and asterisks represent statistical significance. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004003#pgen.1004003.s004" target="_blank">Figure S4</a>.</p

Increased levels of neoblast transcripts and their increased frequency of long poly(A) tails are irradiation sensitive.

<p>(A) WMISH of the neoblast marker <i>Smedtud-1</i> in <i>control(RNAi)</i> animals (left panels) and <i>Smed-not1(RNAi)</i> animals (right panels) non irradiated (top panels) and irradiated 24 hours (bottom panels) prior to data collection time points 10 and 15 days after RNAi. A consistent qualitative difference is observed in non irradiated animals, however, no qualitative differences are observed in irradiated animals, showing that <i>Smed-not1(RNAi)</i> animals do not overexpress ectopically <i>Smedtud-1</i>. Anterior is to the left. Scale bars: 500 µm. (B) Quantification of the level of expression by qRT-PCR of the neoblast markers <i>Smedtud-1</i>, <i>Smed-vasa-1</i> and <i>Smed-pcna</i> in <i>control(RNAi)</i> (c) and <i>Smed-not1(RNAi)</i> (n) animals non-irradiated and irradiated 24 hours prior to data collection time points 10 and 15 days after RNAi, normalized expression and relative to respective <i>control(RNAi)</i> samples. Error bars represent standard deviation, asterisks represent statistical significance. <i>Smedtud-1</i>, <i>Smed-vasa-1</i> and <i>Smed-pcna</i> transcripts accumulate progressively after 10 and 15 days of RNAi, but this accumulation is eliminated 24 hours post irradiation, with <i>Smed-not1(RNAi)</i> irradiated animals showing levels similar to <i>control(RNAi)</i> irradiated animals. (C–F) PAT assays reflecting the distribution of mRNA poly(A) tail lengths for the neoblast specific mRNAs <i>Smedwi-1</i> and <i>Smed-pcna</i> (C), the neoblast and CNS expressed mRNAs <i>Smed-vasa-1</i> and <i>Smedtud-1</i> (D), the progeny specific mRNA <i>Smed-nb.21.11e</i> and <i>Smed-agat-1</i> (E) and the housekeeping and tissue specific mRNAs <i>Smed-ef-2</i>, <i>Smed-eif-3</i>, <i>Smed-mhc</i> and a spiked-in control in <i>control(RNAi)</i> (c) and <i>Smed-not1(RNAi)</i> (n) animals non irradiated and irradiated 24 hours prior to data collection time points 15 days after RNAi. Size markers used are represented in blue, the theoretical length of the amplicon corresponding to the deadenylated mRNA species given the primers used in each assay is given in green. Neoblast specific markers are not detected after irradiation (C). The marked differences in poly(A) tail length distribution detected for the neoblast and CNS mRNAs <i>Smed-vasa-1</i> and <i>Smedtud-1</i> are eliminated by irradiation, showing that the fractions of these mRNA populations localised in the CNS show no differences after <i>Smed-not1</i> knock down (D). The differences in poly(A) tail length distribution detected for the progeny specific mRNAs <i>Smed-nb.21.11e</i> and <i>Smed-agat-1</i> are not affected by irradiation, as these cells are not eliminated after 24 hours of irradiation (E). No differences are detected for <i>Smed-ef-2</i>, <i>Smed-eif-3</i>, <i>Smed-mhc</i> and a spike-in control RNA.</p

The CCR4-NOT Complex Mediates Deadenylation and Degradation of Stem Cell mRNAs and Promotes Planarian Stem Cell Differentiation

<div><p>Post-transcriptional regulatory mechanisms are of fundamental importance to form robust genetic networks, but their roles in stem cell pluripotency remain poorly understood. Here, we use freshwater planarians as a model system to investigate this and uncover a role for CCR4-NOT mediated deadenylation of mRNAs in stem cell differentiation. Planarian adult stem cells, the so-called neoblasts, drive the almost unlimited regenerative capabilities of planarians and allow their ongoing homeostatic tissue turnover. While many genes have been demonstrated to be required for these processes, currently almost no mechanistic insight is available into their regulation. We show that knockdown of planarian Not1, the CCR4-NOT deadenylating complex scaffolding subunit, abrogates regeneration and normal homeostasis. This abrogation is primarily due to severe impairment of their differentiation potential. We describe a stem cell specific increase in the mRNA levels of key neoblast genes after <i>Smed-not1</i> knock down, consistent with a role of the CCR4-NOT complex in degradation of neoblast mRNAs upon the onset of differentiation. We also observe a stem cell specific increase in the frequency of longer poly(A) tails in these same mRNAs, showing that stem cells after <i>Smed-not1</i> knock down fail to differentiate as they accumulate populations of transcripts with longer poly(A) tails. As other transcripts are unaffected our data hint at a targeted regulation of these key stem cell mRNAs by post-transcriptional regulators such as RNA-binding proteins or microRNAs. Together, our results show that the CCR4-NOT complex is crucial for stem cell differentiation and controls stem cell-specific degradation of mRNAs, thus providing clear mechanistic insight into this aspect of neoblast biology.</p></div

<i>Smed-not1(RNAi)</i> animals have increasing numbers of <i>Smed-agat-1</i> transcripts with increased frequency of long poly(A) tails but decreasing numbers of <i>Smed-agat-1</i>-positive cells.

<p>(A) Quantification of the level of expression by qRT-PCR of the progeny markers <i>Smed-nb.21.11e</i> and <i>Smed-agat-1</i> in <i>Smed-not1(RNAi)</i> animals 10 and 15 days after RNAi, normalized expression and relative to respective <i>control(RNAi)</i> samples. Error bars represent standard deviation, asterisks represent statistical significance. <i>Smed-agat-1</i> transcripts accumulate progressively after 10 and 15 days of RNAi. (B) Quantification of the number of <i>Smed-agat-1</i>-positive cells in <i>control(RNAi)</i> and <i>Smed-not1(RNAi)</i> worms 10 and 15 days after RNAi. Animals (N = 8 per time point and treatment) were stained by FWMISH of <i>Smed-agat-1</i> and analyzed by confocal microscopy. Multiple squares corresponding to 250 µm² in both the dorsal and ventral parts of the animals were selected for counting along the length of the animals. 130 squares were counted, 4 representative squares are shown. Error bars represent standard error of the mean, asterisk represents statistical significance. A significant accumulation of <i>Smed-agat-1</i>-positive cells is detected in <i>Smed-not1(RNAi)</i> animals 10 days after RNAi but a decrease is observed at 15 days (C) Confocal Z-projection of FWMISHs of <i>Smed-agat1</i> in <i>control(RNAi)</i> (top panels) and <i>Smed-not1(RNAi)</i> (bottom panels) animals corresponding to anterior dorsal (left) and anterior ventral (right) regions of representative worms. <i>Smed-agat-1</i>-positive cells are shown in green, nuclei counterstaining in magenta. Scale bar: 100 µm. (D) PAT assays reflecting the distribution of mRNA poly(A) tail lengths for <i>Smed-agat-1</i> and <i>Smed-nb.21.11e</i>, the housekeeping mRNAs <i>Smed-ef-2</i> and <i>Smed-eif-3</i>, the tissue specific mRNA <i>Smed-mhc</i> and a spiked-in control mRNAs in <i>control(RNAi)</i> (c) and <i>Smed-not1(RNAi)</i> (n) animals 10 and 15 days after RNAi. Size markers used are represented in blue, the theoretical length of the amplicon corresponding to the deadenylated mRNA species given the primers used in each assay is given in green. Products above this length originate from polyadenylated mRNA molecules. Marked differences in poly(A) tail length distribution are detected for <i>Smed-agat-1</i>, showing an increased frequency of long poly(A) tails after <i>Smed-not1</i> knock down. Slight differences are observed for <i>Smed-nb.21.11e</i> and <i>Smed-eif-3</i>.</p

<i>Smed-not1</i> is expressed in CNS and throughout the parenchyma in an irradiation-sensitive manner.

<p>(A–D) WMISH of <i>Smed-not1</i> (A), <i>Smedtud-1</i> (B), <i>Smed-vasa-1</i> (C), <i>Smed-pcna</i> (D) and the control marker <i>Smed-eye53</i> (E), in non-irradiated and irradiated animals. <i>Smed-not1</i> signals are detected as a broad staining pattern in non-irradiated animals (A, top panel, non irrad), and decrease 3 days (A, middle panel, irrad 3d) and 5 days (A, bottom panel, irrad 5d) after lethal irradiation. <i>Smed-not1</i> signal is detected in the CNS (arrows). The neoblast specific signals of other mRNAs expressed in neoblasts disappear by day 3 of irradiation (B, C, D, irrad 3d), while signals are still detectable in the CNS for <i>Smedtud-1</i> (B, arrows), and <i>Smed-vasa-1</i> (C, arrows). No <i>Smed-pcna</i> signal is detected in the CNS (D). The differentiated cell control marker <i>Smed-eye53</i> shows no differences upon irradiation (E). Anterior is to the left. Scale bars: 500 µm.</p

Dynamics of neoblasts and their progeny in <i>Smed-not1(RNAi)</i> animals.

<p>(A–L) WMISH of the neoblast marker <i>Smedwi-1</i> (A, D, G, J), the early neoblast progeny marker <i>Smed-nb.21.11e</i> (B, E, H, K) and the late neoblast progeny marker <i>Smed-agat-1</i> (C, F, I, L) in <i>control(RNAi)</i> animals (A–C) and <i>Smed-not1(RNAi)</i> animals 10 (D–F), 15 (G–I) and 20 (J–L) days after RNAi. <i>Smed-not1(RNAi)</i> animals have detectable expression of <i>Smedwi-1</i> in all time points (A, D, G, J), although a decline in the level of <i>Smedwi-1</i> signals is detected 20 days after RNAi (J). The dynamics of progeny markers is also abnormal, with a progressive decline of <i>Smed-nb.21.11e</i> signals (H, K) and an accumulation (F) followed by a decline (I, L) of <i>Smed-agat-1</i> signals. Anterior is to the left. Scale bars: 500 µm. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004003#pgen.1004003.s005" target="_blank">Figure S5</a>.</p

<i>Smed-not1(RNAi)</i> animals have increased levels of neoblast transcripts with increased frequency of long poly(A) tails.

<p>(A–I) WMISH of the neoblast marker <i>Smedtud-1</i> (A), <i>Smed-vasa-1</i> (B) and <i>Smed-pcna</i> (C) in <i>control(RNAi)</i> animals (upper panels) and <i>Smed-not1(RNAi)</i> animals 10 (middle panels) and 15 (bottom panels) days after RNAi, showing normal expression of these mRNAs after <i>Smed-not1</i> knock down, though qualitative differences in the level of expression are suggested. Anterior is to the left. Scale bars: 500 µm. (D) Quantification of the level of expression by qRT-PCR of the neoblast markers <i>Smedwi-1</i>, <i>Smedtud-1</i>, <i>Smed-vasa-1</i> and <i>Smed-pcna</i> in <i>control(RNAi)</i> (c) and <i>Smed-not1(RNAi)</i> animals (n) 10 and 15 days after RNAi, normalized expression and relative to respective <i>control(RNAi)</i> samples. Error bars represent standard deviation, asterisks represent statistical significance. <i>Smedtud-1</i>, <i>Smed-vasa-1</i> and <i>Smed-pcna</i> transcripts accumulate progressively after 10 and 15 days of RNAi, while <i>Smedwi-1</i> only accumulates significantly after 15 days. (E) PAT assays reflecting the distribution of mRNA poly(A) tail lengths for <i>Smedwi-1</i>, <i>Smedtud-1</i>, <i>Smed-vasa-1</i> and <i>Smed-pcna</i> in <i>control(RNAi)</i> (c) and <i>Smed-not1(RNAi)</i> animals (n) 10 and 15 days after RNAi. Size markers used are represented in blue, the theoretical length of the amplicon corresponding to the deadenylated mRNA species given the primers used in each assay is given in green. Marked differences in poly(A) tail length distribution are detected for all four mRNAs, showing an increased frequency of long poly(A) tails after <i>Smed-not1</i> knock down.</p

Additional file 3: Figure S3. of Cell fixation and preservation for droplet-based single-cell transcriptomics

Single-cell data from Drosophila embryos are reproducible and correlate well with bulk mRNA-seq data. Related to Fig. 3. (a) Identification of cell barcodes associated with single-cell transcriptomes for single-cell libraries from Drosophila embryos, a complex primary tissue harbouring small, low RNA content cells. (For methods details, see Additional file 1: Figure S1a.) Four of seven replicates are shown. (b) Correlations between gene expression measurements from bulk mRNA-seq and seven Drop-seq runs with methanol-fixed single cells (expressing >1000 UMIs). Cells were from two independent biological samples representing dissociated Drosophila embryos (75% stages 10 and 11). Bulk mRNA-seq data were generated with total RNA extracted directly from whole, intact, live embryos. (Sample 1: rep 1, 2, 7 and bulk 1; sample 2: rep 3–6 and bulk 2). Non-single cell bulk mRNA-seq data were expressed as reads per kilobase per million (RPKM). Drop-seq expression counts were converted to average transcripts per million (ATPM) and plotted as log2 (ATPM + 1). Upper right panel depicts Pearson correlations. The intersection (common set) of genes between all samples was high (~10,000 genes). (PDF 162 kb

Additional file 5: Table S1. of Cell fixation and preservation for droplet-based single-cell transcriptomics

Top 50 marker genes expressed in 4873 fixed, primary cells from Drosophila embryos. Related to Fig. 3. Tables S1 and S2 contain the top 50 marker genes per cluster, provided by Seurat's function 'FindAllMarkers' [17]. We additionally ordered them per cluster in decreasing log2-fold change (log2FC). The log2FC was computed for a given gene by dividing its average normalized expression for a given cluster over the average normalized expression in the rest of the clusters and taking the logarithm of the fold change. (XLSX 214 kb