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, 20 (11), 2527-2537

Piwi Is Required to Limit Exhaustion of Aging Somatic Stem Cells

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Piwi Is Required to Limit Exhaustion of Aging Somatic Stem Cells

Pedro Sousa-Victor et al. Cell Rep.

Abstract

Sophisticated mechanisms that preserve genome integrity are critical to ensure the maintenance of regenerative capacity while preventing transformation of somatic stem cells (SCs), yet little is known about mechanisms regulating genome maintenance in these cells. Here, we show that intestinal stem cells (ISCs) induce the Argonaute family protein Piwi in response to JAK/STAT signaling during acute proliferative episodes. Piwi function is critical to ensure heterochromatin maintenance, suppress retrotransposon activation, and prevent DNA damage in homeostasis and under regenerative pressure. Accordingly, loss of Piwi results in the loss of actively dividing ISCs and their progenies by apoptosis. We further show that Piwi expression is sufficient to allay age-related retrotransposon expression, DNA damage, apoptosis, and mis-differentiation phenotypes in the ISC lineage, improving epithelial homeostasis. Our data identify a role for Piwi in the regulation of somatic SC function, and they highlight the importance of retrotransposon control in somatic SC maintenance.

Keywords: aging transcriptome; chromatin stability; epigenetics; intestinal stem cell function; stem cell aging; transposable elements.

Figures

Figure 1
Figure 1. Infection induced JAK/STAT signaling promotes Piwi expression in ISCs/EBs
A, Setup and timeline for Ecc15 infection experiments. B, Proliferative activity of ISCs after Ecc15 infection, evaluated by the number of pH3+ cells/midgut (n=8 /time point) C, Scatter plots with GFP intensity (vertical axis) and cell size (horizontal axis) show a GFPhigh population of smaller cells (P3, ISCs) and a GFPhigh population of larger cells (P4, EBs). D, FPKM values of progenitor-specific genes in FACS-sorted ISCs. E, Relative expression of a set of genes induced by Ecc15 infection in a STAT-dependent manner measured in FACS sorted ISCs. Genes labeled in red are associated with DNA replication and repair (n=7 for mock condition) F, Venn diagram showing the proportion of genes induced by Ecc15 infection in ISCs that require STAT function for the induction. Piwi is one of these genes. G–H, FPKM values (G) and RT-qPCR (H) showing Piwi expression in FACS sorted ISCs, 4h (G) and 16h (G-H) after Ecc15 infection in the presence or absence of STAT RNAi (for RTqPCR, n=3 sorted samples/condition, 50100 midguts/sorting). Error bars indicate s.e.m. and p-values are from student’s t-test. See also Fig. S1 and Table S1.
Figure 2
Figure 2. Piwi expression in ISCs/EBs of the Drosophila midgut
A–B, Piwi expression in ISCs/EBs of the Drosophila midgut, detected by in situ hybridization using an anti-sense probe (A, left panel) or by immunohistochemistry (B). mRNA signal specificity was confirmed by the lack of signal using a sense probe (A, right panel). ISCs/EBs were identified by esg::GFP expression. STAT-dependent induction of Piwi protein in ISCs/EBs after Ecc15 infection is shown and quantified (B, n=25 cells quantified/condition using ImageJ. Quantification is of the mean intensity in the Piwi channel normalized to the DAPI channel for each ISC/EB area defined by the GFP channel). Arrowheads indicate Piwi nuclear signal. Error bars indicate s.e.m. and p-values are from student’s t-test. Scale bars are 20μm. See also Fig. S2.
Figure 3
Figure 3. Piwi is required for maintenance of ISC function
A, Representative images of Drosophila posterior midguts isolated from wt animals or animals expressing Piwi-RNAi in the ISCs/EBs, after Ecc15 infection, showing ISC density by Dl staining and posterior midgut morphology by DAPI staining. Quantifications of average number of Dl+ cells/field are shown (n=8–10/condition). B, Representative images of Drosophila posterior midguts infected with Ecc15, showing wt (FRT40A) and Piwi null (piwi3) clones. Quantifications of average number of cells/clone are shown (n=8 /condition, 6–10 clones/gut). C, Representative images of Drosophila posterior midguts isolated from wt animals or animals expressing Piwi-RNAi in the ISCs/EBs, 72h after P.e. infection. DAPI staining shows the altered posterior midgut morphology in Piwi-deficient animals. D, Percent of animal survival after P.e. infection. E, Representative images of Drosophila posterior midguts isolated from wt animals (or animals expressing mCherry-RNAi), and animals expressing Piwi-RNAi in the ISCs/EBs for 14 days. ISC are identified by esg::GFP. On the two left panels cell boundaries are labeled by immunostaining against Armadillo (membrane red), and EE cells are labeled by nuclear pros staining (nuclear red). Error bars indicate s.e.m. and p-values are from student’s t-test. Scale bars are 50μm. See also Fig. S2 and S3.
Figure 4
Figure 4. Piwi is required for chromatin maintenance and transposon silencing
A, Reporter locus in the ln(3L)BL1 line used to detect relative levels of heterochromatin (red). B, Representative images and relative levels of LacZ transcripts quantified by RT-qPCR (n=3, 8 guts/sample) of Drosophila midguts isolated from 20 day old wt animals (w1118) or piwi heterozygous animals (piwi3/+) carrying the reporter locus in (A) and isolated 1 hour after heat-shock. Guts were stained for Dl to identify ISCs and βGal to identify LacZ-expressing cells. C, Relative levels of TE transcripts quantified by RT-qPCR in midguts isolated from animals expressing RNAi against Piwi, Aub or Ago3 in the ISCs/EBs for 7 days, compared to animals expressing a control hairpin (n=4, 8 guts/sample). D, Gypsy-TRAP line used to detect Gypsy integration events. E, Representative images of Drosophila posterior midguts isolated from 20 days old wt animals (w1118) or piwi heterozygous animals (piwi3/+) carrying the reporter in (D). Quantification of average number of GFP-positive cells/midgut (n=8/condition) is shown. Error bars indicate s.e.m. and p-values are from student’s t-test. Scale bars are 20μm. See also Fig. S4 and Table S1.
Figure 5
Figure 5. Piwi overexpression prevents age-associated decline in ISC function
A, Relative levels of TE transcripts quantified by RT-qPCR in midguts isolated from young (4 days) and old (45 days) wild type animals (n=4–6, 8 guts/sample). B, Representative images of Drosophila posterior midguts isolated from young (7 days) and old (35 days) wt animals carrying the Gypsy-TRAP reporter. C, Quantification of the average number of GFP-positive cells/midgut at different ages in flies carrying the Gypsy-TRAP reporter (left, n=8/condition). Right graph is a similar quantification but in flies carrying the Gypsy-TRAP reporter with mutated ovo-sites, where Gypsy cannot integrate (right, n=8/condition). D, GO analysis of the data set of genes down-regulated in ISCs from 60 days old flies. Graphs on the bottom show the relative levels of expression of some of these genes in ISCs at different ages. E–F, Representative images of Drosophila posterior midguts isolated from 60 days-old flies overexpressing Piwi in ISCs/EBs and corresponding wt controls, showing esg::GFP, armadillo and prospero (E) or Pdm1 (F). G, Quantification of the average number of pH3+ cells/midgut (left; n=7/condition) and the average fraction of Pdm1+/GFP+ cells (right; n=17/condition) for the different ages and genotypes. H–J, Representative images of Drosophila posterior midguts isolated from 60 days-old flies overexpressing Piwi in ISCs/EBs and corresponding 60 days old and young wt controls (I–J), showing the expression of esg::GFP (I) or Dl (H and J) in green to identify progenitor cells/ISCs, respectively. Co-staining with p4EBP (H), H2AvD (I) and cDCP-1 (J) in red is shown. Quantifications on the right are for the percentage of mis-differentiated ISCs (H, n=4–7/condition); progenitor cells with signs of DNA damage (I, n=5–7/condition) and apoptotic ISCs (J, n=5–6/condition). Error bars indicate s.e.m. and p-values are from student’s t-test. Scale bars are 50μm. See also Fig. S5 and Table S1.

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