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. Author manuscript; available in PMC: 2018 Sep 12.
Published in final edited form as: Cell Rep. 2017 Sep 12;20(11):2527–2537. doi: 10.1016/j.celrep.2017.08.059

Piwi is required to limit exhaustion of aging somatic stem cells

Pedro Sousa-Victor 1, Arshad Ayyaz 1, Rippei Hayashi 2, Yanyan Qi 1, David T Madden 1,3, Victoria V Lunyak 4, Heinrich Jasper 1,5,6,*
PMCID: PMC5901960  NIHMSID: NIHMS957572  PMID: 28903034

SUMMARY

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 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 highlight the importance of retrotransposon control in somatic SC maintenance.

INTRODUCTION

Stem cells in high-turnover tissues require high precision in genome maintenance mechanisms to ensure their long-term maintenance. In vertebrate stem cell populations, such mechanisms include effective cell cycle checkpoints and DNA repair machineries (Behrens et al., 2014). Deficiency in these mechanisms can result in stem cell exhaustion, mis-differentiation, and cancers (Adams et al., 2015). Especially during periods of high proliferative pressure, genome integrity becomes quickly compromised, increasing the potential for stem cell exhaustion and cancers. DNA repair pathways play a critical role in limiting stem cell failure and exhaustion during replicative pressure, and their loss contributes to the development of age–associated phenotypes (Murga et al., 2009; Walter et al., 2015). It can be anticipated that dynamically-regulated genome maintenance strategies are employed to ensure resilience of proliferating stem cell populations. The identity and regulation of these strategies remain to be established in vivo.

Potential dangers to genome integrity include replication-related DNA damage and telomere dysfunction, but also deficiencies in higher-order chromatin regulation and dysfunction in retro-transposon control. The cell autonomous activation and integration of transposable elements (TEs) can lead to insertional mutagenesis and genome rearrangements (Burns and Boeke, 2012). Such phenomena have recently been implicated in the aging process of multiple organisms (De Cecco et al., 2013; Maxwell et al., 2011; Wang et al., 2011). In the aging fly brain, somatic transposition increases and exacerbated TE expression results in age-associated impairment of memory and shorter lifespan (Li et al., 2013).

Given the mutagenic potential and the impact at the level of genomic instability of transposition events (Wang et al., 2011), organisms have evolved mechanisms to repress the activity of their endogenous TEs. In the germline, this is primarily achieved by the Piwi-interacting RNA pathway (Siomi et al., 2011), which represses TE activity by promoting post-transcriptional processing of TE mRNAs and mediating transcriptional silencing of TE rich regions through chromatin modifications. Piwi is the founding member of the evolutionarily conserved Ago/Piwi protein family and is required for the self-renewal of germline stem cells (GSCs) in flies and mice (Carmell et al., 2007; Cox et al., 1998; Unhavaithaya et al., 2009). In both species, loss of Piwi is associated with transposon desilencing and increased apoptosis (Juliano et al., 2011). The functions of Piwi outside the germline are only beginning to be explored (Ross et al., 2014). In differentiated somatic tissues, a physiologic function for the Piwi pathway has been reported in the brain and fat body of Drosophila (Janic et al., 2010; Jones et al., 2016). Piwi protein orthologues are also expressed in neoblasts of planaria (Reddien et al., 2005) and blastemal cells of salamander (Zhu et al., 2012), where they are required for efficient regeneration. However, the physiological function of the pathway and its relevance for stem cell maintenance and regenerative capacity has not been explored. Intestinal stem cells (ISCs) of the Drosophila posterior midgut epithelium constitute an experimentally accessible system to address these questions (Ayyaz and Jasper, 2013).

ISCs are the main mitotically competent cell type in the intestinal epithelium of flies, giving rise to an enteroblast (EB) daughter cell that further differentiates into an enterocyte (EC) or an enteroendocrine cell (EE). A complex network of local and systemic signals regulates ISC maintenance and proliferative homeostasis (Ayyaz and Jasper, 2013). In response to an acute stress signal, such as the infection by a pathogen, ISCs readily increase their proliferative activity to fulfill regenerative demands, and subsequently shut down the proliferative response to avoid hyperplasia and reinstate epithelial homeostasis (Ayyaz et al., 2015). Damage to the epithelium induces the expression of cytokines of the Unpaired family (Upd 2 and 3) in ECs, which in turn activate JAK/STAT signaling in ISCs to promote the shift from quiescence to proliferation. JAK/STAT signaling cooperates with EGF Receptor (EGFR) activation to promote proliferation of ISCs, and the return to quiescence is associated with inhibition of JAK/STAT signaling by negative feedback regulators like SOCS36E (Ayyaz and Jasper, 2013).

The control of proliferative activity is de-regulated in old flies, resulting in dysplasia that is reminiscent of hyper-proliferative phenotypes acquired in young animals in response to chronic stress (Ayyaz and Jasper, 2013). This is accompanied by widespread mis-differentiation of ISC daughter cells, caused by ectopic activation of Delta/Notch signaling (Biteau et al., 2008).

Due to the frequent exposure of ISCs to regenerative pressure, mechanisms that control genomic integrity of ISCs are likely to be critical for long-term functional maintenance of the intestinal epithelium. However, how this is achieved remains unclear. Here we show that the dynamic control of Piwi expression in ISCs is one such mechanism. Piwi is transcriptionally activated in ISCs that are induced to proliferate, and is required for regenerative capacity and maintenance of ISCs during the regenerative process. Our findings suggest that a decline in the ability of Piwi to maintain heterochromatin contributes to the age-associated deregulation of ISC function and loss of tissue homeostasis.

RESULTS

ISCs respond to infection-induced JAK/STAT signaling by increasing Piwi expression

In response to infection with the mild enteropathogen Erwinia carotovora carotovora 15 (Ecc15), ISCs undergo transient activation followed by a return to quiescence when the infection is cleared (Fig. 1A and B). To characterize molecular changes that are associated with this transient activation of ISCs, we analyzed transcriptomes of wild-type (wt) and STAT-deficient ISCs isolated at various time points after infection by fluorescence-activated cell sorting (FACS; compare (Dutta et al., 2015), Fig. 1A and C). STAT was knocked down by expression of a dsRNA against STAT using the ISC/EB specific driver escargot::Gal4 (esg::Gal4, UAS::GFP), combined with ubiquitous expression of temperature sensitive Gal80 (tub::Gal80ts, TARGET system). RNAi was induced in young (3 day old) adults for 3 days prior to infection (Fig. 1A) to knock down STAT expression (Fig. S1A).

Figure 1. Infection induced JAK/STAT signaling promotes Piwi expression in ISCs/EBs.

Figure 1

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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, 50–100 midguts/sorting). Error bars indicate s.e.m. and p-values are from student’s t-test. See also Fig. S1 and Table S1.

ISCs were identified in the FACS experiment by their expression of GFP, and were differentiated from EBs by their smaller size (Fig. 1C; note that inhibition of Gal4 in EBs by expression of Gal80 under the control of a Su(H) promoter (Wang et al., 2014b) selectively inhibits GFP in the larger cell population). This strategy allows faithful purification of ISCs, as confirmed by expression of the ISC marker Delta (Dl) and enrichment for progenitor-specific genes (Fig. 1D and S1B). This cell population is also depleted for genes expressed in ECs (Fig. S1B).

Using RNAseq (Fig. S1C), we identified over 655 genes induced in wt ISCs at 4 hours after Ecc15 infection. About one half of those genes required JAK/STAT activity for their induction, and this subset was highly enriched for genes associated with DNA replication and repair (Fig. 1E–F and Table S1). piwi was found among these genes (Fig. 1G), and analysis of independent samples collected in the same conditions, but analyzed by qRT-PCR, confirmed STAT-dependent Piwi induction in ISCs upon regenerative pressure (Fig. 1H).

Using in situ hybridization (Fig. 2A), immunohistochemistry (Fig. 2B), a lacZ enhancer trap line (Fig. S2A) and a reporter line expressing an N-terminally EGFP tagged Piwi from the endogenous piwi locus (Fig. S2B, (Sienski et al., 2012)), we found that Piwi was specifically expressed in ISCs and EBs of the posterior midgut, but not in differentiated cell types. In these experiments, ISCs/EBs were detected by GFP expression driven by esg::Gal4 (Fig. 2A–B), or by co-staining with Dl (Fig. S2A–B), which co-localized with piwi mRNA or protein signal. We confirmed the induction of Piwi protein after Ecc15 infection (Fig. 2B and Fig. S2A–D) and the dependence on STAT signaling (Fig. 2B). Piwi protein detected using immunohistochemistry (Fig. 2B) or with the GFP signal of the tagged Piwi protein (Fig. S2B), further revealed that Piwi localizes both to the cytoplasm and to the nucleus.

Figure 2. Piwi expression in ISCs/EBs of the Drosophila midgut.

Figure 2

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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.

Piwi expression was also induced, in a STAT dependent manner, in ISCs/EBs of flies exposed to other damaging insults, such as bleomycin (Fig. S2E), and in conditions where proliferative pressure is imposed by the overexpression of constitutively active Ras (Rasv12, Fig. S2F). Furthermore, activation of STAT signaling, through over-expression of a constitutively active form of the JAK kinase hopscotch (hopTumL, (Hanratty and Dearolf, 1993)) was sufficient to induce Piwi expression in progenitor cells (Fig. S2F). Conversely, blocking EGFR signaling prevented Ecc15-dependent Piwi induction (Fig. S2F). Thus, Piwi is induced in ISCs/EBs of the Drosophila midgut under proliferative pressure, independently of the type of regenerative stimulus.

To test if Piwi activity is required to regulate or maintain ISC function during regeneration, we analyzed the effects of Piwi knockdown (Fig. 3A). Expression of an RNAi against Piwi for 3 days in ISCs/EBs of young adult flies was sufficient to significantly reduce its expression in the midgut (Fig. S2G–H). Loss of Piwi impaired regenerative capacity of the gut (Fig. 3A), leading to reduced density of both ECs and progenitor cells in infected midguts containing Piwi-deficient progenitors. This was likely associated with the inability to maintain ISCs rather than with defects in ISC activation, as the number of Dl+ cells was significantly reduced in midguts containing Piwi-deficient ISCs, while mitotic activity (determined using phosphorylated Histone H3, pH3) was similar to wild-type guts at earlier time points (Fig. 3A and Fig. S3A). Similarly, piwi2 homozygous flies had significantly lower numbers of Dl+ ISCs already 48h after Ecc15 infection, and a large number of these ISCs also expressed the EC marker PDM1, indicating mis-differentiation (Fig. S3B).

Figure 3. Piwi is required for maintenance of ISC function.

Figure 3

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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.

The requirement of Piwi for ISC function under regenerative pressure was further confirmed by lineage-tracing ISCs homozygous for the piwi3 loss of function allele (Lin and Spradling, 1997), using mosaic analysis with a repressible cell marker (MARCM) during regeneration. Consistent with the previous results, piwi3 mutant ISCs generated smaller MARCM clones than wild-type controls (Fig. 3B). piwi-deficient MARCM clones were generally composed of 2–3 cells rather than single ISCs, indicating that before arresting, ISCs underwent a few rounds of division after induction of piwi3 homozygosity.

Survival after enteropathogen infection is an indicator of effective regeneration, as inability to restore epithelial integrity can result in animal’s death. Thus, we monitored survival following infection with the strong enteropathogen Pseudomonas entomophila (P.e.). Flies with Piwi-deficient ISCs and EBs died faster after infection with P.e. (Fig. 3C–D), consistent with an inability to efficiently regenerate the intestinal epithelium (Fig. 3C).

The role of Piwi in maintaining ISC function during proliferative pressure was not limited to infection conditions, as loss of Piwi limited the growth of ISC tumors generated by Notch deficiency (Fig. S3C). Our results suggest that even in these conditions, Piwi-deficient ISCs cannot sustain a high rate of proliferative activity.

Nuclear Piwi is required for long-term maintenance of ISC function

We asked if Piwi was also required for long-term maintenance of ISCs and midgut homeostasis. We knocked down Piwi in ISCs of young adults and analyzed the effects 7 and 14 days later. While Piwi-deficient intestines did not exhibit major defects at 7 days after Piwi knockdown (not shown), loss of epithelial homeostasis became apparent at 14 days after Piwi depletion, as evidenced by a reduction in the density of ECs (Fig. 3E, large polyploid nuclei stained with DAPI) and progenitor cells (Fig. 3E). These effects of Piwi loss are region-specific, with most of ISC/EB loss occurring in the R4bc morphological subdomain (Buchon et al., 2013) of the midgut (Fig. 3E).

To determine the cell-type specificity of this effect, we used ISC-specific and EB-specific drivers to knock down Piwi. ISC-specific knock down of Piwi resulted in a significant loss of ISCs (Fig. S3D) while EB specific knock down of Piwi had no effect on ISC or EB numbers (Fig. S3E).

Altogether, these data suggested that Piwi acts in an ISC-autonomous fashion to maintain ISC function and that, accordingly, loss of Piwi is accompanied by a progressive loss of ISC function in the posterior midgut. This observation was reproduced in piwi2 mutant flies (Fig. S3F) and by lineage-tracing piwi-mutant ISCs: clones derived from piwi3 homozygous ISCs were significantly smaller than clones derived from wild-type ISCs (Fig. S3G–I). This phenotype was region specific (Fig. S3H, also after Ecc15 infection Fig. S3J) and recapitulated the loss of ISCs observed after Piwi knock down, as there was a significantly higher proportion of clones derived from piwi3 homozygous ISCs which were completely depleted of Dl+ ISCs (Fig. S3I).

Piwi proteins have both cytoplasmic and nuclear functions, having been implicated in the maintenance of the chromatin state in the nucleus (Rozhkov et al., 2013). To assess if Piwi nuclear function is critical for ISC maintenance, we lineage traced ISCs that were homozygous for a piwi allele in which the 26 N-terminal amino acids containing the nuclear localization signal are absent (piwiNT). This mutant Piwi lacks the ability to translocate to the nucleus, thus preventing its ability to regulate chromatin structure, while retaining its cytoplasmic function (Klenov et al., 2011). piwiNT MARCM clones phenocopied the piwi3 loss of function mutant, suggesting that the nuclear function of Piwi is required for the maintenance of ISC function (Fig. S3K).

Piwi regulates heterochromatin maintenance in ISCs

The silencing of transposons in the Drosophila germline relies in part on the formation and maintenance of heterochromatic regions by Piwi family proteins, through interaction with heterochromatin-forming pathways (Sienski et al., 2012). To test this function of nuclear Piwi in ISCs, we assessed the extent of heterochromatinization globally using a Position-Effect Variegation (PEV) reporter line (Lu et al., 1996). The In(3L)BL1 line contains a heat shock-inducible HS-lacZ gene insertion juxtaposed to pericentric heterochromatin, resulting in variegated expression of lacZ. Thus, lacZ expression can be evaluated at the level of single cells as an indicator of heterochromatin status (Fig. 4A). piwi3 heterozygous mutants showed an increase in the number of lacZ expressing progenitor cells in the posterior midgut at 20 days of age (Fig. 4B) and in conditions of acute regenerative pressure caused by infection with Ecc15 (Fig. S4A), associated with a significant increase in LacZ transcript abundance (Fig. 4B and S4A). This indicates that a significant loss of constitutive heterochromatin occurred in those animals and that the nuclear function of Piwi that is required for ISC maintenance may involve regulation of heterochromatin. Supporting this view, loss of Piwi was associated with de-repression of TE expression and with TE mobilization: Knockdown of Piwi, but not of Aub or AGO3, was accompanied by an accumulation of several TE transcripts (Fig. 4C). The requirement of Piwi for TE repression was further confirmed in the piwi3 heterozygous background, where a significant induction of Gypsy could be detected, in homeostasis and after Ecc15 infection (Fig. S4B). Furthermore, the number of integration events in piwi3 heterozygous flies, assessed using a Gypsy-TRAP reporter system (Li et al., 2013), was higher than in wt controls (Fig. 4D–E). The Gypsy-TRAP reporter consists of a GAL80 (Gal4 inhibitor) transgene expressed under the control of a tubulin promoter separated by a gypsy target site (Tub::OvoSite::GAL80), so that insertions of gypsy prevent GAL80 expression. Combined with esg::Gal4, UAS::GFP, this system allows the detection of de novo gypsy integration events in ISCs and EBs (Fig. 4D).

Figure 4. Piwi is required for chromatin maintenance and transposon silencing.

Figure 4

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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.

Piwi is required to prevent apoptosis of ISCs

To further characterize the consequences of Piwi loss in ISCs, we analyzed the transcriptome of Piwi-deficient ISCs (Table S1) isolated by FACS. GO (Gene Ontology) term and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway classification analysis of genes up-regulated after Piwi knockdown indicated enrichment for components involved in proteasome-mediated degradation, DNA damage response (DDR) pathways and apoptosis (Fig. Fig. S4C–E). Interestingly, we found several subunits of the proteasome complex overexpressed, in agreement with previously reported effects of piwi loss in the germ line (Le Thomas et al., 2013). The upregulated DDR components and molecules associated with apoptosis suggested an increase in DNA damage, which may result in checkpoint activation and the triggering of apoptotic pathways. Using qRT-PCR in an independent set of samples of ISCs isolated by FACS, we confirmed the up-regulation of several of the genes listed above, as well as of TEs in Piwi-deficient ISCs (Fig. S4F–G).

As predicted by the transcriptome analysis, Piwi deficiency led to an increase in ISC apoptosis: apoptotic cells were detected using either an antibody against the cleaved form of the Drosophila effector caspase, also known as death caspase 1 (cDcp1), or Terminal deoxynucleotidyl transferase dUTP Nick End Labeling (TUNEL) (Sarkissian et al., 2014). We observed a significant increase in the percentage of cDcp1+/Dl+ ISCs expressing an RNAi against Piwi during Ecc15-induced regeneration and in 5 day-old piwi2 homozygous flies (Fig. S4H–I). Similarly, we observed a significant increase of TUNEL+ ISCs/EBs after piwi knock down (Fig. S4J). In addition, we used a GFP-based caspase activity reporter (Apoliner, (Bardet et al., 2008)) comprising two fluorophores, mRFP and eGFP, linked by a specific caspase sensitive site. piwi knock down resulted in increased caspase activity in esg+ progenitor cells, as evaluated by the nuclear translocation of GFP in apoliner flies (Fig. S4K).

TE expression and transposition increases with aging in the Drosophila midgut

Consistent with the de-repression of TE loci with aging in other species and fly tissues (De Cecco et al., 2013; Li et al., 2013; Wang et al., 2011), the transcripts of different classes of TEs were significantly increased in midguts from old flies relative to young animals (Fig. 5A). Because genomic instability and DNA damage are expected to be linked with insertions of TEs into the genome, we examined whether the increase in TE transcription in older flies was associated with physical transposition and increased DNA damage. Using the gypsy-TRAP system (Fig. 4D), we found a progressive accumulation of GFP positive cells in the intestine of aging wt flies (Fig. 5B–C). These effects were specific for TE integration events and not due to age-associated inactivation of Gal80 due to loss of heterozygosity, as a similar system composed of mutated Ovo-sites did not result in increased GFP expression (Fig. 5C).

Figure 5. Piwi overexpression prevents age-associated decline in ISC function.

Figure 5

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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.

Staining for the phosphorylated form of the Drosophila histone H2AX variant (γ-H2Av, (Madigan et al., 2002)), which accumulates near chromosomal break sites, indicated that DNA damage was elevated in intestinal cells of 60 day-old flies (Fig. S5A – note that the epithelial dysplasia that develops in old intestines precludes clear classification of these cells as ISCs or EBs (Biteau et al., 2008)).

The increase in TE expression in old intestines is thus accompanied by an increase in actively integrating transposons and by higher rates of DNA damage, suggesting that a high level of genomic rearrangements and instability could lead to a decline in ISC-maintenance and/or function.

Transcriptome analysis of 60 days old ISCs reveals defects in maintenance of heterochromatin structure and DNA repair capacity

To further detail intrinsic changes associated with ISC aging, we FACS-sorted progenitor cells from midguts of flies at different ages (isolating small, GFP+ cell populations to enrich for ISCs), and analyzed the global gene expression alterations occurring during physiological aging by RNAseq (Table S1). Strikingly, the transcriptomes of ISCs sorted from young flies (4 day old) and mid-aged flies (30 day old) were almost identical, while significant differences were observed between ISCs from 30 day-old and 60 day-old animals (Fig. S5B). Based upon GO term classification analysis, we discovered that pathways responsible for heterochromatin maintenance and DNA repair were significantly reduced in the 60 day-old animals compared to the 30 day-old animals (Fig. 5D). These findings are consistent with our earlier observations that old ISCs accumulate DNA damage and are subject to elevated TE activity. These data suggest that ISC aging is accompanied by robust changes in the highly inter-connected network of genes responsible for chromatin maintenance and repair.

Interestingly, among the GO terms significantly altered in 60 day-old ISCs, we also found pathways known to be involved in the control ISC function, including Wingless and Notch signaling, as well as genes involved in the regulation of stem cell differentiation and cell cycle (Fig. 5D and Fig. S5C). GOs up-regulated in aged ISCs included genes associated with proteolytic processes (Fig. S5D, green lines), similar to what is observed in Piwi loss of function conditions and genes associated with EC differentiation, such as Pdm1 (Fig. S5D, red lines), in agreement with observed mis-differentiation phenotypes (see below).

Piwi overexpression prevents age-associated decline in ISC function

In the Drosophila midgut, aging is associated with intestinal dysplasia and a disruption of epithelial function, caused by a combination of increased ISC proliferation and mis-differentiation (Ayyaz and Jasper, 2013; Biteau et al., 2008). Thus, given that Piwi expression does not increase in old ISCs, as it does during acute regenerative pressure (See Fig. 1 and Table S1), we explored the extent to which we could impact ISC aging by increasing Piwi levels in old progenitor cells. We used esg::Gal4 to drive the expression of a fully functional Piwi protein (using the piwiEP allele, which allows Gal4-mediated over-expression of endogenous Piwi, (Cox et al., 2000)) in ISCs/EBs of aging flies (Fig. S5E). The dysplastic phenotype observed in old flies was reduced in the midgut of animals with elevated expression of Piwi (Fig. 5E–F). However, analysis of the number of pH3+ cells detected at 30 and 60 days revealed that Piwi overexpression did not affect the capacity of ISCs to proliferate (Fig. 5G), suggesting that the rescue of epithelial dysplasia was primarily a consequence of reduced mis-differentiation. Consistently, the percentage of cells co-expressing both progenitor and differentiation markers (esg+/PDM1+; Fig. 5F–G) or ISC and EB markers simultaneously (Dl1+/p4EBP+; Fig. 5H) was high at 60 days, but was significantly rescued in midguts where Piwi was overexpressed in progenitor cells (Fig. 5F–H). In agreement, ISCs sorted from 60 day-old flies overexpressing Piwi in progenitor cells showed significantly lower levels of Pdm1 mRNA compared to wt ISCs at the same age (Fig. S5F). This was accompanied by a significant reduction in TE expression (Fig. S5G). Piwi overexpression could also significantly reduce DNA damage in ISCs, quantified by the presence of γ-H2Av in Dl+ cells (Fig. 5I) and ISC apoptosis, quantified by cDCP1 staining (Fig. 5J).

DISCUSSION

Our study identifies Piwi as a critical regulator of somatic stem cell function that is induced in ISCs in periods of regenerative pressure in which JAK/STAT is active. We propose that Piwi is a critical component of the ISC safeguard machinery that acts throughout life to ensure maintenance of stem cell function (Fig. S5H). Piwi homologs have also been reported to be induced and necessary during regenerative events in other organisms (Reddien et al., 2005; Rizzo et al., 2014; Zhu et al., 2012). Furthermore, other mechanisms controlling genomic integrity, such as telomerase activity, have also been associated with stem cell proliferation and found to be required for stem cell maintenance (Behrens et al., 2014; Flores et al., 2006; Wang et al., 2014a). JAK/STAT mediated regulation of regenerative responses and their de-regulation during aging is conserved in mammals (Neves et al., 2015), highlighting the importance of its downstream effectors in the proper regulation of tissue homeostasis. Interestingly, hTert expression is also activated downstream of STAT signaling in human cancers (Chung et al., 2013; Konnikova et al., 2005), suggesting that JAK/STAT activation leads to the coordinated activation of pathways safeguarding genomic integrity (see also supplemental discussion).

However, analysis of the Piwi locus does not suggest that Piwi is a direct STAT target. Piwi is induced in proliferating ISCs independently of the activating stimulus and its induction is also dependent on other pathways that regulate ISC activation. Piwi induction may thus be a general feature of ISCs under proliferative pressure, strengthening the idea that Piwi plays a key function in dividing ISCs. The molecular mechanism of Piwi induction in ISCs during regenerative events will be an interesting area of future study.

Although our data clearly establishes a role for Piwi in the prevention of TE expression and transposition in ISCs and in the maintenance of somatic stem cell function during the organism’s lifespan, it remains to be determined if there is a causal relation between the two observations. Moreover, it remains to be determined if, as in the germline, Piwi function relies on piRNA -mediated mechanisms to regulate TE silencing and heterochromatin maintenance. piRNA-independent roles for Piwi in somatic stem cells would be unexpected, but of significant interest for further study.

In mouse models, genomic damage and instability are major inducers of the DDR, and defects of the DNA repair machinery can cause phenotypes resembling premature aging (Wong et al., 2003). Our study suggests that targeting Piwi and other chromatin remodeling pathways may be an effective way to delay or prevent age-associated loss of stem cell function. Interestingly, recent reports support the idea that Piwi expression and TE control in other somatic tissues influence organismal homeostasis and lifespan (Jones et al., 2016). Further studies comparing the relative effects of piwi loss of function on lifespan in a tissue-specific manner will contribute to our understanding of the different mechanisms through which the Piwi pathway contributes to tissue homeostasis.

EXPERIMENTAL PROCEDURES

Fly lines and husbandry

Flies were cultured on yeast/molasses-based food at 25 °C with a 12 h light/dark cycle and female animals were used in all experiments. For details and the use of the TARGET and MARCM systems, see Supplemental Experimental Procedures and Table S2.

FACS sorting and RNAseq

For ISC isolation by FACS sorting, 80–100 adult female Drosophila guts were dissected in 1× phosphate-buffered saline with 1% bovine serum albumin (PBS-BSA) and dissociated for 30min with 500ul of 0.5% Trypsin-EDTA twice and then passed through the fine mesh of Polystyrene tubes with Cell-strainer cap (Falcon 352235). Cells were sorted using a FACS Aria II.

Bacterial infection and survival assay

Previously described procedures (Ayyaz et al., 2015) were followed for oral bacterial challenge. For details see Supplemental Experimental Procedures.

qRT-PCR analysis

cDNA was synthesized using an oligo-dT primer. Real-time PCR was performed on a Bio-Rad CFX96 detection system. Relative expression was normalized to Actin5C. For details on primers used see Supplemental Experimental Procedures.

Statistical analysis

Statistical significance of differences between groups was analyzed by Student’s unpaired two-tailed t test using Graphpad prism 5 software. Data are represented as mean ± s.e.m. of the “n” designated for each experiment.

Supplementary Material

supplement. Table S1, related to Figure 1, 4 and 5.

FPKM values obtained by RNA sequencing for the samples as described.

Acknowledgments

Work was supported by NIH grants AG047497, AG050104, GM117412.

Footnotes

AUTHOR CONTRIBUTIONS

Conceptualization: P.S.V., V.V.L. and H.J.; Methodology, Validation, Formal Analysis: P.S.V. and H.J.; Investigation: P.S.V, A.A., R.H., Y.Q. and D.T.M.; Writing – Original Draft: P.S.V and H.J.; Writing – Review & Editing: P.S.V and H.J.; Visualization: P.S.V and H. J.; Supervision: H.J.; Project Administration: P.S.V. and H.J.; Funding Acquisition: H.J.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

supplement. Table S1, related to Figure 1, 4 and 5.

FPKM values obtained by RNA sequencing for the samples as described.

NIHMS957572-supplement-supplement.docx (80.8MB, docx)

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