elife01530.xml

<?xml version="1.0" encoding="UTF-8"?><!DOCTYPE article PUBLIC "-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD v1.1d1 20130915//EN"  "JATS-archivearticle1.dtd"><article article-type="research-article" dtd-version="1.1d1" xmlns:xlink="http://www.w3.org/1999/xlink"><front><journal-meta><journal-id journal-id-type="nlm-ta">elife</journal-id><journal-id journal-id-type="hwp">eLife</journal-id><journal-id journal-id-type="publisher-id">eLife</journal-id><journal-title-group><journal-title>eLife</journal-title></journal-title-group><issn publication-format="electronic">2050-084X</issn><publisher><publisher-name>eLife Sciences Publications, Ltd</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="publisher-id">01530</article-id><article-id pub-id-type="doi">10.7554/eLife.01530</article-id><article-categories><subj-group subj-group-type="display-channel"><subject>Research article</subject></subj-group><subj-group subj-group-type="heading"><subject>Developmental biology and stem cells</subject></subj-group></article-categories><title-group><article-title>p53 activity is selectively licensed in the <italic>Drosophila</italic> stem cell compartment</article-title></title-group><contrib-group><contrib contrib-type="author" equal-contrib="yes" id="author-7881"><name><surname>Wylie</surname><given-names>Annika</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="equal-contrib">†</xref><xref ref-type="other" rid="par-5"/><xref ref-type="fn" rid="con1"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" equal-contrib="yes" id="author-7882"><name><surname>Lu</surname><given-names>Wan-Jin</given-names></name><xref ref-type="aff" rid="aff2"/><xref ref-type="fn" rid="equal-contrib">†</xref><xref ref-type="fn" rid="con2"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-7883"><name><surname>D’Brot</surname><given-names>Alejandro</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="con3"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-7885"><name><surname>Buszczak</surname><given-names>Michael</given-names></name><xref ref-type="aff" rid="aff3"/><xref ref-type="other" rid="par-6"/><xref ref-type="fn" rid="con4"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" corresp="yes" id="author-7748"><name><surname>Abrams</surname><given-names>John M</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="corresp" rid="cor1">*</xref><xref ref-type="other" rid="par-1"/><xref ref-type="other" rid="par-2"/><xref ref-type="other" rid="par-3"/><xref ref-type="other" rid="par-4"/><xref ref-type="fn" rid="con5"/><xref ref-type="fn" rid="conf1"/></contrib><aff id="aff1"><institution content-type="dept">Department of Cell Biology</institution>, <institution>University of Texas Southwestern Medical Center</institution>, <addr-line><named-content content-type="city">Dallas</named-content></addr-line>, <country>United States</country></aff><aff id="aff2"><institution content-type="dept">Institute for Stem Cell Biology and Regenerative Medicine</institution>, <institution>Stanford University School of Medicine</institution>, <addr-line><named-content content-type="city">Stanford</named-content></addr-line>, <country>United States</country></aff><aff id="aff3"><institution content-type="dept">Department of Molecular Biology</institution>, <institution>University of Texas Southwestern Medical Center</institution>, <addr-line><named-content content-type="city">Dallas</named-content></addr-line>, <country>United States</country></aff></contrib-group><contrib-group content-type="section"><contrib contrib-type="editor"><name><surname>Prives</surname><given-names>Carol</given-names></name><role>Reviewing editor</role><aff><institution>Columbia University</institution>, <country>United States</country></aff></contrib></contrib-group><author-notes><corresp id="cor1"><label>*</label>For correspondence: <email>John.Abrams@utsouthwestern.edu</email></corresp><fn fn-type="con" id="equal-contrib"><label>†</label><p>These authors contributed equally to this work</p></fn></author-notes><pub-date date-type="pub" publication-format="electronic"><day>11</day><month>03</month><year>2014</year></pub-date><pub-date pub-type="collection"><year>2014</year></pub-date><volume>3</volume><elocation-id>e01530</elocation-id><history><date date-type="received"><day>13</day><month>09</month><year>2013</year></date><date date-type="accepted"><day>11</day><month>02</month><year>2014</year></date></history><permissions><copyright-statement>© 2014, Wylie et al</copyright-statement><copyright-year>2014</copyright-year><copyright-holder>Wylie et al</copyright-holder><license xlink:href="http://creativecommons.org/licenses/by/3.0/"><license-p>This article is distributed under the terms of the <ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/3.0/">Creative Commons Attribution License</ext-link>, which permits unrestricted use and redistribution provided that the original author and source are credited.</license-p></license></permissions><self-uri content-type="pdf" xlink:href="elife01530.pdf"/><abstract><object-id pub-id-type="doi">10.7554/eLife.01530.001</object-id><p>Oncogenic stress provokes tumor suppression by p53 but the extent to which this regulatory axis is conserved remains unknown. Using a biosensor to visualize p53 action, we find that <italic>Drosophila</italic> p53 is selectively active in gonadal stem cells after exposure to stressors that destabilize the genome. Similar p53 activity occurred in hyperplastic growths that were triggered either by the Ras<sup>V12</sup> oncoprotein or by failed differentiation programs. In a model of transient sterility, p53 was required for the recovery of fertility after stress, and entry into the cell cycle was delayed in p53<sup>-</sup> stem cells. Together, these observations establish that the stem cell compartment of the <italic>Drosophila</italic> germline is selectively licensed for stress-induced activation of the p53 regulatory network. Furthermore, the findings uncover ancestral links between p53 and aberrant proliferation that are independent of DNA breaks and predate evolution of the ARF/Mdm2 axis.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01530.001">http://dx.doi.org/10.7554/eLife.01530.001</ext-link></p></abstract><abstract abstract-type="executive-summary"><object-id pub-id-type="doi">10.7554/eLife.01530.002</object-id><title>eLife digest</title><p>The most common genetic change seen in cancer patients produces a faulty version of the p53 protein, which normally restricts tissue growth. This change promotes cancer because cells can now divide faster and fail to die when they should. Much remains to be learned about how p53 functions to restrain growth. As p53 is found in primitive organisms, and cancer is unlikely to have significantly influenced evolution, suppressing tumor formation was almost certainly not the original function of this gene. Furthermore, p53 works in a different way compared to many other tumour suppressors. Therefore, prevention of cancer is likely to have evolved as a side effect derived from more ancient functions.</p><p>Recently, a link between p53 and stem cells has been uncovered. Stem cells are special because they can develop into many different types of cells, and they are crucial for the growth and repair of tissues. To form a particular type of cell, the stem cell divides to create two daughter cells. Commonly, one daughter cell stays in the stem state, whereas the other becomes a particular type of cell, such as a nerve cell or muscle cell. Because of this special property, scientists hypothesize that stem cells have special mechanisms to protect them from DNA damage that might partially depend on p53. This would prevent the spread of damaged genomes that would otherwise occur among daughter cells.</p><p>To learn more about how p53 influences stem cells, Wylie, Lu et al. monitored its activity in the gonads of fruit flies, which are a powerful genetic model. They found that damaging DNA activates p53 in stem cells and their daughter cells, but not in other types of cells that have been damaged. In addition, p53 is activated by the uncontrolled growth and division of stem cells in the gonad, even when DNA is not damaged. This is unexpected since molecules linking inappropriate growth to p53 were thought to be present only in mammals. Therefore, it appears that the tumor-suppressing behavior of p53 in mammals was adapted from its more ancient ability to regulate stem cell growth—an ability that evolved before organisms divided into vertebrates and invertebrates.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01530.002">http://dx.doi.org/10.7554/eLife.01530.002</ext-link></p></abstract><kwd-group kwd-group-type="author-keywords"><title>Author keywords</title><kwd>stem cells</kwd><kwd>germline</kwd><kwd>p53</kwd><kwd>biosensor</kwd><kwd>oncogenic stress</kwd><kwd>Drosophila</kwd></kwd-group><kwd-group kwd-group-type="research-organism"><title>Research organism</title><kwd><italic>D. melanogaster</italic></kwd></kwd-group><funding-group><award-group id="par-1"><funding-source><institution-wrap><institution>CPRIT</institution></institution-wrap></funding-source><award-id>RP110076</award-id><principal-award-recipient><name><surname>Abrams</surname><given-names>John M</given-names></name></principal-award-recipient></award-group><award-group id="par-2"><funding-source><institution-wrap><institution>Ellison Foundation</institution></institution-wrap></funding-source><award-id>AG-SS-2743-11</award-id><principal-award-recipient><name><surname>Abrams</surname><given-names>John M</given-names></name></principal-award-recipient></award-group><award-group id="par-3"><funding-source><institution-wrap><institution>National Institute of General Medical Sciences</institution></institution-wrap></funding-source><award-id>R01GM072124</award-id><principal-award-recipient><name><surname>Abrams</surname><given-names>John M</given-names></name></principal-award-recipient></award-group><award-group id="par-4"><funding-source><institution-wrap><institution>Welch Foundation</institution></institution-wrap></funding-source><award-id>I-1727</award-id><principal-award-recipient><name><surname>Abrams</surname><given-names>John M</given-names></name></principal-award-recipient></award-group><award-group id="par-5"><funding-source><institution-wrap><institution>Genetics Training Grant</institution></institution-wrap></funding-source><award-id>5T32GM083831</award-id><principal-award-recipient><name><surname>Wylie</surname><given-names>Annika</given-names></name></principal-award-recipient></award-group><award-group id="par-6"><funding-source><institution-wrap><institution>National Institute of General Medical Sciences</institution></institution-wrap></funding-source><award-id>GM086647</award-id><principal-award-recipient><name><surname>Buszczak</surname><given-names>Michael</given-names></name></principal-award-recipient></award-group><funding-statement>The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.</funding-statement></funding-group><custom-meta-group><custom-meta><meta-name>elife-xml-version</meta-name><meta-value>2</meta-value></custom-meta><custom-meta specific-use="meta-only"><meta-name>Author impact statement</meta-name><meta-value>Ancient features of the p53 regulatory network are coupled to the stem cell compartment in normal and pathologic contexts.</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id="s1" sec-type="intro"><title>Introduction</title><p>Throughout the animal kingdom, p53 occupies a central position within conserved stress response networks. The protein integrates diverse signals associated with DNA damage and uncontrolled proliferation to govern adaptive downstream responses such as increased DNA repair, arrested cell cycle, and apoptosis (<xref ref-type="bibr" rid="bib43">Vousden and Lane, 2007</xref>). Where examined, the genes encoding p53 are not essential for viability but have been implicated as regulators of aging (<xref ref-type="bibr" rid="bib9">Derry et al., 2007</xref>; <xref ref-type="bibr" rid="bib10">Donehower et al., 1992</xref>; <xref ref-type="bibr" rid="bib19">Lee et al., 2003</xref>; <xref ref-type="bibr" rid="bib37">Sogame et al., 2003</xref>). It is now well appreciated that ancestral roles for this gene family must have predated functions in tumor suppression. In support of this, members of the p53 gene family are present in unicellular protists and short-lived multicellular organisms (<xref ref-type="bibr" rid="bib24">Lu et al., 2009</xref>; <xref ref-type="bibr" rid="bib29">Mendoza et al., 2003</xref>; <xref ref-type="bibr" rid="bib31">Nordstrom and Abrams, 2000</xref>). Furthermore, cancer was probably a negligible source of selection pressure during the course of human evolution (<xref ref-type="bibr" rid="bib2">Aranda-Anzaldo and Dent, 2007</xref>) and the combined removal of canonical p53 effectors (p21, Puma, and Noxa) does not account for tumor suppression in mice (<xref ref-type="bibr" rid="bib41">Valente et al., 2013</xref>). These and other observations suggest that tumor suppressive roles for the p53 family were co-opted from primordial functions, some of which may have been linked to meiotic recombination (<xref ref-type="bibr" rid="bib23">Lu et al., 2010</xref>).</p><p>In recent years, considerable evidence has surfaced linking p53 action to stem cell biology. For example, in mammary stem cells p53 promotes asymmetric division and cell polarity, thereby helping to limit the population of stem cells in the mammary gland (<xref ref-type="bibr" rid="bib7">Cicalese et al., 2009</xref>). Furthermore, reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) is greatly increased in p53 deficient cells, suggesting that p53 may act as a ‘barrier for induced pluripotency’ (<xref ref-type="bibr" rid="bib18">Krizhanovsky and Lowe, 2009</xref>). Consistent with this, several labs have shown that p53 induces embryonic stem cell differentiation to maintain genomic stability after DNA damage (<xref ref-type="bibr" rid="bib21">Lin et al., 2005</xref>; <xref ref-type="bibr" rid="bib30">Neveu et al., 2010</xref>; <xref ref-type="bibr" rid="bib45">Zhao and Xu, 2010</xref>). Together with recent studies in planaria, these observations indicate that an ancestral focus of p53 action could operate in stem cells (<xref ref-type="bibr" rid="bib34">Pearson and Sanchez Alvarado, 2009</xref>). We directly tested this possibility using a p53 biosensor to visualize <italic>Drosophila</italic> germline stem cells and their progeny. When DNA breaks were exogenously imposed or intrinsically engineered, <italic>Drosophila</italic> p53 (Dp53) was activated selectively in germline stem cells (GSCs) and their immediate daughters, indicating that these cells are uniquely licensed for p53 action. Furthermore, in various germline tumor models Dp53 was constitutively hyperactivated, suggesting that ancient links between p53 and inappropriate growth predate canonical effectors that connect these regulatory networks (e.g., ARF and MDM2).</p></sec><sec id="s2" sec-type="results"><title>Results</title><sec id="s2-1"><title>Damage-induced Dp53 activity in the germline is restricted to stem cells</title><p>The <italic>Drosophila</italic> gonad is a classic system for studying the stem cell compartment since stem cells, their immediate daughters, and the surrounding niche are easily identified. In the ovary, germline stem cells (GSCs) undergo self-renewing divisions that typically produce a GSC and a cystoblast (CB). These GSCs support egg production throughout the lifespan of female adults (<xref ref-type="fig" rid="fig1">Figure 1B</xref>). We used in vivo biosensors (<xref ref-type="bibr" rid="bib23">Lu et al., 2010</xref>; <xref ref-type="bibr" rid="bib3">Brodsky et al., 2000</xref>) to visualize p53 activity as GSCs responded to various sources of stress (<xref ref-type="fig" rid="fig1">Figure 1A</xref>). To exclude technical artifacts, two GFP reporters were used—one localizes to the nucleus (p53R-GFPnls) and the other does not (p53R-GFPcyt). As previously described (<xref ref-type="bibr" rid="bib23">Lu et al., 2010</xref>), programed p53 activity triggered by meiosis was only observed in region 2 (<xref ref-type="fig" rid="fig1">Figure 1B</xref>). After exposure to ionizing radiation (IR) stress, p53 activity was induced in virtually all germaria. However, despite widespread damage to the organ (<xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1</xref>), this unprogrammed response was remarkably restricted to germline stem cells (GSCs) and their immediate progeny (CBs) (<xref ref-type="fig" rid="fig1">Figure 1C,E</xref>). Furthermore, as seen in <xref ref-type="supplementary-material" rid="SD1-data">Figure 1—source data 1A</xref>, this response was highly penetrant. Since we rarely observe reporter activation only in CBs, the signal seen in CBs probably reflects GFP perduring from the parental stem cells. Furthermore, post-irradiation levels of GFP were noticeably more robust than the programed activity during meiosis (compare solid arrows to open arrows in <xref ref-type="fig" rid="fig1">Figure 1C,D</xref>) (<xref ref-type="bibr" rid="bib23">Lu et al., 2010</xref>). As expected, p53 biosensor activity was not observed within the ovary of p53<sup>−/−</sup> animals and was also absent from ovaries lacking the upstream Chk2 kinase (<xref ref-type="fig" rid="fig1">Figure 1E</xref>, <xref ref-type="fig" rid="fig1s2">Figure 1—figure supplement 2A′,A′′</xref>, <xref ref-type="supplementary-material" rid="SD1-data">Figure 1—source data 1A</xref>).<fig-group><fig id="fig1" position="float"><object-id pub-id-type="doi">10.7554/eLife.01530.003</object-id><label>Figure 1.</label><caption><title>Genotoxic stress selectively triggers p53 activity in ovarian stem cells.</title><p>(<bold>A</bold>) Construction of p53 biosensors. A well-characterized p53 enhancer (black line) that contains a p53 consensus binding site (blue box) conserved from flies to humans resides upstream of the <italic>reaper</italic> locus (gray box) (<xref ref-type="bibr" rid="bib3">Brodsky et al., 2000</xref>). A 150-bp fragment containing this enhancer was placed upstream of GFP (p53R-GFP). Transgenic fly strains are made with two reporter constructs, one contains a nuclear localization signal for GFP (p53R-GFPnls) and the other one does not (p53R-GFPcyt). Stimuli that trigger p53 activation induce GFP expression. These biosensors require wild-type p53 and are effective readouts for p53 function. (<bold>B</bold>) Germline stem cells (GSCs) are in contact with cap cells (in gray) at the apical tip of the germarium and undergo self-renewing division to produce a GSC and cystoblast (CBs) (<xref ref-type="bibr" rid="bib40">Spradling et al., 2001</xref>). In unperturbed ovaries, programed activation of the p53R-GFP biosensor is triggered by meiotic recombination in region 2 of the germarium, marked by open arrowhead in (<bold>C</bold>) and (<bold>D</bold>) (<xref ref-type="bibr" rid="bib23">Lu et al., 2010</xref>). (<bold>C</bold>) After radiation challenge (IR) the p53R-GFPcyt biosensor (green) is selectively induced in ovarian GSCs and CBs noted by a solid arrowhead. Bracket denotes the germarium. The open arrowhead and dotted line indicates p53 activation in region 2 prompted by meiosis. Insets (<bold>C’</bold> and <bold>C’’</bold>) are confocal images from different irradiated germaria counterstained with DAPI (blue). p53R-GFPcyt induction (green) initiates in GSCs that exhibit rounded fusomes (<bold>C’</bold> white arrows) labeled by α-HTS (Hu li tai shao, red) and are in contact with cap cells (<bold>C’’</bold> yellow arrows). Cells that activate p53 in (<bold>C’</bold> and <bold>C’’</bold>) were confirmed to be germ cells by α-Vasa staining (shown in <xref ref-type="fig" rid="fig1s2">Figure 1—figure supplement 2C–D′′</xref>). (<bold>D</bold>) An engineered DNA double-stranded break (DSB) mediated by I-SceI (see texts and ‘Materials and methods’) induces the p53R-GFPnls biosensor (green) in GSCs/CBs, noted by a solid arrow. Open arrow indicates meiotic p53. The germarium is counterstained with α-HTS (red) and DAPI (blue). (<bold>E</bold>) Quantifies the percentage of germaria activated for the p53 biosensors in GSCs and their immediate progeny. Note that the perturbation-dependent responses reported here are all highly penetrant. Selective activation is IR (green) and I-SceI (blue) dependent at the 0.001 significance level. Note that biosensor activation did not occur in <italic>p53</italic><sup><italic>−/−</italic></sup> (red) or <italic>chk2</italic><sup><italic>−/−</italic></sup> (orange) mutants (see <xref ref-type="fig" rid="fig1s2">Figure 1—figure supplement 2A′,A′′</xref>). Sample sizes are combined from at least two independent trials (available in <xref ref-type="supplementary-material" rid="SD1-data">Figure 1—source data 1</xref>). All scale bars represent 10 μm. In panels <bold>C</bold>–<bold>C’’</bold> the p53R-GFPcyt reporter was used. In panel <bold>D</bold>, the p53R-GFPnls biosensor was used.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01530.003">http://dx.doi.org/10.7554/eLife.01530.003</ext-link></p><p><supplementary-material id="SD1-data"><object-id pub-id-type="doi">10.7554/eLife.01530.004</object-id><label>Figure 1—source data 1.</label><caption><title>Validation of the p53R-GFP biosensors.</title><p>In <bold>A</bold> and <bold>B</bold> reporter activation is quantified as indicated. (<bold>A</bold>) Reporter activation in female GSCs/CBs is p53 dependent and Chk2 dependent but independent of the topoisomerase, Spo11. Reporter activation in testis is also stimulus dependent and p53 dependent. p53, Chk2, or Spo11 status are noted in column 1. The reporter used (nuclear or cytoplasmic) is indicated in column 2. Column 3 shows unirradiated controls in which the percent reporter activation in GSCs/CBs is provided, as well as the total number of germaria or testis that were assayed. Column 4 shows reporter activation in irradiated tissue at 24 hr post-irradiation with percentage of germaria or testis with GFP positive GSCs/CBs and the number of samples assayed. Quantification of reporter activation is from three independent trials in the ovary and two independent trials in the testis. (<bold>B</bold>) Quantification of p53-GFPnls in region 1 of flies containing I-SceI endonuclease by itself or with the I-SceI cutsite. Reporter activation in I-SceI expressing animals that also have the I-SceI cutsite is comparable to wild-type irradiated flies (<bold>A</bold>). Quantification of reporter activation is from two independent trials. (<bold>C</bold>) Quantification of p53-GFPnls in GSCs and follicle cells of flies heterozygous (ATR<sup>+/−</sup>) or mutant for ATR (ATR<sup>−/−</sup>). After irradiation challenge, p53 activation is highly penetrant in both ATR<sup>+/−</sup> and ATR<sup>−/−</sup> genotypes. ATR mutants show a robust induction of reporter activation in follicle cells after irradiation.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01530.004">http://dx.doi.org/10.7554/eLife.01530.004</ext-link></p></caption><media mime-subtype="xlsx" mimetype="application" xlink:href="elife01530s001.xlsx"/></supplementary-material></p></caption><graphic xlink:href="elife01530f001"/></fig><fig id="fig1s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01530.005</object-id><label>Figure 1—figure supplement 1.</label><caption><title>Wide-spread DNA breaks after irradiation.</title><p>Unirradiated (<bold>A</bold>) and irradiated (<bold>B</bold>) WT germaria were stained for the <italic>Drosophila</italic> counterpart of H2Ax, designated pH2Av (green). <bold>A′</bold> and <bold>B′</bold> show the pH2Av channel alone (white) from <bold>A</bold> to <bold>B</bold> respectively. DNA double-stranded breaks visualized by pH2Av (green) appear throughout germarium within 15 min after irradiation. Germaria in <bold>A</bold> and <bold>B</bold> are counterstained with α-HTS (red) and DAPI (blue). Note that panels A and A′ are the same as in <xref ref-type="fig" rid="fig4s2">Figure 4—figure supplement 2 A-A′</xref>. Panels B and B′ are the same as in <xref ref-type="fig" rid="fig4s2">Figure 4—figure supplement 2 C-C′</xref>. Scale bar, 10 μm.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01530.005">http://dx.doi.org/10.7554/eLife.01530.005</ext-link></p></caption><graphic xlink:href="elife01530fs001"/></fig><fig id="fig1s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01530.006</object-id><label>Figure 1—figure supplement 2.</label><caption><title>Selective p53 action in germline stem cells is detected using a p53 biosensor.</title><p>Irradiated ovaries in panels (<bold>A</bold>–<bold>A′′</bold>) validate the p53R-GFP biosensor, establishing that wild-type p53 and chk2 are required for GFP activation in the germarium (brackets). (<bold>A</bold>) p53R-GFPnls is selectively activated in GSC/CBs (arrowhead) upon irradiation. Reporter activation is not seen in p53 mutants (<bold>A′</bold> p53R-GFPnls;p53<sup>−</sup>) or in Chk2 mutants (<bold>A′′</bold> Chk2<sup>−</sup>;p53R-GFPcyt). Panel (<bold>B</bold>) is a control for <xref ref-type="fig" rid="fig1">Figure 1D</xref> showing that p53R-GFPnls activation is not observed in stem cells (white arrow) that express the I-SceI enzyme but lack the I-SceI restriction site. Note that programed meiotic p53 activation is still observed in these ovaries (open arrowhead). See <xref ref-type="fig" rid="fig1">Figure 1E</xref> and <xref ref-type="supplementary-material" rid="SD1-data">Figure 1—source data 1</xref> for quantification. In panels (<bold>C</bold>–<bold>D′′</bold>) irradiated germarium are stained with α-HTS (<xref ref-type="bibr" rid="bib22">Lin et al., 1994</xref>) and α-VASA (<xref ref-type="bibr" rid="bib14">Hay et al., 1990</xref>) to confirm GFP expression in the GSCs/CBs. (<bold>C</bold> and <bold>D</bold>) p53R-GFPcyt activation (green in <bold>C</bold> and <bold>D</bold>, white arrows in <bold>C</bold>–<bold>D′′</bold>) is restricted to the germline, identified by Vasa staining (white in <bold>C′′</bold> and <bold>D′′</bold>). p53R-GFP activation is also restricted to GSCs and CBs identified by round fusomes (HTS staining, red in <bold>C</bold> and <bold>D</bold>, white in <bold>C′</bold> and <bold>D′</bold>). Rounded yellow fusomes validate co-incidence of p53R-GFP and rounded HTS. (<bold>E–E′′</bold>) p53R-GFPcyt activation in GSCs (green in <bold>E</bold>) was seen in cells that contact the cap cells (yellow arrowheads), which are visualized by location and size of DAPI stained nuclei (blue in <bold>E</bold>, white in <bold>E′′</bold>). Note that the sample in <bold>C</bold> is also shown as the <bold>C′</bold> inset in <xref ref-type="fig" rid="fig1">Figure 1C</xref> stained with DAPI. Likewise, <bold>D</bold> is also shown as the <bold>C′′</bold> inset in <xref ref-type="fig" rid="fig1">Figure 1C</xref> with single channels of α-HTS and DAPI shown in <bold>D′</bold> and <bold>D′′</bold> respectively. Also, <bold>D</bold> and <bold>E</bold> are the same sample but <bold>D</bold> highlights Vasa positive cells while (<bold>E</bold>) highlights contact with cap cells. In panels <bold>A′′</bold>, <bold>C</bold>, <bold>D</bold>, and <bold>E</bold> the p53R-GFPcyt reporter was used and in panels A, A′, and B the p53R-GFPnls reporter was used. All scale bars are 10 μm.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01530.006">http://dx.doi.org/10.7554/eLife.01530.006</ext-link></p></caption><graphic xlink:href="elife01530fs002"/></fig><fig id="fig1s3" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01530.007</object-id><label>Figure 1—figure supplement 3.</label><caption><title>ATR is not rate limiting for p53 activation in the germline.</title><p>p53R-GFPnls activation (green) was examined in ATR mutants (<bold>A</bold>–<bold>B′</bold>) after irradiation. (<bold>A</bold>–<bold>A′</bold>) GSCs (yellow arrowhead) that are in contact with cap cells (yellow arrows in <bold>A</bold> and <bold>A′</bold>) identified by DAPI staining (white in <bold>A</bold>, red in <bold>A′</bold>). These observations show that ATR is not rate limiting for p53 activation in GSCs. Panels (<bold>B</bold>–<bold>B′</bold>) show that induction of the p53R-GFPnls biosensor is not selective in ATR mutants (<bold>B′</bold>) when compared to WT controls (<bold>B</bold>). Genotype for (<bold>A</bold>–<bold>A′</bold>) is <italic>mei-41</italic><sup><italic>[D3]/[D3]</italic></sup> and for (<bold>B′</bold>) is <italic>mei-41</italic><sup><italic>[D3]/[RT]</italic></sup>. The p53R-GFPnls biosensor was used for panels <bold>A</bold>–<bold>B′</bold>. (<bold>C</bold>) Quantification of p53R-GFPnls reporter in GSCs/CBs and follicle cells in ATR heterozygous controls and ATR mutants with and without irradiation. Both the ATR<sup>+/−</sup> control and ATR<sup>−/−</sup> show a robust induction of p53R-GFPnls in GSCs after irradiation. ATR mutants also show a robust induction of reporter activation in follicle cells after irradiation. Scale bars, 10 μm.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01530.007">http://dx.doi.org/10.7554/eLife.01530.007</ext-link></p></caption><graphic xlink:href="elife01530fs003"/></fig></fig-group></p><p>Double stranded DNA breaks (DSBs) are responsible for many of the biological effects associated with IR (<xref ref-type="bibr" rid="bib44">Ward, 1994</xref>). Therefore, to determine whether DSBs are sufficient to induce the p53 reporter, we ubiquitously expressed the I-SceI endonuclease in the germline of flies engineered to harbor a single I-SceI recognition site in each nucleus. As seen with IR exposure, p53 activity occurred only in GSCs/CBs when DSBs were induced (<xref ref-type="fig" rid="fig1">Figure 1D,E</xref>, <xref ref-type="fig" rid="fig1s2">Figure 1—figure supplement 2B</xref>, <xref ref-type="supplementary-material" rid="SD1-data">Figure 1—source data 1B</xref>). Furthermore, it is notable that a single DSB was sufficient to provoke robust p53 activity in GSCs/CBs. Therefore, whether exogenously imposed or intrinsically engineered, DSBs triggered p53 selective activation that was confined to GSCs and their immediate progeny. Furthermore, this stem cell restricted response is clearly under genetic control. For example, in directed tests of chosen mutants we identified a class of lesions that exhibit non-selective p53 action throughout the ovary only after IR challenge (see <xref ref-type="fig" rid="fig1s3">Figure 1—figure supplement 3</xref>, <xref ref-type="supplementary-material" rid="SD1-data">Figure 1—source data 1C</xref>). Therefore, p53 is present and potentially functional in all cells of the ovary but, under normal conditions, its action is somehow confined to GSCs and their immediate progeny.</p><p>To ask whether this pattern might reflect a general property of germline stem cells, we similarly examined the male gonad. As seen in the ovary, we observed selective p53 reporter activation in GSCs and their immediate progeny (gonioblasts) in irradiated testis (<xref ref-type="fig" rid="fig2">Figure 2</xref>). Likewise, stimulus-dependent activity required p53 and was not seen in unchallenged testis (<xref ref-type="fig" rid="fig2">Figure 2C</xref>, <xref ref-type="fig" rid="fig2s1">Figure 2—figure supplement 1A,B</xref>, <xref ref-type="supplementary-material" rid="SD1-data">Figure 1—source data 1A</xref>). Occasionally, the biosensor was also present in early spermatogonial cysts, perhaps reflecting perduring GFP and/or independent activation associated with dying cells (<xref ref-type="fig" rid="fig2">Figure 2D–D′′</xref>, <xref ref-type="fig" rid="fig2s1">Figure 2—figure supplement 1D-E′′</xref>). Collectively, the observations in <xref ref-type="fig" rid="fig1">Figure 1</xref> and <xref ref-type="fig" rid="fig2">Figure 2</xref> demonstrate that selective p53 activation in the stem cell compartment is a general property of germline tissues exposed to genotoxic stress. We note that perturbation-dependent induction of the p53 biosensor in gonadal stem cells was highly penetrant (<xref ref-type="fig" rid="fig1">Figure 1E</xref>, <xref ref-type="fig" rid="fig2">Figure 2C</xref>). However, like all stress responses, the strength of signal and the number of responding cells were variable from animal to animal (<xref ref-type="fig" rid="fig1">Figure 1C,D</xref>, <xref ref-type="fig" rid="fig2">Figure 2D</xref>) perhaps reflecting distinct cell cycle dynamics occurring in GSCs at the time of challenge.<fig-group><fig id="fig2" position="float"><object-id pub-id-type="doi">10.7554/eLife.01530.008</object-id><label>Figure 2.</label><caption><title>Selective p53 activity occurs in male germline stem cells.</title><p>(<bold>A</bold>) p53R-GFPcyt (green) is induced at the apical tip of an irradiated testis (arrowhead), where stem cells are located (see <bold>B</bold>). α-HTS co-staining (red) highlights early stages of germline development. The inset in panel (<bold>A</bold>) shows a higher magnification view from a different irradiated testis. (<bold>B</bold>) Male GSCs are in contact with cap cells (blue flower pattern) at the apical tip of the testis and divide to produce a gonioblast daugther (GB). (<bold>C</bold>) Quantifies the percentage of testis activated for the p53 biosensors in GSCs and their immediate progeny. Selective activation is IR (green) dependent and conditional upon p53 since p53R-GFP activation did not occur in <italic>p53</italic><sup><italic>−/−</italic></sup> mutants (red bar). (<bold>D</bold>–<bold>D’’</bold>) Confocal images from other irradiated testes confirmed that stem cells induced for p53R-GFPcyt (green, <bold>D</bold> and <bold>D’’</bold>) are also positive for rounded HTS staining (red, <bold>D</bold> and <bold>D’’</bold>) and the germline specific marker Vasa (white, <bold>D’</bold> and <bold>D’’</bold>) as expected. The hub (dotted line, <bold>D</bold>) was routinely identified by the characteristic nuclei pattern as illustrated in <bold>B</bold> (blue cells) and by negative Vasa staining (<bold>D’</bold> and <bold>D’’</bold>). Asterisks mark p53R-GFP positive cells that are adjacent to the hub and Vasa positive or Vasa positive with rounded fusomes. Also note that the hub was identified by α-Armadillo staining (<xref ref-type="fig" rid="fig2s1">Figure 2—figure supplement 1C</xref>). Open arrowhead in (<bold>D</bold> and <bold>D’’</bold>) is likely a dying cyst as indicated by pyknotic and condensing nuclei and irregular HTS (<xref ref-type="fig" rid="fig2s1">Figure 2—figure supplement 1D–E′</xref>). In panels <bold>A</bold>, <bold>D</bold>–<bold>D’’</bold> the p53R-GFPcyt reporter was used. All scale bars represent 10 μm.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01530.008">http://dx.doi.org/10.7554/eLife.01530.008</ext-link></p></caption><graphic xlink:href="elife01530f002"/></fig><fig id="fig2s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01530.009</object-id><label>Figure 2—figure supplement 1.</label><caption><title>p53 Reporter activation in the male germline (seen in <xref ref-type="fig" rid="fig2">Figure 2A</xref>) is conditional upon irradiation (A) and is p53 dependent (B).</title><p>Compare <bold>A</bold> and <bold>B</bold> to <xref ref-type="fig" rid="fig2">Figure 2A</xref>) (<bold>C</bold>–<bold>C′</bold>) p53R-GFPnls (green) activation in testis after irradiation. The hub is identified here by α-Armadillo staining (red in <bold>H</bold>, white in <bold>H′</bold>), which is noted by a dotted line in (<bold>H</bold>). (<bold>D</bold>–<bold>D′</bold>) Image in (<bold>D</bold>) is a different z projection from the same irradiated testis shown in <xref ref-type="fig" rid="fig2">Figure 2D</xref>, where HTS (red) and DAPI (blue) are used to identify cells. The p53R-GFP positive dying cyst (green) indicated by the open arrowhead in Figure <bold>D</bold>–<bold>D′′′</bold> exhibits pyknotic nuclei (<bold>D′</bold> arrow), condensing nuclei (<bold>D′</bold> open arrowheads) and irregular HTS. Image in (<bold>D′</bold>) represents a magnified view of the dashed box in (<bold>D</bold>). Compare <bold>D</bold> and <bold>D′</bold> to the unirradiated WT testis control in (<bold>E</bold>–<bold>E′</bold>) that shows a branched fusome detected by α-HTS (red in <bold>E</bold> and <bold>E′</bold>) and nuclei of similar size by DAPI (blue in <bold>E</bold> and <bold>E′</bold>). In panels <bold>A</bold>, <bold>B</bold>, and <bold>E</bold> the p53R-GFPcyt reporter was used. In panels <bold>C</bold>, the p53R-GFPnls reporter was used. All scale bars are 10 μm.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01530.009">http://dx.doi.org/10.7554/eLife.01530.009</ext-link></p></caption><graphic xlink:href="elife01530fs004"/></fig></fig-group></p></sec><sec id="s2-2"><title>Genome instability provokes p53 action in the stem cells</title><p>We tested whether other genome destabilizing factors elicited similar p53 activity in stem cells. To examine the effect of deregulated retrotransposons, we introduced the p53 biosensor into <italic>cutoff</italic> or <italic>aubergine</italic> mutant animals. These genes encode essential components of the piwi-associated RNA (piRNA) pathway, acting to silence retrotransposons in the germline (<xref ref-type="bibr" rid="bib6">Chen et al., 2007</xref>). The corresponding mutants exhibit disregulated retrotransposition, reduced fecundity, and egg shell ventralization (<xref ref-type="bibr" rid="bib6">Chen et al., 2007</xref>). <xref ref-type="fig" rid="fig3">Figure 3A</xref> shows that in <italic>cutoff</italic> mutants induction of p53R-GFP occurs exclusively in GSCs and their progeny at a penetrance comparable to irradiated wild-type animals (<xref ref-type="supplementary-material" rid="SD2-data">Figure 3—source data 1</xref>). Frequent p53 activation in the germline was similarly observed in the GSCs of <italic>aubergine</italic> mutants (<xref ref-type="fig" rid="fig3">Figure 3B</xref>) and <italic>rad54</italic> mutants defective for DNA repair (<xref ref-type="fig" rid="fig3">Figure 3C</xref>). However, in contrast to <italic>cutoff</italic> mutants, the p53 biosensor was not entirely restricted to GSCs/CBs in these mutants (<xref ref-type="fig" rid="fig3s1">Figure 3—figure supplement 1B,C</xref>, <xref ref-type="supplementary-material" rid="SD2-data">Figure 3—source data 1</xref>) perhaps reflecting differences in the kinetics of repair that may occur in these different backgrounds (<xref ref-type="bibr" rid="bib17">Klattenhoff et al., 2007</xref>).<fig-group><fig id="fig3" position="float"><object-id pub-id-type="doi">10.7554/eLife.01530.010</object-id><label>Figure 3.</label><caption><title>Stem cell associated p53 activity in defective DNA repair and retrotransposon silencing mutants.</title><p>(<bold>A</bold>–<bold>B</bold>) Activation of the p53 biosensor (green) in the germarium of piRNA mutants, (<bold>A</bold>) <italic>cutoff</italic><sup>[QQ}/[WM]</sup> and (<bold>B</bold>) <italic>aubergine</italic><sup>[HN]/[QC]</sup>. (<bold>C</bold>) Activation of the p53 biosensor in <italic>rad54,</italic> a meiotic DNA repair mutant. (<bold>D</bold>–<bold>F</bold>) Germaria were found to express p53R-GFPcyt in GSCs/CBs with a penetrance of 90% for <italic>cutoff</italic> mutants (<bold>D</bold>, p&lt;0.0001), 80% for <italic>aubergine</italic> mutants (<bold>E</bold>, p=0.0018), and 33% for <italic>rad54</italic> mutants (<bold>F</bold>, p=0.0039). Asterisks indicate significant differences between heterozygous controls and homozygous mutants. GSCs/CBs were identified by rounded fusomes detected with α-HTS (red in merge <bold>A</bold>, <bold>B</bold>, <bold>C</bold> and white in <bold>A’</bold>, <bold>B’</bold>, <bold>C’</bold>). Arrowheads indicate that p53R-GFP positive cells are also germ cells identified by Vasa staining (blue in <bold>A</bold>, <bold>B</bold>, <bold>C</bold> and white split channel in <bold>A’’</bold>, <bold>B’’</bold>, <bold>C’’</bold>). Note that this particular α-Vasa antibody cross-reacts against the muscle sheath that surrounds each ovariole. If the sheath is not fully dissected and removed, then background staining is evident, as seen in <xref ref-type="fig" rid="fig2">Figure 2B′′</xref>. Control genotypes were <italic>cuff</italic><sup><italic>[WM]</italic></sup><italic>/CyO</italic>, <italic>aub</italic><sup><italic>[HN]</italic></sup><italic>/CyO</italic>, <italic>rad54</italic><sup><italic>[AA]</italic></sup><italic>/CyO</italic>. Note that <italic>aub</italic> and <italic>rad54</italic> mutants occasionally showed p53 activation beyond region 2 of the germarium (arrow in <bold>C</bold>), quantified in <xref ref-type="fig" rid="fig3s1">Figure 3—figure supplement 1</xref>, <xref ref-type="supplementary-material" rid="SD2-data">Figure 3—source data 1</xref>. All scale bars represent 10 μm. In panels <bold>A</bold>, <bold>B</bold>, and <bold>C</bold>, the p53R-GFPcyt reporter was used.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01530.010">http://dx.doi.org/10.7554/eLife.01530.010</ext-link></p><p><supplementary-material id="SD2-data"><object-id pub-id-type="doi">10.7554/eLife.01530.011</object-id><label>Figure 3—source data 1.</label><caption><title>Quantification of p53 activation in defective DNA repair and retrotransposon silencing mutants.</title><p>Mutants defective for (<bold>A</bold>) meiotic repair (<italic>rad54</italic> and <italic>rad50</italic>) and (<bold>B</bold>) retrotransposon silencing (<italic>aubergine</italic> and <italic>cutoff</italic>) have increased spontaneous reporter activation compared to heterozygous controls. The percentage of ovarioles positive for p53R-GFP in the regions indicated (GSC/CB, region 3, stage 2–8 egg chamber) was calculated and the number of ovarioles assayed per region is indicated. Quantification of reporter activation is from three independent trials.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01530.011">http://dx.doi.org/10.7554/eLife.01530.011</ext-link></p></caption><media mime-subtype="xlsx" mimetype="application" xlink:href="elife01530s002.xlsx"/></supplementary-material></p></caption><graphic xlink:href="elife01530f003"/></fig><fig id="fig3s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01530.012</object-id><label>Figure 3—figure supplement 1.</label><caption><title>Quantification of p53 activation in defective DNA repair and retrotransposon silencing mutants in region 3 and stage 2–8 egg chambers.</title><p><bold>A</bold>, <bold>B</bold>, and <bold>C</bold> show quantification of reporter activation in region 3 of the germarium. For <italic>rad54</italic> mutants, p53 activation was significantly different from the heterozygous control for region 3 (<bold>C′</bold>, p=0.0157). <bold>A′</bold>, <bold>B′</bold>, <bold>C′</bold> show quantification of p53 reporter activation in stage 2–8 egg chambers. For <italic>aubergine</italic> mutants, p53 activation was significantly different from the heterozygous control for stage 2–8 egg chambers (<bold>B′′</bold>, p=0.0437). Control genotypes were <italic>cuff</italic><sup><italic>[WM]</italic></sup><italic>/CyO</italic>, <italic>aub</italic><sup><italic>[HN]</italic></sup><italic>/CyO</italic>, <italic>rad54</italic><sup><italic>[AA]</italic></sup><italic>/CyO</italic>. See <xref ref-type="supplementary-material" rid="SD2-data">Figure 3—source data 1</xref> for number of ovarioles quantified. Samples shown here are combined from three independent trials. The p53R-GFPcyt reporter was used for these studies.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01530.012">http://dx.doi.org/10.7554/eLife.01530.012</ext-link></p></caption><graphic xlink:href="elife01530fs005"/></fig></fig-group></p></sec><sec id="s2-3"><title>p53 enables recovery from stress-induced sterility and proper exit from proliferative arrest</title><p>In somatic cells, Dp53 promotes stress-induced apoptosis (<xref ref-type="bibr" rid="bib37">Sogame et al., 2003</xref>). Therefore, we examined the germarium for evidence of cell death by detecting cleaved caspase-3. In the 24-hr period post challenge, over 90% of GSCs induce the reporter but the average incidence of apoptosis was less than 4% (<xref ref-type="fig" rid="fig4s1">Figure 4—figure supplement 1</xref>, <xref ref-type="supplementary-material" rid="SD3-data">Figure 4—source data 1A</xref>). Furthermore, we did not observe an obvious role for p53 in regulating stem cell numbers in the <italic>Drosophila</italic> ovary in the presence or absence of stress (<xref ref-type="supplementary-material" rid="SD1-data">Figure 1—source data 1</xref>, <xref ref-type="supplementary-material" rid="SD3-data">Figure 4—source data 1A</xref>). We also used α-pH2Av immunostaining, the <italic>Drosophila</italic> counterpart of mammalian pH2AX (<xref ref-type="bibr" rid="bib28">Mehrotra and McKim, 2006</xref>), to follow the repair of DSBs after IR and found that resolution of these lesions was unaffected in the germaria of p53 mutants (<xref ref-type="fig" rid="fig4s2">Figure 4—figure supplement 2</xref>). Similarly, in BrdU incorporation studies, the rates at which wild-type and p53<sup>−/−</sup> GSCs/CBs entered proliferative arrest were also indistinguishable (<xref ref-type="fig" rid="fig4">Figure 4A</xref>). However, in the post-stress period, we did observe that p53 mutants were significantly delayed for re-entry into the cell cycle (<xref ref-type="fig" rid="fig4">Figure 4A</xref>). Furthermore, this defect is reversed in p53 genomic rescue strains confirming an assignment of this phenotype to the p53 locus (<xref ref-type="supplementary-material" rid="SD3-data">Figure 4—source data 1B</xref>).<fig-group><fig id="fig4" position="float"><object-id pub-id-type="doi">10.7554/eLife.01530.013</object-id><label>Figure 4.</label><caption><title>p53 mutants exhibit impaired fertility and delayed re-entry into the cell cycle after irradiation.</title><p>(<bold>A</bold>) BrdU incorporation in GSCs after 4 krad of IR. The percentage of germaria containing BrdU positive GSCs/CBs was plotted on the Y axis. WT and p53<sup>−/−</sup> GSCs arrest with similar kinetics but p53<sup>−/−</sup> GSCs were significantly delayed for re-entry into the cell cycle. Error bars represent standard deviation from tests of three independent cohorts. WT and two rescue strains are significantly different from p53<sup>−/−</sup> at the 0.05 level at the x24 hr time point. Percentages and number of germaria assayed are included in <xref ref-type="supplementary-material" rid="SD3-data">Figure 4—source data 1B</xref>. In panels <bold>A</bold> and <bold>B</bold>, p53<sup>−/−</sup> represents animal transheterozygous for two p53 null alleles, p53<sup>ns</sup> and p53<sup>K1</sup>. (<bold>B</bold>) Fertility in wild-type (WT) and p53<sup>−/−</sup> females was measured after exposure to 11.5 krad of IR (see ‘Materials and methods’), which induces persisting sterility in p53 mutants. WT fertility is significantly different from p53<sup>−/−</sup> during time points 7–10, 10–15, and 15- at the 0.05 level (see ‘Materials and methods’). Two rescue strains showed partial restoration of fertility. Rescue 1A strain showed restored fertility is significantly different from p53<sup>−/−</sup> at the 0.05 level at days 10–15 and 15-. Note that after 15 days post irradiaton, fertility was monitored for at least 9 more days as indicated by 15-. Error bars represent standard deviation from five independent trials.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01530.013">http://dx.doi.org/10.7554/eLife.01530.013</ext-link></p><p><supplementary-material id="SD3-data"><object-id pub-id-type="doi">10.7554/eLife.01530.014</object-id><label>Figure 4—source data 1.</label><caption><title>Quantification of proliferative potential and apoptosis of germaria challenged with irradiation.</title><p>(<bold>A</bold>) Quantification of germaria that have cleaved-caspase3 (CC3) positive GSCs/CBs. Columns indicate the time points after irradiation. Rows indicate the genotype. The percentage of germaria that have CC3 positive GSCs/CBs and the number of germaria is quantified from three independent trials for the x4 hr time point and from two independent trials for the non-irradiated, x2 hr, x8 hr, and x24 hr time points. (<bold>B</bold> and <bold>C</bold>) Quantification of germaria that have BrdU positive cells in region 1. The percentage of germaria that have BrdU positive cells in region 1 and the number of germaria assayed are quantified from three independent trials in <bold>B</bold> and two trials in <bold>C</bold> per time point.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01530.014">http://dx.doi.org/10.7554/eLife.01530.014</ext-link></p></caption><media mime-subtype="xlsx" mimetype="application" xlink:href="elife01530s003.xlsx"/></supplementary-material></p></caption><graphic xlink:href="elife01530f004"/></fig><fig id="fig4s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01530.015</object-id><label>Figure 4—figure supplement 1.</label><caption><title>Reporter activation after irradiation does not lead to purging of GSCs through apoptosis.</title><p>Time course analysis of stainings for cleaved-caspase3 (CC3) in GSCs/CBs after 4.0 krad of irradiation. The percent of germaria with CC3 positive GSCs/CBs are plotted on the Y axis. The highest incidence of CC3 was only 8% at 4 hr post irradiation in wild-type flies. This is considerably different from the incidence of p53R-GFP positive stem cells after irradiation (∼90%, see <xref ref-type="fig" rid="fig1">Figure 1E</xref>). The inset is a magnified view of the same graph to better appreciate the error bars. Error bars represent standard deviations from two trials for no irradiation, 2 hr, 8 hr, 24 hr and three trials for 4 hr for both genotypes. Percentages and number of germaria assayed are provided in <xref ref-type="supplementary-material" rid="SD3-data">Figure 4—source data 1B</xref>.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01530.015">http://dx.doi.org/10.7554/eLife.01530.015</ext-link></p></caption><graphic xlink:href="elife01530fs006"/></fig><fig id="fig4s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01530.016</object-id><label>Figure 4—figure supplement 2.</label><caption><title>Radiation-induced DNA double-stranded breaks appear and disappear with similar kinetics in WT and p53<sup>−/−</sup> GSCs.</title><p>Time course of α-pH2Av (green) clearance after irradiation of wild-type (<bold>A</bold>, <bold>C</bold>, <bold>E</bold>, <bold>G</bold>, <bold>I</bold>) and p53 mutants <bold>(B</bold>, <bold>D</bold>, <bold>F</bold>, <bold>H</bold>, <bold>J</bold>) at a dose of 4 krad. Little or no pH2Av staining is observed in unirradiated WT (<bold>A</bold>) or p53<sup>−/−</sup> stem cells (<bold>B</bold>). Similar pH2Av staining is observed in WT and p53<sup>−/−</sup> stem cells 15 min after irradiation (<bold>C</bold> and <bold>D</bold>). In both cases damage was generally cleared from GSCs within 24 hr <bold>(I</bold> and <bold>J)</bold>. Note that many cells are damaged after irradiation (compare <bold>A</bold> to <bold>G</bold>) yet p53 biosensor activation is restricted to GSCs/CBs (<xref ref-type="fig" rid="fig1">Figure 1</xref>). White circles indicate stem cells. Insets are magnified views of tip of the germarium from the same image for better GSC visualization. HTS (red) and DAPI (blue) are used to highlight cells in the germarium. Note that the samples in A, A′, C and C′ are included in <xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1 A-B′</xref>. Scale bars, 10 μm.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01530.016">http://dx.doi.org/10.7554/eLife.01530.016</ext-link></p></caption><graphic xlink:href="elife01530fs007"/></fig><fig id="fig4s3" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01530.017</object-id><label>Figure 4—figure supplement 3.</label><caption><title>Fertility recovery correlates with proliferation by GSCs and their progeny.</title><p>(<bold>A</bold>) Fertility recovery after 9.0 krad of IR. A similar pattern is observed as seen at higher doses (<xref ref-type="fig" rid="fig4">Figure 4B</xref>, 11.5 krad). WT fertility is significantly different from p53<sup>−/−</sup> at all time points (p&lt;0.05). The A1 Rescue and p53<sup>−/−</sup> are significantly different at 10–15 and 15- days after IR (p&lt;0.05). Error bars represent standard deviation from four independent trials. Note that fertility was monitored for a total of 25+ days after irradiation as indicated by 15−. To link the fertility defect to cell cycle kinetic differences we observe at lower doses (<xref ref-type="fig" rid="fig4">Figure 4A</xref>, 4.0 krad), we performed 2 trials where we assayed fertility (<bold>B</bold>) and BrdU incorporation in region 1 of the germarium (<bold>C</bold>) after 9.0 krad of irradiation. Panel (<bold>C</bold>) shows that GSCs and CBs in p53<sup>−/−</sup> flies have a reduced proliferation potential at 2 and 7 days post irradiation (<xref ref-type="supplementary-material" rid="SD3-data">Figure 4—source data 1C</xref>). Fertility recovery suggests a radiation sensitivity phenotype since p53<sup>−/−</sup> flies recover fertility in a dose dependent manner (compare to <xref ref-type="fig" rid="fig4">Figure 4B</xref>).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01530.017">http://dx.doi.org/10.7554/eLife.01530.017</ext-link></p></caption><graphic xlink:href="elife01530fs008"/></fig></fig-group></p><p>To examine how the action of p53 might coordinate adaptive stress responses in GSCs, we developed a fertility recovery assay. In this study, females were irradiated to induce transient sterility and the recovery of fertility was scored over time (see ‘Materials and methods’). <xref ref-type="fig" rid="fig4">Figure 4B</xref> shows that wild-type females recovered from infertility within 1 week post-exposure to IR at a dose of 11.5 krad. In contrast, females lacking p53 remained permanently infertile even when tracked over 2 weeks after IR (<xref ref-type="fig" rid="fig4">Figure 4B</xref>). To confirm that p53 gene function is responsible for this phenotype, we tested p53<sup>−</sup> females carrying a genomic rescue fragment spanning the p53 gene (see ‘Materials and methods’). We tested two rescue strains and in both cases the sterility defect was reversed (<xref ref-type="fig" rid="fig4">Figure 4B</xref>). However, neither rescue strain fully restored fertility to wild type levels, possibly reflecting incomplete restoration of wild type regulation in the transgenes.</p><p>To test whether we could link the fertility defect (<xref ref-type="fig" rid="fig4">Figure 4B</xref>) to the cell cycle defects observed at a lower dose (<xref ref-type="fig" rid="fig4">Figure 4A</xref>), we examined fertility and cell cycle kinetics at an intermediate dose (9 krad) of IR. After this challenge, p53<sup>−/−</sup> females exhibit impaired fertility, whereas WT flies remained fertile (<xref ref-type="fig" rid="fig4s3">Figure 4—figure supplement 3</xref>). We performed BrdU incorporation studies over 7 days with females irradiated at 9 krad and assayed the number of germaria that had BrdU positive cells in region 1. Under these conditions, we observed persistently reduced proliferative activity in p53<sup>−/−</sup> stem cells even 7 days after IR (<xref ref-type="fig" rid="fig4s3">Figure 4—figure supplement 3C</xref>, <xref ref-type="supplementary-material" rid="SD3-data">Figure 4—source data 1C</xref>). This result is consistent with the possibility that fertility defects seen in p53<sup>−/−</sup> flies are linked to the impaired cell cycle kinetics found in GSCs. Furthermore, the data in <xref ref-type="fig" rid="fig4">Figure 4B</xref> and <xref ref-type="fig" rid="fig4s3">Figure 4—figure supplement 3A</xref> suggest that radiosensitivity associated with the p53<sup>−/−</sup> genotype, previously been documented for larval stages (<xref ref-type="bibr" rid="bib37">Sogame et al., 2003</xref>), also applies to germline tissue.</p></sec><sec id="s2-4"><title>Uncontrolled stem cell proliferation activates p53</title><p>Oncogenic properties are thought to simulate ‘stemness’ and oncogenic signals frequently result in p53 activation (<xref ref-type="bibr" rid="bib43">Vousden and Lane, 2007</xref>). However, it is not known whether this regulatory axis is conserved beyond mammals. To test whether inappropriate growth triggers <italic>Drosophila</italic> p53 function, we examined the p53 biosensors in various germline tumor models. First, we expressed an oncogenic form of RAS commonly found in human cancers together with the p53 biosensor (<xref ref-type="bibr" rid="bib20">Lee et al., 1996</xref>). Transient expression of the <italic>Drosophila</italic> Ras<sup>V12</sup> counterpart provoked robust p53 activation mainly in the GSCs and CBs (<xref ref-type="fig" rid="fig5">Figure 5B</xref>, <xref ref-type="supplementary-material" rid="SD4-data">Figure 5—source data 1</xref>). <xref ref-type="fig" rid="fig5">Figure 5E</xref> shows that another oncoprotein, Cyclin E, produced similar results. We also examined these biosensors in <italic>bam</italic> mutants, where a block in differentiation causes extensive hyperplasia (<xref ref-type="bibr" rid="bib26">McKearin and Ohlstein, 1995</xref>) and in these tumors extensive reporter activity was also seen (<xref ref-type="fig" rid="fig5">Figure 5C,D</xref>). Likewise, expanded BMP (bone morphogenic protein) signaling (<xref ref-type="bibr" rid="bib5">Chen and McKearin, 2003</xref>) or reduced <italic>Lsd1</italic> (lysine-specific demethylase 1) activity (<xref ref-type="bibr" rid="bib11">Eliazer et al., 2011</xref>) in neighboring somatic cells can also cause inappropriate growth and robust p53 activity was similarly observed in these germline tumors as well (<xref ref-type="fig" rid="fig5">Figure 5F,G,H</xref>). Therefore, whether caused by forced oncoprotein expression (panels B, E), failed differentiation programs (panels C, D), or expansion of the stem cell niche (panels F–H), inappropriate growth of <italic>Drosophila</italic> tissues was consistently accompanied by p53 activity. As seen with genotoxic stress, biosensor responses seen in these contexts was somewhat variable, perhaps reflecting complex signaling and/or cell cycle dynamics that occur in these tumor models. Technical sources of variation linked the UAS-GAL4 driver system and/or non-uniform accumulation of the oncogenic product could also contribute to variability in these contexts.<fig-group><fig id="fig5" position="float"><object-id pub-id-type="doi">10.7554/eLife.01530.018</object-id><label>Figure 5.</label><caption><title>Deregulated growth in the stem cell compartment provokes p53 action.</title><p>(<bold>A</bold>) In an unperturbed wild-type (WT) germarium, the p53R-GFPnls biosensor is absent from GSCs/CBs, marked here by rounded fusomes stained with α-HTS (red). The modest signal in region 2 reflects meiotic p53 activity (dotted bracket) (<xref ref-type="bibr" rid="bib23">Lu et al., 2010</xref>). When perturbed by Ras<sup>V12</sup> (<bold>B</bold>) the p53 biosensor (green) is induced in GSCs/CBs (solid bracket in <bold>B</bold>, see <xref ref-type="supplementary-material" rid="SD4-data">Figure 5—source data 1</xref>). Perturbation from failed differentiation programs caused by the <italic>bam</italic> mutation <bold>(C</bold>–<bold>D)</bold> or Cyclin E over-expression (<bold>E</bold>) provokes similar p53 biosensor activity. Likewise, increased DPP signaling caused by a constitutively active Tkv receptor (<bold>F</bold>) or ectopic DPP ligand expression (<bold>G</bold>) also prompts induction of the p53 reporter. Induction of the p53 reporter is also seen, when the stem cell niche is expanded by silencing of <italic>Lsd1</italic> (<bold>H</bold>) (<xref ref-type="bibr" rid="bib11">Eliazer et al., 2011</xref>). Insets in panels <bold>E</bold>–<bold>H</bold> are magnified views of tumor cysts showing that p53R-GFP positive cells exhibit stem-like properties with rounded fusomes detected by α-HTS co-staining (red). Note in panels <bold>B</bold>, <bold>E</bold> and <bold>F</bold>, the indicated UAS transgenes were expressed using the germline specific driver, nanos-GAL4VP16 (<xref ref-type="bibr" rid="bib35">Rorth, 1998</xref>). For panels <bold>G</bold> and <bold>H</bold>, expression was achieved by the driver c587-GAL4 in somatic cells of the ovariole tip (<xref ref-type="bibr" rid="bib38">Song et al., 2004</xref>). All images shown are immunostainings for the p53R-GFPnls biosensor (green), HTS (red), and/or DAPI (blue) except for panel <bold>D</bold> which was co-stained with α-Vasa (red) to show that p53 activated cells retain the germline marker in <italic>bam</italic> mutants. All other panels (<bold>A</bold>–<bold>C</bold>, <bold>E</bold>–<bold>H</bold>) were stained with α-HTS (red). Note that panel <bold>D</bold> stained with α-Vasa is the same <italic>bam</italic> ovariole shown in <bold>C</bold> with α-HTS. Relevant quantification including the nanosGAL4 driver alone is shown in <xref ref-type="supplementary-material" rid="SD4-data">Figure 5—source data 1</xref>. Scale bars = 10 μm.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01530.018">http://dx.doi.org/10.7554/eLife.01530.018</ext-link></p><p><supplementary-material id="SD4-data"><object-id pub-id-type="doi">10.7554/eLife.01530.019</object-id><label>Figure 5—source data 1.</label><caption><title>Quantification of biosensor activity in germline tumors.</title><p>This table quantifies the number of p53R-GFPnls positive stem-like cells associated with a rounded fusome (α-HTS). Ovaries containing the nanosGAL4 driver alone (control) or the UAS oncogene indicated (Ras<sup>v12</sup> or CyclinE) and the same GAL4 driver were scored. Note that the numbers of stem cells activated for p53 is much greater when either Ras<sup>v12</sup> or CyclinE are present when compared to the control alone.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01530.019">http://dx.doi.org/10.7554/eLife.01530.019</ext-link></p></caption><media mime-subtype="xlsx" mimetype="application" xlink:href="elife01530s004.xlsx"/></supplementary-material></p></caption><graphic xlink:href="elife01530f005"/></fig><fig id="fig5s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01530.020</object-id><label>Figure 5—figure supplement 1.</label><caption><title>Reporter induction during forced proliferation signals is independent of DNA damage.</title><p>(<bold>A</bold>) Immunostaining for α-pH2Av (red) and p53R-GFPnls (green) in <italic>bam</italic><sup><italic>Δ86</italic></sup> ovaries. (<bold>A′</bold>) shows α-pH2Av channel from (<bold>A</bold>). Note the incidence of pH2Av (arrows) is rare and infrequently colocalizes with GFP.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01530.020">http://dx.doi.org/10.7554/eLife.01530.020</ext-link></p></caption><graphic xlink:href="elife01530fs009"/></fig></fig-group></p><p>We considered the possibility that inappropriate growth might indirectly activate p53 by provoking DNA damage. To test this, we stained <italic>bam</italic><sup><italic>Δ86</italic></sup> ovaries for pH2Av (<xref ref-type="bibr" rid="bib16">Joyce et al., 2011</xref>). We observed very few pH2Av foci in <italic>bam</italic> tumors and, notably, these foci did not co-localize with p53 biosensor activity (<xref ref-type="fig" rid="fig5s1">Figure 5—figure supplement 1</xref>). Therefore, p53 activity in these tumors is not triggered by DSBs but instead, appears to be directly triggered by signals associated with hyperplastic growth.</p><p>As seen in <xref ref-type="fig" rid="fig5">Figure 5</xref>, diverse types of hyperplastic growth triggered constitutive p53 activity. To ask how p53 functions in these tumors, we examined bam<sup>−/−</sup> ovaries that were either WT or null for p53. Tumor size was not significantly altered in the absence of p53, but we did observe dramatically altered cytology in tumors that lacked p53. As seen in <xref ref-type="fig" rid="fig6">Figure 6A</xref>, bam<sup>−/−</sup> ovarian cysts are typically filled with stem-like cells that exhibit round or dumbbell-shaped fusomes when stained with α-HTS (<xref ref-type="bibr" rid="bib22">Lin et al., 1994</xref>). As documented in <xref ref-type="fig" rid="fig6">Figure 6C</xref>, defective fusomes were seen in all bam<sup>−/−</sup>;p53<sup>−/−</sup> cysts and, in nearly half of these unusually large nuclei were observed. Though not quantified, micronuclei were also prevalent in these samples. Since defective fusome morphologies and irregular nuclei are consistent with aberrant mitosis, our data suggests a role for p53 in promoting proper cell cycle progression in these stem-like tumors.<fig id="fig6" position="float"><object-id pub-id-type="doi">10.7554/eLife.01530.021</object-id><label>Figure 6.</label><caption><title>Abnormal fusomes and irregular nuclei are seen in bam<sup>−/−</sup>p53<sup>−/−</sup> tumors.</title><p>(<bold>A–A’</bold>) Cells in bam<sup>−/−</sup> tumors have rounded fusomes normally associated with the undifferentiated GSC fate. These are detected by α-HTS staining (red in <bold>B</bold>, white in <bold>B’</bold>). The nuclei of these cells counterstained with DAPI have diameters less than 10 μm (blue in <bold>B</bold>). (<bold>B</bold>–<bold>B’</bold>) bam<sup>−/−</sup>;p53<sup>−/−</sup> tumors frequently exhibit disorganized fusomes detected here by α-HTS staining (red in <bold>C</bold>, white in <bold>C’</bold>, yellow arrowhead). These tumors also have many fragmented and enlarged nuclei with a diameter significantly greater than 10 μm (blue in <bold>C</bold>, yellow arrow). (<bold>C</bold>) Quantification of altered fusome structure and irregular nuclei in bam<sup>−/−</sup> and bam<sup>−/−</sup>;p53<sup>−/−</sup> tumors. Note that in panel <bold>C</bold>, counts for irregular nuclei do not include micronuclei. A total of 14 cysts were assayed in bam<sup>−/−</sup>;p53<sup>−/−</sup> and 8 cysts were assayed for bam<sup>−/−</sup>. All scale bars, 10 μm.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01530.021">http://dx.doi.org/10.7554/eLife.01530.021</ext-link></p></caption><graphic xlink:href="elife01530f006"/></fig></p><p>To further examine the functional role of p53 in this context, we examined gene expression profiles of bam<sup>−/−</sup> ovaries that were WT or null for p53 by microarray. In total, we found that 297 gene transcripts were altered by at least twofold or greater in the absence of p53. <xref ref-type="table" rid="tbl1">Table 1</xref> lists the top 20 genes that are affected (upregulated or downregulated) by p53 in these tumors. Using the Gene Expression Commons (GEXC) tool, we compared these gene sets to existing germline, embryonic, and somatic expression profiles. We did not find a coherent pattern among the top 20 genes that are normally upregulated by p53. However, among the top 20 genes that are normally suppressed by p53 in these germline tumors, we observed a modest enrichment for transcripts that were absent in either the embryonic stages or other somatic tissues (<xref ref-type="supplementary-material" rid="SD5-data">Table 1—source data 1</xref>). These data, together with our histological studies (<xref ref-type="fig" rid="fig6">Figure 6</xref>), establish that p53 exerts functional activities that impact cellular and molecular properties of <italic>Drosophila</italic> stem cell tumors.<table-wrap id="tbl1" position="float"><object-id pub-id-type="doi">10.7554/eLife.01530.022</object-id><label>Table 1.</label><caption><p>p53 status impacts expression profiles in <italic>bam</italic><sup><italic>−/−</italic></sup> tumors</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01530.022">http://dx.doi.org/10.7554/eLife.01530.022</ext-link></p><p><supplementary-material id="SD5-data"><object-id pub-id-type="doi">10.7554/eLife.01530.023</object-id><label>Table 1—source data 1.</label><caption><title>Expression features of the top 20 genes suppressed by p53.</title><p>The top 20 genes that were suppressed by p53 in <italic>bam</italic><sup><italic>−/−</italic></sup><italic>;p53</italic><sup><italic>−/−</italic></sup> tumors (see <xref ref-type="table" rid="tbl1">Table 1</xref>) were examined using GEXC (<xref ref-type="bibr" rid="bib36">Seita et al., 2012</xref>) to identify enriched pathways. Using this collection we observed a mild enrichment for genes that were absent in embryos or absent in adult somatic tissues relative to all genes in the fly genome.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01530.023">http://dx.doi.org/10.7554/eLife.01530.023</ext-link></p></caption><media mime-subtype="xlsx" mimetype="application" xlink:href="elife01530s005.xlsx"/></supplementary-material></p></caption><table frame="hsides" rules="groups"><thead><tr><th/><th colspan="2">Downregulated by p53</th><th colspan="2">Upregulated by 53</th></tr><tr><th/><th>Gene symbol</th><th>Fold change</th><th>Gene symbol</th><th>Fold change</th></tr></thead><tbody><tr><td>1</td><td>CG31681</td><td>8.7</td><td>CG31809</td><td>−7.2</td></tr><tr><td>2</td><td>CG5156</td><td>8.0</td><td>CG31810</td><td>−5.6</td></tr><tr><td>3</td><td>LysX</td><td>7.9</td><td>CG2177</td><td>−5.2</td></tr><tr><td>4</td><td>CG31901</td><td>7.6</td><td>CG7106</td><td>−5.1</td></tr><tr><td>5</td><td>CG16762</td><td>7.5</td><td>CG1504</td><td>−4.5</td></tr><tr><td>6</td><td>CG32277</td><td>7.3</td><td>CG15614</td><td>−4.3</td></tr><tr><td>7</td><td>CG17239</td><td>7.2</td><td>unpg</td><td>−4.2</td></tr><tr><td>8</td><td>CG17012</td><td>7.1</td><td>CG7329</td><td>−4.2</td></tr><tr><td>9</td><td>CG9897</td><td>7.1</td><td>CG15236</td><td>−4.1</td></tr><tr><td>10</td><td>Ser12</td><td>6.8</td><td>CG9294</td><td>−4.1</td></tr><tr><td>11</td><td>CG2191</td><td>6.8</td><td>esg</td><td>−3.9</td></tr><tr><td>12</td><td>CG33258</td><td>6.6</td><td>Ugt36Ba</td><td>−3.7</td></tr><tr><td>13</td><td>CG18125</td><td>6.5</td><td>CG14297</td><td>−3.6</td></tr><tr><td>14</td><td>CG12780</td><td>6.4</td><td>CG17129</td><td>−3.6</td></tr><tr><td>15</td><td>CG4783</td><td>6.3</td><td>Cyp6a14</td><td>−3.6</td></tr><tr><td>16</td><td>Cyp6a18</td><td>6.3</td><td>CG5568</td><td>−3.4</td></tr><tr><td>17</td><td>CG17234</td><td>6.2</td><td>CG1077</td><td>−3.3</td></tr><tr><td>18</td><td>CG18063</td><td>6.2</td><td>CG11226</td><td>−3.3</td></tr><tr><td>19</td><td>CG9568</td><td>6.1</td><td>CG33105</td><td>−3.1</td></tr><tr><td>20</td><td>CG32834</td><td>6.0</td><td>CG3328</td><td>−3.1</td></tr></tbody></table><table-wrap-foot><fn><p>We performed microarray analysis on bam<sup>−/−</sup> and bam<sup>−/−</sup>;p53<sup>−/−</sup> tumors. The genes that are altered by p53 status in bam<sup>−/−</sup> tumors are recorded. Listed on the left are the top 20 genes whose abundance is directly or indirectly suppressed by p53. Listed on the right are the top 20 genes whose abundance is directly or indirectly induced by p53. The gene symbol is listed on the left and the fold change in gene expression between bam<sup>−/−</sup> and bam<sup>−/−</sup>;p53<sup>−/−</sup> tumors is listed on the right. Many genes listed here are dramatically affected when p53 is absent.</p></fn></table-wrap-foot></table-wrap></p></sec></sec><sec id="s3" sec-type="discussion"><title>Discussion</title><p>We found that adult <italic>Drosophila</italic> exposed to genotoxic stress or genome destabilizers selectively activated p53 in GSCs and their immediate progeny. This striking specificity was observed despite widespread Dp53 expression (<xref ref-type="bibr" rid="bib12">FBgn0039044 m.</xref>; <xref ref-type="bibr" rid="bib15">Jin et al., 2000</xref>; <xref ref-type="bibr" rid="bib33">Ollmann et al., 2000</xref>) and widespread tissue damage (<xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1</xref>). We note that stem cell specificity was not an artifact intrinsic to the biosensors, since independent reporters behaved similarly in both the female and male germline and required the wild-type Dp53 locus in both cases. Furthermore, in certain mutant backgrounds stress-induced activity restricted to GSCs was lost and non-selective p53 activation was widespread throughout the ovary (<xref ref-type="fig" rid="fig1s3">Figure 1—figure supplement 3B′</xref>). Therefore, despite the fact that it is present and activatable throughout the gonad, functional p53 is restricted to stem cells and their immediate progeny by specific genetic determinants.</p><p>Collectively, our work supports previous indications that there is an intimate and ancient link between p53 and stem cell biology (<xref ref-type="bibr" rid="bib34">Pearson and Sanchez Alvarado, 2009</xref>). Our findings also offer rare and novel opportunities to operationally mark the stem cells in the fly germline, as visualized by p53R-GFP. This marker is distinct from conventional stem cell labels (<xref ref-type="bibr" rid="bib8">Deng and Lin, 1997</xref>) since it is not constitutively expressed but, instead, represents a functional output that is conditional upon a perturbation. We further note that like all reporter systems, our p53 biosensors may not reflect the full scope of effector output regulated by this network, and activities visualized here could transmit only subsets of p53-mediated responses. Nevertheless, despite this possible limitation, our results are consistent with suggestions that stem cells may be acutely sensitive to sources of genomic instability with a higher propensity for engaging adaptive responses relative to other cells (<xref ref-type="bibr" rid="bib25">Mandal et al., 2011</xref>; <xref ref-type="bibr" rid="bib39">Sperka et al., 2012</xref>). We propose that in reproductive tissues, the p53 regulatory network is either preferentially licensed in stem cells or selectively blocked outside of this compartment.</p><p>What upstream regulators might specify p53 activation in GSCs/CBs? Given that stem cells have unique properties, p53 activation in these cells might lie downstream of a novel pathway. Consistent with this idea, ATR expression, was not rate limiting for p53 activation in the germline (<xref ref-type="fig" rid="fig1s3">Figure 1—figure supplement 3A′</xref>, <xref ref-type="supplementary-material" rid="SD1-data">Figure 1—source data 1C</xref>). Furthermore, unlike meiotic induction, p53 induction in GSCs/CBs was independent of the topoisomerase, Spo11 (<xref ref-type="supplementary-material" rid="SD1-data">Figure 1—source data 1A</xref>; <xref ref-type="bibr" rid="bib23">Lu et al, 2010</xref>). Chk2 could contribute to the selective activation in stem cells seen here, but since Chk2 is also broadly expressed and functionally associated with oocyte development throughout the ovary (<xref ref-type="bibr" rid="bib1">Abdu et al., 2002</xref>; <xref ref-type="bibr" rid="bib32">Oishi et al., 1998</xref>) any potential role in GSCs must extend beyond a simple presence or absence of this kinase.</p><p>Our findings also imply stimulus-dependent effectors of p53 in stem cells that are not yet appreciated. For example, within detection limits, we observed no obvious connection between p53 status and apoptosis, DNA double-strand break repair, or cell cycle arrest. However, irradiated <italic>p53</italic><sup><italic>−/−</italic></sup> GSCs were significantly delayed in the re-entry phase for cell cycle. Future studies will explore this defect and also examine progeny derived from stressed GSCs for transgenerational phenotypes that might be adaptive.</p><p>Our discovery that p53 action is coupled to hyperplasia in a non-vertebrate species was unexpected for two reasons. First, the role of this gene family as a tumor suppressor is thought to be a derived feature that evolved only in vertebrate lineages. Second, the canonical ARF/MDM2 pathway that links aberrant growth to p53 is absent outside of higher vertebrates (<xref ref-type="bibr" rid="bib24">Lu et al., 2009</xref>). Surprisingly, our combined results suggest that ancient pathways linking p53 to aberrant stem cell proliferation may predate the divergence between vertebrates and invertebrates.</p></sec><sec id="s4" sec-type="materials|methods"><title>Materials and methods</title><sec id="s4-1"><title>Fly stocks and genetics</title><p>All fly stocks were maintained at 22–25°C on standard food media. We obtained <italic>rad54</italic>, <italic>aubergine</italic> and <italic>cutoff</italic> mutants: <italic>rad54</italic><sup><italic>RU</italic></sup>, <italic>rad54</italic><sup><italic>AA</italic></sup>, <italic>aub</italic><sup><italic>HN</italic></sup>, <italic>aub</italic><sup><italic>QC</italic></sup>, <italic>cuff</italic><sup><italic>WM</italic></sup>, and <italic>cuff</italic><sup><italic>QQ</italic></sup> from T Schupbach (Princeton University, Princeton, NJ, USA); <italic>c587-</italic>GAL4, UAS<italic>-dpp</italic>, UAS- <italic>Lsd1</italic><sup><italic>KD</italic></sup> (<xref ref-type="bibr" rid="bib11">Eliazer et al., 2011</xref>), homozygous viable allele of <italic>bam</italic><sup><italic>Δ86</italic></sup> (<xref ref-type="bibr" rid="bib27">McKearin and Spradling, 1990</xref>; <xref ref-type="bibr" rid="bib26">McKearin and Ohlstein, 1995</xref>), <italic>nanos-</italic>GAL4VP16, and UASp-<italic>tkvCA</italic> (<xref ref-type="bibr" rid="bib4">Chen and McKearin, 2003</xref>) have been described previously. All other stocks were obtained from Bloomington Stock Center (Indiana University, Bloomington, IN, USA). The Dp53 rescue strain was engineered by ϕC31 integration of a 20-kb genomic fragment BAC containing the Dp53 locus into an attP site on the X chromosome of the PBac{y + -attP-9A}VK00006 line (Bloomington #9726). The parent BAC CH322-15D03 was obtained from the P[acman] resource library (<xref ref-type="bibr" rid="bib42">Venken et al., 2009</xref>) and Rainbow Transgenic Flies performed the injection and screening for recombinants. The I-SceI endonuclease strain was generated by K Galindo (<xref ref-type="bibr" rid="bib13">Galindo et al., 2009</xref>), which was crossed to p53R-GFPnls(STI150); HS-(70Flp)(70 I- Sce I)/TM6 for heat-inducible I-SceI endonuclease expression. Adult females were fattened for 2–3 days after eclosion and then subjected to heat shock in a circulating water bath at 37°C for 90 min and repeated for three consecutive days. 24 hr after the last heat shock, ovaries were dissected for immunostaining. For forced proliferation assays, two GAL4 lines were used: <italic>nanos</italic>-GAL4VP16 was used to achieve overexpression in the germline with UAS constructs for Ras<sup>V12</sup>, CyclinE, and Thickveins (<xref ref-type="bibr" rid="bib35">Rorth, 1998</xref>). <italic>c587</italic>-GAL4 was used to achieve overexpression of UAS constructs of <italic>Dpp</italic> or <italic>Lsd1-RNAi</italic> in the somatic cells of the ovariole tip (<xref ref-type="bibr" rid="bib38">Song et al., 2004</xref>). For cyclinE overexpression, stocks were maintained at 25°C and female virgins were collected upon eclosion, shifted to 29°C for 4–5 days then subjected to immunostaining. For the Ras<sup>V12</sup> studies, female virgins were shifted to 29°C for 1 day and then shifted down to 25°C for 3 days prior to immunostaining. The Gal4-UAS system (adapted from yeast) often produces optimal expression at temperatures higher than 25°C. Since the UAS-Rasv12 and UAS-CyclinE constructs were not optimized for expression in the germline we applied these temperature shifts to produce more penetrant phenotypes.</p></sec><sec id="s4-2"><title>Irradiation assay</title><p>Well-fed flies were exposed to ionizing radiation using a Cs-137 Mark 1-68A irradiator (JL Shepherd &amp; Associates, San Ferando, CA, USA) at a dose of 4 krad unless otherwise noted. When irradiating several genotypes, each genotype was placed in an individual vial, and all vials were exposed to IR at the same time on a rotating turntable inside the irradiator. For visualizing reporter activation after IR, flies were dissected 24 hr post-IR to allow for stable GFP expression.</p></sec><sec id="s4-3"><title>Immunostaining of fly tissue</title><p>3- to 5-days-old well-fed females were dissected in PBS and fixed in 4% EM-grade formaldehyde (Polysciences, Warrington, PA) diluted in PBS-0.1% tween-20, with three times the volume of heptane. After washing, tissues were blocked in 1.5% BSA, then incubated with primary antibodies at 4°C overnight. Antibodies used: rabbit α-GFP (Invitrogen, Carlsbad, CA); rabbit α-pH2Av (kindly provided by K McKim with specific staining protocols), rabbit α-cleaved caspase 3 (Asp175) (Cell Signaling, Danvers, MA); mouse α-Armadillo, mouse α-BrdU (Sigma, St. Louis, MO), mouse α-HTS clone 1B1 (Developmental Studies Hybridoma Bank, Iowa City, IA), and rat α-Vasa (Developmental Studies Hybridoma Bank). For fluorescence visualization, Alexa-488, 568 (Invitrogen), and DyLight 649 (Jackson ImmunoResearch, West Grove, PA) secondary antibodies were used and 0.1 μg/ml of DAPI (Invitrogen) for DNA staining was added in the first wash step. After three washes, ovaries were further hand dissected and mounted in VECTASHIELD (Vector Laboratories, Burlingame, CA) for microscopy imaging. For validating stimulus-dependent p53 action as visualized by the reporters, we routinely confirmed absence of GFP expression using flies null for Dp53. We note that p53R-GFPnls shows constitutive expression independent of p53 in a subset of gut cells and in the region of the testis containing elongated spermatids, reflecting position effects upon this transgene.</p></sec><sec id="s4-4"><title>Fertility tracking and proliferative arrest assay</title><p>In fertility assays, two <italic>p53</italic> null alleles, 238H (ns) and 5A-1-4 (k1) were used in trans-combination to reduce genetic background influences. Two wild-type strains, <italic>yw</italic> and <italic>w</italic><sup><italic>1118</italic></sup> were used for comparison. p53 rescue transgenes were tested in a transheterozygous p53<sup>−/−</sup> background (A1; ns/k1 and A2; ns/k1) to exclude contributions from background modifiers. 5- to 7-day-old females were irradiated at desired doses (11.5 krad for <xref ref-type="fig" rid="fig4">Figure 4B</xref> and 9 krad for <xref ref-type="fig" rid="fig4s3">Figure 4—figure supplement 3</xref>) and fertility was tracked over time in groups. Each group contained 10 females and five unirradiated wild-type Canton-S males. The animals were transferred to a new vial at designated time points, and fertility was scored by the presence of larvae 10 days after the parents were removed. Each trial contained 2 to 15 replicates per genotype. For <xref ref-type="fig" rid="fig4">Figure 4B</xref> percentages of fertile samples are plotted based on five trials. In the proliferative arrest assay, ovaries were dissected and immersed in Grace’s media containing BrdU (10 µM) for 1 hr at room temperature. After fixation, ovaries were treated with 2N HCl for 30 min then 100 mM of borax was added for 2 min to neutralize the pH. Tissues were then processed for blocking and regular immunostaining.</p></sec><sec id="s4-5"><title>Statistical analysis</title><p>For all statistical analysis, data were placed into GraphPad Prism software. For statistics on the IR and Isce-I reporter activation (<xref ref-type="fig" rid="fig1">Figure 1G</xref>, <xref ref-type="supplementary-material" rid="SD1-data">Figure 1—source data 1</xref>), one-way ANOVA test was performed on all genotypes with a Tukey’s Multiple Comparison post-test. Reporter activation in <italic>aubergine</italic>, <italic>cutoff</italic>, and <italic>rad54</italic> mutants (<xref ref-type="fig" rid="fig3">Figure 3</xref>, <xref ref-type="supplementary-material" rid="SD2-data">Figure 3—source data 1</xref>) was analyzed using a two-tailed unpaired t-test comparing the transheterozygous mutant to the heterozygous control. The same analysis was carried out for region 3 and stage 2–8 (<xref ref-type="fig" rid="fig3s1">Figure 3—figure supplement 1</xref>, <xref ref-type="supplementary-material" rid="SD2-data">Figure 3—source data 1</xref>). For statistical analysis on fertility and BrdU incorporation assays (<xref ref-type="fig" rid="fig4">Figure 4</xref>), one-way ANOVA test was performed for each time point with a Dunnett post-test in which p53<sup>−/−</sup> data was the control. For cleaved-caspase 3 analysis (<xref ref-type="fig" rid="fig4s1">Figure 4—figure supplement 1</xref>), the data was analyzed using a two-tailed unpaired <italic>t</italic> test. In cases where replicates produce identical values incompatible with the prism two-tailed unpaired t-test tool, one value was negligibly revised to enable computation by this software (e.g., when both values were 0, one value was changed to 1.0<sup>e−12</sup>).</p></sec><sec id="s4-6"><title>Microarray and Gene Expression Commons (GEXC) analysis</title><p>About 200 ovaries from bam or bamp53 adult females were dissected in batches and pooled together to extract total RNA using Trizol (Invitrogen). After verifying RNA integrity using Bioanalyzer (2100; Agilent), whole-genome expression of each genotype was analyzed using Affymetrix Drosophila Genome 2.0 Array at UTSW Genomics &amp; Microarray core facility. Microarray data sets were uploaded to Gene Expression Commons (<ext-link ext-link-type="uri" xlink:href="https://gexc.stanford.edu">https://gexc.stanford.edu</ext-link>) and analyzed with 17 other public available data sets. In Gene Expression Commons, raw microarray data is individually normalized against a large-scale common reference (for <italic>Drosophila</italic> genome, n = 2687 as of Nov 2013), mapped onto the probeset meta profile. This strategy enables profiling of absolute expression levels of all genes on the microarray, instead of conventional methods where differences in gene expression are compared only between samples within an individual experiment (<xref ref-type="bibr" rid="bib36">Seita et al., 2012</xref>).</p></sec></sec></body><back><ack id="ack"><title>Acknowledgements</title><p>We thank the following for kindly providing materials: Dr Trudi Schupbach for <italic>rad54</italic>, <italic>aubergine</italic> and <italic>cutoff</italic> mutants, Dr Dennis McKearin for bam, nanos-GAL4VP16, and UASp-TkvCA strains, and Dr Kim McKim for α-pH2Av and staining protocols. We thank Dr Susan Eliazer for providing unpublished strains and information. We greatly appreciate Ashley Aguilar for help with dissections and immunohistochemistry and Jessica Alatorre for maintaining fly stocks.</p></ack><sec sec-type="additional-information"><title>Additional information</title><fn-group content-type="competing-interest"><title>Competing interests</title><fn fn-type="conflict" id="conf1"><p>The authors declare that no competing interests exist.</p></fn></fn-group><fn-group content-type="author-contribution"><title>Author contributions</title><fn fn-type="con" id="con1"><p>AW, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con2"><p>W-JL, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con3"><p>AD’B, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con4"><p>MB, Conception and design, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents</p></fn><fn fn-type="con" id="con5"><p>JMA, Conception and design, Analysis and interpretation of data, Drafting or revising the article</p></fn></fn-group></sec><ref-list><title>References</title><ref id="bib1"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Abdu</surname><given-names>U</given-names></name><name><surname>Brodsky</surname><given-names>M</given-names></name><name><surname>Schupbach</surname><given-names>T</given-names></name></person-group><year>2002</year><article-title>Activation of a meiotic checkpoint during Drosophila oogenesis regulates the translation of Gurken through Chk2/Mnk</article-title><source>Current Biology: CB</source><volume>12</volume><fpage>1645</fpage><lpage>1651</lpage><pub-id 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An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent (see <ext-link ext-link-type="uri" xlink:href="http://elife.elifesciences.org/review-process">review process</ext-link>). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.</p></boxed-text><p>Thank you for sending your work entitled “p53 activity is selectively licensed in the <italic>Drosophila</italic> stem cell compartment” for consideration at <italic>eLife</italic>. Your article has been evaluated by a Senior editor, a Reviewing editor, and 3 reviewers.</p><p>The Reviewing editor and the three reviewers discussed their comments and the Reviewing editor has assembled the following comments that require your response before we can make a determination on the suitability of this work for publication in <italic>eLife</italic>.</p><p>The referees feel that your findings are intriguing and potentially of sufficient interest for the broad audience of <italic>eLife</italic> because of the provocative finding that p53 may be activated exclusively in germline stem cells after myriad forms of stress. Yet, it was felt that your findings as described are somewhat preliminary and that further details on the roles of p53 in stem cells need to be provided. The reviewers also felt that the text has to be clarified in several instances. Please consider the following points: </p><p>1) The authors observe a phenotype of p53 mutants on recovery from sterility and re-entry into the cell cycle after irradiation. However, it's not clear if the delay in cell cycle re-entry (which is quite modest, especially after 72 hours after irradiation) causes the infertility. For example, at higher doses of irradiation (<xref ref-type="fig" rid="fig4s3">Figure 4–figure supplement 3</xref>), wild type flies did not become sterile and p53 mutants showed a reduced fertility (but not complete infertility). Does this correlate with an accelerated re-entry into the cell cycle (when compared with lower doses of irradiation)? </p><p>2) The role of apoptosis in these phenotypes is also insufficiently explored. Staining with cleaved caspase 3 was performed but did not implicate apoptosis. And is it clear whether inhibition of apoptosis would produce any phenotype in these germaria? Further, what is measured is caspase 3 activation per germarium, which doesn't take into account the number of GSCs or CBs per germarium. C3 is a good marker for induction of apoptosis but doesn't indicate how many cells may have already died and been cleared. </p><p>3) What is the role of p53 in stem cell tumors? The authors show activation of p53 in different tumor models. But what is the phenotype of these tumors when they are also mutant for p53? Would these tumors grow even bigger? Also, in the different tumor models that they use, there seems to be a different pattern of p53 activation. With Ras overexpression they observe a pattern very similar to the one they see after irradiation. However, the rest of oncogenic stresses (<italic>bam</italic><sup><italic>-/-</italic></sup><italic>, nanos</italic>&gt;<italic>CyE, nanos</italic>&gt;<italic>TkvCA, c587</italic>&gt;DPP and <italic>c587</italic>&gt;<italic>Lsd1KD</italic>) show a totally different pattern, and in many cases it looks that p53 activation is not restricted to stem cells. In the case of <italic>bam</italic><sup><italic>-/-</italic></sup> they show that there is an expansion of the stem cell population, and that cells that activate p53 also express the germline marker Vasa. Is that also the case for the rest of them? Or otherwise, what would be the explanation for the different pattern of p53 activation? </p><p>4) What is the evidence that the reporter transgene, which contains an enhancer from the known p53 target gene reaper fused to GFP, actually reports all activation of p53? That is, is this reaper enhancer activated in response to p53 activity in all cell types and under all conditions that p53 is “activated”? Without such knowledge, which is admittedly nontrivial to obtain, the main conclusion that “adult <italic>Drosophila</italic> exposed to genotoxic stress or genome destabilizers selectively activated p53 in GSCs and their immediate progeny” cannot be made. The data in <xref ref-type="fig" rid="fig1s3">Figure 1–figure supplement 3B'</xref> may actually address this point somewhat. These data show that the reporter can be activated in non-GSCs of the ovary when ATR is mutated (both follicle cells and nurse cells it appears, although a better image is needed to make this call). Is this activation p53 dependent? If so, then the restriction of reporter activity to GSCs in response to IR is ATR dependent, which would be somewhat counter intuitive since ATR normally activates p53. What is the activity of the reporter in ATR mutants in the absence of IR? In the absence of additional experiments, a more prudent or expansive interpretation of the current data seems warranted. </p><p>5) There are also problems with the presentation and description of the results. As some examples: – In vertebrates there is strong evidence for a role of p53 as a master regulation of stem cell proliferation and differentiation. Here it appears that they don't see a significant change of stem cell numbers in p53 mutants, but this is never clearly stated. </p><p>– Often an experimental result is given but only cursory information is given to assist the reader in interpreting that result. Some of this is because explanations that belong in the main text are only found in supplementary figure legends. </p><p>– Another example occurs with the introduction of the <italic>cuff</italic> and <italic>aub</italic> mutations, where the phenotype of these mutants in ovaries is not described other than to say they are involved in retrotransposon silencing. </p><p>– There are also inconsistencies in some of their results (i.e., biosensor activation outside of the germline) that are not addressed except for the suggestion they result from lack of penetrance or GFP perdurance, etc. How often are these deviations from their model observed? </p></body></sub-article><sub-article article-type="reply" id="SA2"><front-stub><article-id pub-id-type="doi">10.7554/eLife.01530.025</article-id><title-group><article-title>Author response</article-title></title-group></front-stub><body><p><italic>1) The authors observe a phenotype of p53 mutants on recovery from sterility and re-entry into the cell cycle after irradiation. However, it's not clear if the delay in cell cycle re-entry (which is quite modest, especially after 72 hours after irradiation) causes the infertility. For example, at higher doses of irradiation (</italic><xref ref-type="fig" rid="fig4s3"><italic>Figure 4–figure supplement 3</italic></xref><italic>), wild type flies did not become sterile and p53 mutants showed a reduced fertility (but not complete infertility). Does this correlate with an accelerated re-entry into the cell cycle (when compared with lower doses of irradiation)</italic>?</p><p>Good suggestion. Transient sterility and delayed re-entry into the cell cycle are phenotypes that we clearly link to p53<sup>-</sup> status but whether the relationship between these defects is direct or indirect is an open question. The studies suggested here provided a way to extend our correlations between defective cell cycle and reduced fertility. Our revision includes the suggested Brdu labeling experiments on samples at informative time points challenged as in original Figure S3 (now <xref ref-type="fig" rid="fig4s3">Figure 4–figure supplement 3</xref>). Also, please note that the experiments in current <xref ref-type="fig" rid="fig4s3">Figure 4–figure supplement 3</xref> applied lower irradiation doses when compared to original <xref ref-type="fig" rid="fig3">Figure 3A</xref> (now <xref ref-type="fig" rid="fig4">Figure 4B</xref>). In retrospect, we realize that our presentation was confusing and the revised manuscript clarifies these issues.</p><p>At an IR dose of 4 krad (current <xref ref-type="fig" rid="fig4">Figure 4A</xref>) we observe a modest delay in cell cycle re- entry but no effect on fertility. At an IR dose of 11.5 krad, WT flies transiently become infertile while p53-/- flies become permanently infertile (current <xref ref-type="fig" rid="fig4">Figure 4B</xref>). In our revised submission we performed Brdu labeling experiments at 4hrs, 2 days, and 7 days post-irradiation on an intermediate dose where WT flies remain fertile and p53<sup>-/-</sup> flies show decreased fertility (current <xref ref-type="fig" rid="fig4s3">Figure 4–figure supplement 3</xref>). Here we observe that p53<sup>-/-</sup> flies have a persisting delay in cell cycle re-entry, even over 7 days. We have added this new data to the supplement (current <xref ref-type="fig" rid="fig4s3">Figure 4–figure supplement 3 B, C</xref>) and have expanded on these observations in the results section.</p><p><italic>2) The role of apoptosis in these phenotypes is also insufficiently explored. Staining with cleaved caspase 3 was performed but did not implicate apoptosis. And is it clear whether inhibition of apoptosis would produce any phenotype in these germaria? Further, what is measured is caspase 3 activation per germarium, which doesn't take into account the number of GSCs or CBs per germarium. C3 is a good marker for induction of apoptosis but doesn't indicate how many cells may have already died and been cleared</italic>.</p><p>p53 genes often control damage-induced apoptotic responses and examining cell death in these germaria was strongly justified. We were surprised that no cell death role was indicated but we also note here that defective apoptosis does not appear to adequately explain tumor suppression by p53 in mice (see <xref ref-type="bibr" rid="bib41">Valente 2013</xref>, Martins 2006). In our experience, immunohistochemistry with the CC3 antibody is a reliable and robust marker for apoptotic cells. However, like all cell death markers, labeling is transient because the affected cells and their corpses ultimately disappear. For this reason – and to insure a comprehensive examination for possible apoptogenic defects – we completed a well- resolved time course at 2hr, 4hr, 8hr and 24hr post-irradiation (see current <xref ref-type="fig" rid="fig4s1">Figure 4–figure supplement 1</xref>). Because we obtained a negative result from these exhaustive studies, we did not pursue this point further. Furthermore, tests could be conducted to ask whether inhibition of apoptosis produces a phenotype in the germarium as suggested – however, the results would not help us deconstruct p53 action in the context of stem cells, since a role for cell death was not indicated. Therefore, in our view, the results could be of interest in their own right but would not advance the story we are driving at here.</p><p><italic>3) What is the role of p53 in stem cell tumors? The authors show activation of p53 in different tumor models. But what is the phenotype of these tumors when they are also mutant for p53? Would these tumors grow even bigger</italic>?</p><p>Good questions. We carefully examined these tumors to address precisely these points and we included new experiments establishing that p53 does have a role. We did not observe consistent size differences but we did find characteristic affects on cytology and altered gene expression profiles governed by p53 status. These new findings are included in the revision (see <xref ref-type="fig" rid="fig6">Figure 6</xref>, <xref ref-type="table" rid="tbl1">Table 1</xref>, and <xref ref-type="supplementary-material" rid="SD5-data">Table 1–source data 1</xref>).</p><p><italic>Also, in the different tumor models that they use, there seems to be a different pattern of p53 activation. With Ras overexpression they observe a pattern very similar to the one they see after irradiation. However, the rest of oncogenic stresses (bam</italic><sup><italic>-/-</italic></sup><italic>, nanos&gt;CyE, nanos&gt;TkvCA, c587&gt;DPP and c587&gt;Lsd1KD) show a totally different pattern, and in many cases it looks that p53 activation is not restricted to stem cells. In the case of bam</italic><sup><italic>-/-</italic></sup> <italic>they show that there is an expansion of the stem cell population, and that cells that activate p53 also express the germline marker Vasa. Is that also the case for the rest of them? Or otherwise, what would be the explanation for the different pattern of p53 activation</italic>?</p><p>We find it remarkable that p53 reporter is consistently activated in fly tumor models. The dissimilarities in activation patterns seen here most likely trace to differences in the underlying histology and/or differentially deranged signaling in the hyperplasias themselves. In the revised submission we discuss these differences and consider possible explanations where appropriate. The dissimilarities could be related to signaling dynamics, response dynamics, cell cycle dynamics etc. Technical sources of variation linked to subtle differences in the action of the UAS-GAL4 driver system and/or non-uniform accumulation of the oncogenic product are also possible. Please note that none of these considerations detract from the compelling positive findings that we are documenting here. In each of the models tested here (original <xref ref-type="fig" rid="fig4">Figure 4</xref>, now <xref ref-type="fig" rid="fig5">Figure 5</xref>) supernumerary and/or ectopic germline stem cells are already very well documented (Chen 2003; Elazier 2011; Xie 1998; Song 2004). Furthermore, we are confident that p53-activated cells are indeed supernumerary germline cells and, in many cases, a rounded fusome is visible in GFP+ cells. In response to this concern, we revised current <xref ref-type="fig" rid="fig5">Figure 5</xref> so that it now includes higher magnification insets in <xref ref-type="fig" rid="fig5">Figure 5E-H</xref>. These images show that p53R-GFP+ cells also have a rounded fusome, which marks germline stem cells. We also substituted panel F in <xref ref-type="fig" rid="fig5">Figure 5</xref> with an improved DAPI stained confocal image.</p><p><italic>4) What is the evidence that the reporter transgene, which contains an enhancer from the known p53 target gene reaper fused to GFP, actually reports all activation of p53? That is, is this reaper enhancer activated in response to p53 activity in all cell types and under all conditions that p53 is “activated”? Without such knowledge, which is admittedly nontrivial to obtain, the main conclusion that “adult Drosophila exposed to genotoxic stress or genome destabilizers selectively activated p53 in GSCs and their immediate progeny” cannot be made</italic>.</p><p>The reviewers suggest that our interpretation is unreasonable because our sensors might not be perfect. We strongly disagree with this argument. The comment cites limitations that are inherent to virtually all tools that are used in the cell sciences. In fact, the same concern can be raised for any reporter, any biosensor, any antibody, any phospho-specific antibodies, etc. While they may not reflect the entire scope of output from the p53 regulatory network, our reporters are advantageous because they are validated functional sensors for p53 action in vivo. Furthermore, it is not apparent how one might produce idealized sensors that are immune to this caveat. In the revised text (Discussion, paragraph 2), we explicitly consider this limitation.</p><p><italic>The data in</italic> <xref ref-type="fig" rid="fig1s3"><italic>Figure 1–figure supplement 3B'</italic></xref> <italic>may actually address this point somewhat. These data show that the reporter can be activated in non-GSCs of the ovary when ATR is mutated (both follicle cells and nurse cells it appears, although a better image is needed to make this call). Is this activation p53 dependent? If so, then the restriction of reporter activity to GSCs in response to IR is ATR dependent, which would be somewhat counter intuitive since ATR normally activates p53</italic>.</p><p>The relationship between ATR and the p53 network had not been previously examined in flies. Also, given that ATR is not universally required for p53 in mammalian systems (see Nghiem, 2002) the findings are not unexpected. The fact that we see ectopic activation is actually consistent with the probability that ATR exerts constitutive genome stabilizing activities. Where possible, the biosensors are routinely validated by testing them in the p53<sup>-</sup> background. However, we are unable to recover viable animals that are simultaneously homozygous for the biosensor, homozygous for ATR<sup>-</sup> and also homozygous for p53<sup>-</sup>. Ectopic induction of the biosensor here (and in other mutants) raises intriguing implications. But, for purposes of discussion here, the findings have strong merit because they also exclude trivial explanations for the restricted stem cell activity that we observe in the wild type context. For example, it was possible that p53 was simply confined to stem cells or was somehow inert in other cells. It was also possible, for technical reasons, that the biosensor was somehow unable function in somatic cells of the ovary. Our rationale for including these results was twofold: first, we felt it was important to demonstrate that p53 and the biosensor are normally present and ‘activatable’ throughout the ovary; second, we felt it was useful to include additional examples showing that stem cell selectivity is under genetic control.</p><p><italic>What is the activity of the reporter in ATR mutants in the absence of IR? In the absence of additional experiments, a more prudent or expansive interpretation of the current data seems warranted</italic>.</p><p>The revised manuscript adds a new graph (current <xref ref-type="fig" rid="fig1s3">Figure 1–figure supplement 3</xref>) that quantifies reporter activity in GSCs and in follicle cells of ATR mutants. This information will enable readers to appreciate reporter status in the presence and absence of IR.</p><p><italic>5) There are also problems with the presentation and description of the results. As some examples</italic>:</p><p>We extensively revised the text to clarify our presentation and our interpretations as requested.</p><p><italic>– In vertebrates there is strong evidence for a role of p53 as a master regulation of stem cell proliferation and differentiation. Here it appears that they don't see a significant change of stem cell numbers in p53 mutants, but this is never clearly stated</italic>.</p><p>Good point. The revision adds a section in the results where this topic is explicitly discussed. Modestly altered stem cell pools in certain tissues of p53 mutant mice have been reported to be affected by age (Dumble, 2007). However, to our knowledge, stem cell pools in p53 mutant mice are not universally elevated (Reviewed in Solozobova and Blattner, 2011). Instead, it seems that the master regulation concept mostly derives the effects of p53 upon iPS cell production (Krizhanovsky, 2009) and there are alternative interpretations for the this effect (e.g., prevention of apoptosis).</p><p><italic>– Often an experimental result is given but only cursory information is given to assist the reader in interpreting that result. Some of this is because explanations that belong in the main text are only found in supplementary figure legends</italic>.</p><p>In retrospect, we agree. This is a valid criticism. The revised manuscript comprehensively addresses this through improved transitions and additional background information where needed. For example, we divided the ovary and testis data in original <xref ref-type="fig" rid="fig1">Figure 1</xref> into two figures (current <xref ref-type="fig" rid="fig1 fig2">Figures 1 and 2</xref>). We also moved the description of the p53 biosensors from the supplement to current <xref ref-type="fig" rid="fig1">Figure 1</xref>.</p><p><italic>– Another example occurs with the introduction of the cuff and aub mutations, where the phenotype of these mutants in ovaries is not described other than to say they are involved in retrotransposon silencing</italic>.</p><p>The revised manuscript includes additional background and context on these mutants.</p><p><italic>– There are also inconsistencies in some of their results (i.e., biosensor activation outside of the germline) that are not addressed except for the suggestion they result from lack of penetrance or GFP perdurance, etc. How often are these deviations from their model observed</italic>?</p><p>To address this concern, we have improved our presentation of these issues. We emphasize that our ‘licensing’ model refers to the stimulus-dependent action of p53 in a wild type setting. When wild type ovaries are radiation challenged, functional p53 activation is seen only in the stem cell and their immediate progeny – under these conditions, the p53 biosensor is never observed outside of the germ stem cell compartment. This could reflect independent activation in stem cells and their immediate progeny or, alternatively, the GFP signal in cystoblasts could reflect perdurance of GFP from parental stem cells (we favor this for reasons discussed in the text). However, in the ovaries of mutants that affect genome stability, we do observe reporter activation outside of the germline and this observation provides us with important insights. In these cases, we propose that the corresponding genes normally function in ways that ultimately restrict widespread activation. In mutants that produce oncogenic stress, p53 is more broadly activated but the ectopic activation here still occurs within germline cells.</p></body></sub-article></article>