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| <?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">03626</article-id><article-id pub-id-type="doi">10.7554/eLife.03626</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>Pvr expression regulators in equilibrium signal control and maintenance of <italic>Drosophila</italic> blood progenitors</article-title></title-group><contrib-group><contrib contrib-type="author" id="author-15223"><name><surname>Mondal</surname><given-names>Bama Charan</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="con1"/><xref ref-type="fn" rid="conf2"/></contrib><contrib contrib-type="author" id="author-15224"><name><surname>Shim</surname><given-names>Jiwon</given-names></name><contrib-id contrib-id-type="orcid">http://orcid.org/0000-0003-2409-1130</contrib-id><xref ref-type="aff" rid="aff1"/><xref ref-type="aff" rid="aff2"/><xref ref-type="other" rid="par-2"/><xref ref-type="fn" rid="con2"/><xref ref-type="fn" rid="conf2"/></contrib><contrib contrib-type="author" corresp="yes" id="author-15225"><name><surname>Evans</surname><given-names>Cory J</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="corresp" rid="cor1">*</xref><xref ref-type="fn" rid="con3"/><xref ref-type="fn" rid="conf2"/></contrib><contrib contrib-type="author" corresp="yes" id="author-1042"><name><surname>Banerjee</surname><given-names>Utpal</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="aff" rid="aff3"/><xref ref-type="aff" rid="aff4"/><xref ref-type="aff" rid="aff5"/><xref ref-type="corresp" rid="cor2">*</xref><xref ref-type="other" rid="par-1"/><xref ref-type="fn" rid="con4"/><xref ref-type="fn" rid="conf1"/></contrib><aff id="aff1"><institution content-type="dept">Department of Molecular, Cell and Developmental Biology</institution>, <institution>University of California, Los Angeles</institution>, <addr-line><named-content content-type="city">Los Angeles</named-content></addr-line>, <country>United States</country></aff><aff id="aff2"><institution content-type="dept">Department of Life Science</institution>, <institution>Hanyang University</institution>, <addr-line><named-content content-type="city">Seoul</named-content></addr-line>, <country>Republic of Korea</country></aff><aff id="aff3"><institution content-type="dept">Department of Biological Chemistry</institution>, <institution>University of California, Los Angeles</institution>, <addr-line><named-content content-type="city">Los Angeles</named-content></addr-line>, <country>United States</country></aff><aff id="aff4"><institution content-type="dept">Molecular Biology Institute</institution>, <institution>University of California, Los Angeles</institution>, <addr-line><named-content content-type="city">Los Angeles</named-content></addr-line>, <country>United States</country></aff><aff id="aff5"><institution content-type="dept">Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research</institution>, <institution>University of California, Los Angeles</institution>, <addr-line><named-content content-type="city">Los Angeles</named-content></addr-line>, <country>United States</country></aff></contrib-group><contrib-group content-type="section"><contrib contrib-type="editor"><name><surname>Ohlstein</surname><given-names>Benjamin</given-names></name><role>Reviewing editor</role><aff><institution>Columbia University Medical Center</institution>, <country>United States</country></aff></contrib></contrib-group><author-notes><corresp id="cor1"><label>*</label>For correspondence: <email>cjevans@ucla.edu</email> (CJE);</corresp><corresp id="cor2"><label>*</label>For correspondence: <email>banerjee@mbi.ucla.edu</email> (UB)</corresp></author-notes><pub-date date-type="pub" publication-format="electronic"><day>08</day><month>09</month><year>2014</year></pub-date><pub-date pub-type="collection"><year>2014</year></pub-date><volume>3</volume><elocation-id>e03626</elocation-id><history><date date-type="received"><day>07</day><month>06</month><year>2014</year></date><date date-type="accepted"><day>05</day><month>09</month><year>2014</year></date></history><permissions><copyright-statement>© 2014, Mondal et al</copyright-statement><copyright-year>2014</copyright-year><copyright-holder>Mondal et al</copyright-holder><license xlink:href="http://creativecommons.org/licenses/by/4.0/"><license-p>This article is distributed under the terms of the <ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/4.0/">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="elife03626.pdf"/><abstract><object-id pub-id-type="doi">10.7554/eLife.03626.001</object-id><p>Blood progenitors within the lymph gland, a larval organ that supports hematopoiesis in <italic>Drosophila melanogaster</italic>, are maintained by integrating signals emanating from niche-like cells and those from differentiating blood cells. We term the signal from differentiating cells the ‘equilibrium signal’ in order to distinguish it from the ‘niche signal’. Earlier we showed that equilibrium signaling utilizes Pvr (the <italic>Drosophila</italic> PDGF/VEGF receptor), STAT92E, and adenosine deaminase-related growth factor A (ADGF-A) (<xref ref-type="bibr" rid="bib43">Mondal et al., 2011</xref>). Little is known about how this signal initiates during hematopoietic development. To identify new genes involved in lymph gland blood progenitor maintenance, particularly those involved in equilibrium signaling, we performed a genetic screen that identified <italic>bip1</italic> (<italic>bric à brac interacting protein 1</italic>) and <italic>Nucleoporin 98</italic> (<italic>Nup98</italic>) as additional regulators of the equilibrium signal. We show that the products of these genes along with the Bip1-interacting protein RpS8 (Ribosomal protein S8) are required for the proper expression of Pvr.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03626.001">http://dx.doi.org/10.7554/eLife.03626.001</ext-link></p></abstract><abstract abstract-type="executive-summary"><object-id pub-id-type="doi">10.7554/eLife.03626.002</object-id><title>eLife digest</title><p>Progenitor cells are cells that can either multiply to make new copies of themselves or mature into different specialized cell types—such as blood cells. In the fruit fly <italic>Drosophila</italic>, new blood cells are formed in several different locations, including in an organ called the lymph gland.</p><p>In 2011, researchers found that the fate of blood progenitor cells within the lymph gland is controlled by signals from two nearby sources—one from specialized, supportive (‘niche’) cells and the other from maturing blood cells. The signal from the maturing blood cells ensures that the relative amounts of progenitor and maturing blood cells are kept in the right balance. As a result, this signaling process has been called ‘equilibrium signaling’.</p><p>Questions remain as to how equilibrium signaling is regulated, and how it interacts with signals from the niche. To investigate this, Mondal et al.—including some of the researchers involved in the 2011 work—used various genetic techniques to create <italic>Drosophila</italic> larvae in which the tissues that become blood cells are made visible with fluorescent proteins. This meant that these tissues could be examined in live, whole animals by using a microscope. Mondal et al. then searched for the <italic>Drosophila</italic> genes involved in generating new blood cells in the lymph gland—particularly those involved in equilibrium signaling. This was done by switching on and off hundreds of genes, one by one, in the lymph gland, and any genes that caused changes to the generation of new blood cells were then investigated further.</p><p>Following these investigations, Mondal et al. focused on three genes—and when each of these genes was switched off in maturing blood cells, the result was that fewer progenitor cells remained in the lymph gland. This effect was not seen when the genes were switched off in the progenitor or the niche cells, which suggested that the genes are likely to be components of the equilibrium signaling pathway. Switching off these genes in maturing blood cells also dramatically reduced the levels of a protein called Pvr, a key equilibrium signaling protein known from the 2011 study and an important player in blood cell development in several species.</p><p>How the newly identified genes actually control Pvr protein levels to maintain proper equilibrium signaling in the lymph gland remains to be explored. However, this work provides a basis for investigating the role of related genes in blood cell development in vertebrate systems, namely humans.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03626.002">http://dx.doi.org/10.7554/eLife.03626.002</ext-link></p></abstract><kwd-group kwd-group-type="author-keywords"><title>Author keywords</title><kwd>hematopoiesis</kwd><kwd>lymph gland</kwd><kwd>hemocyte</kwd><kwd>equilibrium signaling</kwd><kwd>niche signaling</kwd><kwd>macrophage</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-id institution-id-type="FundRef">http://dx.doi.org/10.13039/100000050</institution-id><institution>National Heart, Lung, and Blood Institute</institution></institution-wrap></funding-source><award-id>R01 HL067395</award-id><principal-award-recipient><name><surname>Banerjee</surname><given-names>Utpal</given-names></name></principal-award-recipient></award-group><award-group id="par-2"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/100000900</institution-id><institution>California Institute for Regenerative Medicine</institution></institution-wrap></funding-source><award-id>TG2-01169</award-id><principal-award-recipient><name><surname>Shim</surname><given-names>Jiwon</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>The THAP-domain protein Bip1, along with other proteins Nup98 and RpS8, controls the expression of the protein Pvr, a critical non-cell-autonomous regulator of <italic>Drosophila</italic> blood progenitor maintenance.</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id="s1" sec-type="intro"><title>Introduction</title><p>Similar to vertebrates, blood cell differentiation in <italic>Drosophila</italic> is regulated in multiple hematopoietic environments, which include the head mesoderm of the embryo (<xref ref-type="bibr" rid="bib71">Tepass et al., 1994</xref>; <xref ref-type="bibr" rid="bib31">Lebestky et al., 2000</xref>; <xref ref-type="bibr" rid="bib42">Milchanowski et al., 2004</xref>), the specialized, tissue-associated microenvironments of the larval periphery (e.g, body wall hematopoietic pockets) (<xref ref-type="bibr" rid="bib39">Markus et al., 2009</xref>; <xref ref-type="bibr" rid="bib37">Makhijani et al., 2011</xref>), and the larval lymph gland, an organ dedicated to the development of blood cells that normally contribute to the pupal and adult stages (<xref ref-type="bibr" rid="bib55">Rizki, 1978</xref>; <xref ref-type="bibr" rid="bib60">Shrestha and Gateff, 1982</xref>; <xref ref-type="bibr" rid="bib30">Lanot et al., 2001</xref>; <xref ref-type="bibr" rid="bib25">Jung et al., 2005</xref>). Understanding how blood cell development is regulated in the lymph gland is the primary goal underlying the work presented here. Differentiating blood cells (hemocytes) of the lymph gland are derived from multipotent progenitors (<xref ref-type="bibr" rid="bib25">Jung et al., 2005</xref>; <xref ref-type="bibr" rid="bib38">Mandal et al., 2007</xref>; <xref ref-type="bibr" rid="bib40">Martinez-Agosto et al., 2007</xref>). These blood progenitors readily proliferate during the early growth phases of lymph gland development, which is followed by a period in which many of these cells slow their rate of division and are maintained without differentiation in a region termed the medullary zone (MZ, <xref ref-type="fig" rid="fig1">Figure 1</xref>) (<xref ref-type="bibr" rid="bib25">Jung et al., 2005</xref>; <xref ref-type="bibr" rid="bib38">Mandal et al., 2007</xref>). During the same period, other progenitor cells begin to differentiate along the peripheral edge of the lymph gland to give rise to a separate cortical zone (CZ) (<xref ref-type="bibr" rid="bib25">Jung et al., 2005</xref>). How progenitor cell maintenance and differentiation are regulated during the course of lymph gland development has become a major area of exploration in recent years, and several different signaling pathways have been identified that maintain progenitor cells through the larval stages (<xref ref-type="bibr" rid="bib32">Lebestky et al., 2003</xref>; <xref ref-type="bibr" rid="bib38">Mandal et al., 2007</xref>; <xref ref-type="bibr" rid="bib49">Owusu-Ansah and Banerjee, 2009</xref>; <xref ref-type="bibr" rid="bib63">Sinenko et al., 2009</xref>; <xref ref-type="bibr" rid="bib43">Mondal et al., 2011</xref>; <xref ref-type="bibr" rid="bib44">Mukherjee et al., 2011</xref>; <xref ref-type="bibr" rid="bib72">Tokusumi et al., 2011</xref>; <xref ref-type="bibr" rid="bib15">Dragojlovic-Munther and Martinez-Agosto, 2012</xref>; <xref ref-type="bibr" rid="bib52">Pennetier et al., 2012</xref>; <xref ref-type="bibr" rid="bib59">Shim et al., 2012</xref>; <xref ref-type="bibr" rid="bib64">Sinenko et al., 2012</xref>). Wingless (Wg; Wnt in vertebrates) is expressed by blood progenitor cells in the lymph gland and has an important role in promoting their maintenance (<xref ref-type="bibr" rid="bib63">Sinenko et al., 2009</xref>), and reactive oxygen species (ROS) function in these cells to potentiate blood progenitor differentiation both in the context of normal development and during oxidative stress (<xref ref-type="bibr" rid="bib49">Owusu-Ansah and Banerjee, 2009</xref>). Progenitor cell maintenance at late developmental stages is also dependent upon Hedgehog (Hh) signaling from a small population of cells called the posterior signaling center that functions as a hematopoietic niche (PSC) (<xref ref-type="bibr" rid="bib32">Lebestky et al., 2003</xref>; <xref ref-type="bibr" rid="bib25">Jung et al., 2005</xref>).<fig id="fig1" position="float"><object-id pub-id-type="doi">10.7554/eLife.03626.003</object-id><label>Figure 1.</label><caption><title>Equilibrium signaling maintains hematopoietic progenitors in the developing lymph gland.</title><p>The lymph gland primary lobe consists of three distinct cellular populations or zones. The medullary zone (MZ) contains blood progenitor cells while the nearby cortical zone (CZ) contains differentiating and mature blood cells. The posterior signaling center (PSC) functions as a supportive population (a niche) that expresses Hedgehog (Hh) and maintains the progenitor cells utilizing this ‘niche signal’. The receptor tyrosine kinase (RTK) Pvr and the STAT (STAT92E) transcriptional activator are required in CZ cells for the proper expression and secretion of the extracellular enzyme ADGF-A, which keeps the extracellular adenosine levels relatively low by converting it to inosine. The Pvr ligand Pvf1 is made in PSC cells and is transported through the lymph gland to activate Pvr in CZ cells. Collectively, we refer to the system that generates ADGF-A from the differentiating cells as ‘equilibrium signaling’, which is required independently of the niche-derived Hh signaling for the maintenance of progenitor blood cells in the MZ. Signaling events downstream of both ADGF-A and Hh (dashed arrows) cause the inhibition of Protein Kinase A (PKA) within progenitor blood cells, thereby promoting their maintenance. The individual components are color coded to match the schematic of the lymph gland. The equilibrium signal ADGF-A is blue, originating from the CZ; the niche signal Hh is magenta, originating in the PSC; PKA is gray, functioning in the MZ progenitor cells. Full details of this molecular pathway can be found in <xref ref-type="bibr" rid="bib43">Mondal et al., (2011)</xref>.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03626.003">http://dx.doi.org/10.7554/eLife.03626.003</ext-link></p></caption><graphic xlink:href="elife03626f001"/></fig></p><p>More recently, it has been discovered that the maintenance of lymph gland blood progenitors also requires a backward signal arising from the differentiating cells (<xref ref-type="bibr" rid="bib43">Mondal et al., 2011</xref>). This signal is controlled by a novel pathway that combines the function of the receptor tyrosine kinase Pvr and JAK-independent STAT (STAT92E) activation in differentiating cells, followed by the expression of ADGF-A (<xref ref-type="fig" rid="fig1">Figure 1</xref>), a secreted enzyme that converts adenosine to inosine (<xref ref-type="bibr" rid="bib43">Mondal et al., 2011</xref>). Extracellular adenosine is a well-established signal in mammalian systems in various contexts, particularly stress conditions (<xref ref-type="bibr" rid="bib19">Fredholm, 2007</xref>; <xref ref-type="bibr" rid="bib58">Sheth et al., 2014</xref>), and an elevated adenosine level in <italic>Drosophila</italic> causes extensive blood cell proliferation (<xref ref-type="bibr" rid="bib14">Dolezal et al., 2005</xref>; <xref ref-type="bibr" rid="bib43">Mondal et al., 2011</xref>). It has been demonstrated that differentiating and mature cells express (and are the primary source of) ADGF-A, and that its enzymatic activity (which converts adenosine to inosine) is required for progenitor cell maintenance (<xref ref-type="bibr" rid="bib43">Mondal et al., 2011</xref>). As differentiation proceeds, ADGF-A expression (activity) increasingly promotes the maintenance of extant blood progenitors through the reduction of stimulatory adenosine. In this way, the differentiating cell population helps balance the progenitor/differentiating cell ratio and is the basis for our referring to ADGF-A as an ‘equilibrium signal’.</p><p>Loss of ADGF-A (or STAT or Pvr) from differentiating cells increases extracellular adenosine level and thereby increases Adenosine Receptor (AdoR) signaling and downstream Protein Kinase A (PKA) activity in progenitors, which causes these cells to proliferate (<xref ref-type="bibr" rid="bib14">Dolezal et al., 2005</xref>; <xref ref-type="bibr" rid="bib43">Mondal et al., 2011</xref>). PKA is a central regulator of progenitor maintenance because it integrates input from both the equilibrium signal (ADGF-A) and the niche signal (Hh). PKA mediates the conversion of the transcriptional regulator Cubitus interruptus (Ci, a homolog of vertebrate Gli) from its full length form (Ci<sup>155</sup>), required for progenitor maintenance (<xref ref-type="bibr" rid="bib38">Mandal et al., 2007</xref>), to a cleaved form (Ci<sup>75</sup>) that promotes proliferation. Signaling by Hh inhibits PKA and promotes Ci<sup>155</sup> stabilization whereas adenosine/AdoR signaling activates PKA and promotes Ci<sup>75</sup> conversion. Thus, both Hh (the niche signal) and ADGF-A (the equilibrium signal which removes adenosine) limit PKA activity and promote progenitor cell maintenance.</p><p>Although niche and equilibrium signaling are both clearly important, details of their regulation and interaction are less clear. Thus, we performed a loss-of-function genetic screen to identify new genes involved in lymph gland blood progenitor maintenance, particularly those involved in equilibrium signaling. In this study, we report the results of this screen and the identification of three genes, <italic>bip1</italic>, <italic>RpS8</italic>, and <italic>Nup98</italic>, as new components of the equilibrium signaling pathway.</p></sec><sec id="s2" sec-type="results|discussion"><title>Results and discussion</title><sec id="s2-1"><title>HHLT-gal4 and its use for whole-animal genetic screening during hematopoietic development</title><p>Unlike the adult eye or wing, analysis of internal larval tissues such as the lymph gland requires laborious dissection and processing. To circumvent this barrier to genetic screening, we generated a line of flies termed the <italic>Hand-Hemolectin Lineage Traced-gal4</italic> line (<italic>HHLT-gal4 UAS-2XEGFP</italic>, <xref ref-type="fig" rid="fig2">Figure 2A</xref>; see ‘Materials and methods’ for precise genotype) in which the hematopoietic system is labeled by Gal4-dependent expression of EGFP, such that it can be visualized in live, whole animals (<xref ref-type="fig" rid="fig2">Figure 2B–C</xref>). This line makes use of two <italic>gal4</italic> drivers to target early lymph gland blood cells (hemocytes; <italic>Hand-gal4</italic>) and circulating and sessile blood cells (<italic>Hemolectin-gal4</italic> or <italic>Hml-gal4</italic>) and incorporates a Gal4/FLP recombinase-dependent cell lineage tracing cassette to maintain Gal4 expression in the lymph gland after the <italic>Hand-gal4</italic> driver itself is down-regulated during the first instar. The <italic>Hand-gal4</italic> driver is expressed in the embryonic cardiogenic mesoderm from which the lymph gland is derived. Therefore, the dorsal vessel (heart) cardioblasts and the pericardial nephrocytes are also marked by the cell lineage tracing cassette (<xref ref-type="fig" rid="fig2">Figure 2B</xref>). EGFP is not expressed in other larval tissues, except in the late third-instar salivary glands that are readily discernible from the hematopoietic system (<xref ref-type="fig" rid="fig2">Figure 2B,E</xref>).<fig-group><fig id="fig2" position="float"><object-id pub-id-type="doi">10.7554/eLife.03626.004</object-id><label>Figure 2.</label><caption><title>The <italic>Hand-Hemolectin Lineage Tracing-gal4</italic> line (<italic>HHLT-gal4 UAS-2XEGFP</italic>) and its use as an in vivo screening tool.</title><p>(<bold>A</bold>) Schematic describing the key elements of the <italic>HHLT-gal4</italic> driver line. (<bold>B</bold>) Image showing the hematopoietic system within a wandering stage third-instar <italic>HHLT > GFP</italic> larva (dorsal view). Primary, secondary, and tertiary lobes of the lymph gland are readily discernible through overlying musculature, epidermal cells, and cuticle. Lymph gland lobes develop bilaterally, flanking the larval heart (dorsal vessel, DV). Non-blood pericardial cells (PC) also express GFP due to early expression of <italic>Hand-gal4</italic>. Circulating/sessile blood cells also express GFP due to <italic>Hml-gal4</italic> and sessile groups are easily observable. GFP is also seen in ventrally located salivary glands (SG, out of focus) of larvae beyond the third-instar transition (due to <italic>Hand-gal4</italic>). (<bold>C</bold>) <italic>HHLT > GFP</italic> control larvae; (<bold>D</bold>) <italic>HHLT > GFP</italic> larvae overexpressing <italic>Ras85D</italic> (<italic>LA 527</italic>) exhibit hyperproliferative lymph glands; (<bold>E</bold>) <italic>HHLT > GFP</italic> larvae overexpressing <italic>combgap</italic> (<italic>LA 630</italic>) show little or no GFP expression in the lymph gland region. Arrows indicate GFP fluorescence from salivary glands (SG).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03626.004">http://dx.doi.org/10.7554/eLife.03626.004</ext-link></p></caption><graphic xlink:href="elife03626f002"/></fig><fig id="fig2s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.03626.005</object-id><label>Figure 2—figure supplement 1.</label><caption><title>As a ‘proof-of-principle’ approach and to assess the effectiveness of <italic>HHLT-gal4</italic> as a screening tool, <italic>HHLT-gal4</italic> was crossed to lines harboring gain-of-function <italic>UAS</italic> transgenes known to cause excessive cellular proliferation, with the expectation that such transgenes would cause significant expansion of the hematopoietic tissues.</title><p>(<bold>A</bold>) <italic>HHLT > GFP</italic> larvae (outcrossed to <italic>w</italic><sup><italic>1118</italic></sup>) showing baseline fluorescence. (<bold>B–B′</bold>) <italic>HHLT > GFP</italic> larvae expressing <italic>UAS-human activated Raf</italic> (<italic>h-Raf</italic><sup><italic>ACT</italic></sup>) show increased GFP fluorescence with the same exposure (<bold>B</bold>) as for control larvae (<bold>A</bold>) and exhibit enlarged lymph glands with reduced exposure time (<bold>B′</bold>). (<bold>C</bold>) <italic>HHLT > GFP</italic> larvae expressing <italic>UAS-activated Drosophila Alk</italic> (<italic>DAlk</italic><sup><italic>ACT</italic></sup>) also exhibit enlarged lymph glands. (<bold>D</bold> and <bold>E</bold>) Relative bleed cell densities from control animals (<bold>D</bold>) and animals expressing <italic>UAS-DAlk</italic><sup><italic>ACT</italic></sup> (<bold>E</bold>) which show an expansion. (<bold>F</bold>) <italic>HHLT > GFP</italic> larvae expressing <italic>UAS-diap1-RNAi</italic> exhibit reduced fluorescence in lymph gland and circulating cells. The obvious changes in the level of <italic>HHLT-gal4</italic>-mediated EGFP expression observed in these backgrounds shows that <italic>HHLT-gal4</italic> is indeed a useful tool with which to assess the hematopoietic system in vivo, in the context of a genetic screen.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03626.005">http://dx.doi.org/10.7554/eLife.03626.005</ext-link></p></caption><graphic xlink:href="elife03626fs001"/></fig></fig-group></p></sec><sec id="s2-2"><title>Screening for progenitor maintenance genes in the developing lymph gland</title><p>A screen was conducted in which <italic>HHLT-gal4</italic> was used to independently misexpress 503 unique <italic>UAS</italic>-controlled <italic>Drosophila</italic> genes, with their effects on the hematopoietic system assessed in whole animals based upon EGFP expression. The particular collection of <italic>UAS</italic>-based gene misexpression lines used (termed LA lines) are mapped insertions (against Flybase release 5.7) of the <italic>P{Mae-UAS.6.11}</italic> element into endogenous gene loci that have been previously shown to cause developmental phenotypes upon misexpression (<xref ref-type="bibr" rid="bib10">Crisp and Merriam, 1997</xref>; <xref ref-type="bibr" rid="bib4">Bellen et al., 2004</xref>). When crossed to <italic>HHLT-gal4</italic>, 281 of these lines cause a scorable phenotype in either lymph glands or circulating blood cells of late third-instar larvae (<xref ref-type="supplementary-material" rid="SD1-data">Supplementary file 1</xref>). As an example, LA line 527, which is predicted to misexpress <italic>Ras85D</italic>, causes a robust expansion of the lymph gland (<xref ref-type="fig" rid="fig2">Figure 2D</xref>), consistent with the previously identified role of <italic>Ras85D</italic> in controlling hemocyte proliferation (<xref ref-type="bibr" rid="bib2">Asha et al., 2003</xref>; <xref ref-type="bibr" rid="bib62">Sinenko and Mathey-Prevot, 2004</xref>). By contrast, LA line 630 misexpresses the gene <italic>combgap</italic> (encoding a zinc finger transcription factor) and causes a strong reduction in lymph gland size (<xref ref-type="fig" rid="fig2">Figure 2E</xref>).</p><p>We used these results from the misexpression screen as a means to select potentially relevant genes for the subsequent loss-of-function analyses by RNA interference (using <italic>UAS-RNAi</italic> lines). We were able to obtain RNAi lines targeting 251 of the candidate genes identified by misexpression and found that 73 RNAi lines targeting 69 genes alter lymph gland size or morphology when crossed to <italic>HHLT-gal4</italic> (<xref ref-type="supplementary-material" rid="SD2-data">Supplementary file 2</xref>)<italic>.</italic></p><p>To characterize the RNAi phenotypes in more detail, the level of blood cell differentiation within the lymph gland was evaluated by immunostaining with anti-Peroxidasin (Pxn) antibodies (<xref ref-type="bibr" rid="bib47">Nelson et al., 1994</xref>). In wild-type lymph glands, expression of mature cell markers such as Pxn is restricted to the periphery of the primary lobe (the cortical zone) (<xref ref-type="bibr" rid="bib25">Jung et al., 2005</xref>). By contrast, when niche signaling or equilibrium signaling are compromised, progenitor cells are lost and differentiation markers, including Pxn, are expressed throughout the lymph gland primary lobes (<xref ref-type="bibr" rid="bib38">Mandal et al., 2007</xref>; <xref ref-type="bibr" rid="bib43">Mondal et al., 2011</xref>). Rescreening the 73 identified RNAi lines using <italic>HHLT-gal4</italic> identified 20 genes (21 RNAi lines) that, when knocked down, cause the expression of Pxn in cells throughout the lymph gland primary lobe (<xref ref-type="fig" rid="fig3">Figure 3</xref>; <xref ref-type="table" rid="tbl1">Table 1</xref> and <xref ref-type="supplementary-material" rid="SD2-data">Supplementary file 2</xref>). Compared to controls, the progenitor population (Pxn negative) is either strongly reduced or absent in each RNAi background. This ‘expanded’ Pxn phenotype is interpreted as a loss-of-progenitor cell phenotype.<fig id="fig3" position="float"><object-id pub-id-type="doi">10.7554/eLife.03626.006</object-id><label>Figure 3.</label><caption><title>Identification of RNAi lines that cause an expanded Peroxidasin phenotype when expressed throughout the lymph gland.</title><p>Peroxidasin (Pxn, <italic>red</italic>) is normally restricted to cortical zone cells (near the periphery) (<bold>A</bold>, control) but is seen throughout the lymph gland in RNAi backgrounds (<bold>B</bold>–<bold>V</bold>) expressed by <italic>HHLT-gal4</italic>. Line identifiers and gene targets are shown; additional details listed in <xref ref-type="table" rid="tbl1">Table 1</xref>. Images represent a single middle confocal section taken from a Z-plane series through the entire primary lobe.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03626.006">http://dx.doi.org/10.7554/eLife.03626.006</ext-link></p></caption><graphic xlink:href="elife03626f003"/></fig><table-wrap id="tbl1" position="float"><object-id pub-id-type="doi">10.7554/eLife.03626.007</object-id><label>Table 1.</label><caption><p>RNAi lines and target genes causing an ‘expanded’ Peroxidasin expression phenotype with <italic>HHLT-gal4</italic></p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03626.007">http://dx.doi.org/10.7554/eLife.03626.007</ext-link></p></caption><table frame="hsides" rules="groups"><thead><tr><th align="center">Line #</th><th align="center">UAS-RNAi ID</th><th align="center">RNAi target</th><th align="center"><italic>Gene</italic></th><th align="center">Off targets</th><th align="center">LG size/quality</th><th align="center">Protein function</th></tr></thead><tbody><tr><td align="center">1</td><td align="center">3859</td><td align="center">CG4214</td><td align="center"><italic>Syx5</italic></td><td align="center">0</td><td align="center">Small/missing</td><td align="center">Golgi SNARE</td></tr><tr><td align="center">2</td><td align="center">6543</td><td align="center">CG7398</td><td align="center"><italic>Trn</italic></td><td align="center">1</td><td align="center">Large/baggy</td><td align="center">hnRNP nuclear import</td></tr><tr><td align="center">3</td><td align="center">9572</td><td align="center">CG5738</td><td align="center"><italic>lolal</italic></td><td align="center">0</td><td align="center">Small</td><td align="center">Transcription factor</td></tr><tr><td align="center">4</td><td align="center">12574</td><td align="center">CG12052</td><td align="center"><italic>lola</italic></td><td align="center">0</td><td align="center">Large/baggy</td><td align="center">Transcription factor</td></tr><tr><td align="center">5</td><td align="center">12759</td><td align="center">CG6854</td><td align="center"><italic>CTPsyn</italic></td><td align="center">0</td><td align="center">Small/baggy</td><td align="center">CTP synthase</td></tr><tr><td align="center">6</td><td align="center">15886</td><td align="center">CG6376</td><td align="center"><italic>E2f</italic></td><td align="center">1</td><td align="center">Small/normal</td><td align="center">Transcription factor</td></tr><tr><td align="center">7</td><td align="center">17954</td><td align="center">CG10009</td><td align="center"><italic>Noa36</italic></td><td align="center">0</td><td align="center">Small/missing</td><td align="center">Zinc finger nucleolar protein</td></tr><tr><td align="center">8</td><td align="center">19485</td><td align="center">CG10009</td><td align="center"><italic>Noa36</italic></td><td align="center">0</td><td align="center">Small/missing</td><td align="center">Zinc finger nucleolar protein</td></tr><tr><td align="center">9</td><td align="center">22836</td><td align="center">CG10267</td><td align="center"><italic>Zif</italic></td><td align="center">0</td><td align="center">Small/missing</td><td align="center">Transcription factor</td></tr><tr><td align="center">10</td><td align="center">24215</td><td align="center">CG8149</td><td align="center"><italic>CG8149</italic></td><td align="center">0</td><td align="center">Baggy</td><td align="center"><italic>DNA binding protein</italic></td></tr><tr><td align="center">11</td><td align="center">26176</td><td align="center">CG3363</td><td align="center"><italic>CG3363</italic></td><td align="center">0</td><td align="center">Small</td><td align="center">Unknown</td></tr><tr><td align="center">12</td><td align="center">26370</td><td align="center">CG4036</td><td align="center"><italic>CG4036</italic></td><td align="center">1</td><td align="center">Large</td><td align="center"><italic>Oxidoreductase</italic></td></tr><tr><td align="center">13</td><td align="center">38472</td><td align="center">CG1129</td><td align="center"><italic>CG1129</italic></td><td align="center">0</td><td align="center">Small/missing</td><td align="center"><italic>Peptide transferase</italic></td></tr><tr><td align="center">14</td><td align="center">40306</td><td align="center">CG31938</td><td align="center"><italic>Rrp40</italic></td><td align="center">0</td><td align="center">Small/normal</td><td align="center">RNA exosome</td></tr><tr><td align="center">15</td><td align="center">41009</td><td align="center">CG3836</td><td align="center"><italic>stwl</italic></td><td align="center">0</td><td align="center">Small/normal</td><td align="center">Transcription factor</td></tr><tr><td align="center">16</td><td align="center">44606</td><td align="center">CG6778</td><td align="center"><italic>Aats-gly</italic></td><td align="center">0</td><td align="center">Small/missing</td><td align="center">Glycyl-tRNA synthetase</td></tr><tr><td align="center">17</td><td align="center">49753<xref ref-type="table-fn" rid="tblfn1">*</xref></td><td align="center">CG33155</td><td align="center"><italic>CG33155</italic></td><td align="center">4</td><td align="center">Small/normal</td><td align="center">Unknown</td></tr><tr><td align="center">18</td><td align="center">7574R-2</td><td align="center">CG7574</td><td align="center"><italic>bip1</italic></td><td align="center">0</td><td align="center">Small/baggy</td><td align="center"><italic>Transcription factor</italic></td></tr><tr><td align="center">19</td><td align="center">10198R-1</td><td align="center">CG10198</td><td align="center"><italic>Nup98-96</italic></td><td align="center">0</td><td align="center">Small/missing</td><td align="center">Nucleoporin</td></tr><tr><td align="center">20</td><td align="center">12030R-2</td><td align="center">CG12030</td><td align="center"><italic>Gale</italic></td><td align="center">0</td><td align="center">Small</td><td align="center">UDP-galactose 4'-epimerase</td></tr><tr><td align="center">21</td><td align="center">12765R-3</td><td align="center">CG12765</td><td align="center"><italic>fsd</italic></td><td align="center">0</td><td align="center">Small/normal</td><td align="center">F-box protein</td></tr></tbody></table><table-wrap-foot><fn id="tblfn1"><label>*</label><p>This RNAi line targeting sequence overlaps with the putative <italic>mRpL53</italic> gene in the same locus. Lines 1–17 from VDRC, lines 18–21 from NIG Japan.</p></fn></table-wrap-foot></table-wrap></p></sec><sec id="s2-3"><title>Zone-specific screening identifies putative equilibrium signaling genes</title><p>Using the pan-lymph gland <italic>HHLT-gal4</italic> driver, we identified 21 RNAi lines that cause a loss of progenitor cells in the primary lobes at late stages of lymph gland development. In order to discern whether any of the associated candidate genes have a specific progenitor-maintenance function that is restricted to cells belonging to a single zone, we rescreened the 21 RNAi lines using cell-type-specific Gal4-expressing lines. Targeting RNAi to differentiating and mature cells using <italic>Hml-gal4</italic> (<xref ref-type="bibr" rid="bib62">Sinenko and Mathey-Prevot, 2004</xref>) identified six genes (CG6854 [<italic>CTPsyn</italic>], CG7398 [<italic>Transportin</italic>], CG7574 [<italic>bip1</italic>], CG10009 [<italic>Noa36</italic>], CG10198 [<italic>Nup98</italic>, also known as <italic>Nup98-96</italic>], and CG31938 [<italic>Rrp40</italic>]) that cause an expansion of Pxn (<xref ref-type="fig" rid="fig4">Figure 4A–G</xref>) and <italic>Hml-gal4, UAS EGFP</italic> (<xref ref-type="fig" rid="fig4">Figure 4H–M</xref>) expression<italic>.</italic> Since the function of these genes is needed in the CZ for the maintenance of the MZ progenitors, these six genes encode likely candidates for new components of the equilibrium signaling pathway.<fig-group><fig id="fig4" position="float"><object-id pub-id-type="doi">10.7554/eLife.03626.008</object-id><label>Figure 4.</label><caption><title>Identification of candidate genes that cause an expanded Peroxidasin expression phenotype within the lymph gland when knocked down by RNAi in differentiating and mature cells.</title><p>RNAi from identified lines (<xref ref-type="fig" rid="fig3">Figure 3</xref>/<xref ref-type="table" rid="tbl1">Table 1</xref>) was expressed in lymph glands using <italic>Hml-gal4 UAS-GFP</italic> (<italic>Hml > GFP</italic>). In the control, Pxn (<bold>A</bold>) and GFP (<bold>A′</bold>) are restricted to the cortical zone (periphery). By contrast, knock down of six candidate genes causes extensive expression of Pxn (<bold>B</bold>–<bold>G</bold>) and <italic>Hml</italic> (<italic>Hml > GFP</italic>) (<bold>B′</bold>–<bold>G′</bold>) throughout the lymph gland, indicating a loss of progenitors in these genetic backgrounds. The combined Pxn and <italic>Hml</italic> expression patterns for each genetic background are shown (MERGE, <bold>A″</bold>–<bold>G″</bold>). DNA (blue) is stained to mark nuclei.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03626.008">http://dx.doi.org/10.7554/eLife.03626.008</ext-link></p></caption><graphic xlink:href="elife03626f004"/></fig><fig id="fig4s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.03626.009</object-id><label>Figure 4—figure supplement 1.</label><caption><title>RNAi lines causing an expanded Peroxidasin expression phenotype when expressed in progenitor cells using <italic>dome-gal4</italic>.</title><p>(<bold>A</bold>) Control (<italic>dome-gal4</italic> with no <italic>UAS-dsRNA</italic>) with Pxn expression (red) limited to the cortical zone of the lymph gland primary lobe; DNA (blue). (<bold>B</bold>–<bold>L</bold>) Individual candidate genes identified by RNAi knockdown directly in progenitor cells using <italic>dome-gal4</italic>. RNAi for each gene causes the expansion of Pxn expression (red) throughout the primary lobes.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03626.009">http://dx.doi.org/10.7554/eLife.03626.009</ext-link></p></caption><graphic xlink:href="elife03626fs002"/></fig></fig-group></p><p>Screening with <italic>dome-gal4</italic> (<xref ref-type="bibr" rid="bib25">Jung et al., 2005</xref>) to target RNAi to the progenitor cells identified eleven genes (<xref ref-type="fig" rid="fig4s1">Figure 4—figure supplement 1A–L</xref>), three of which (<italic>Transportin</italic>, <italic>Noa36</italic>, and <italic>Rrp40</italic>) are in common with those identified using <italic>Hml-gal4</italic>. By contrast, use of <italic>Antennapedia-gal4</italic> (<italic>Antp-gal4</italic>) (<xref ref-type="bibr" rid="bib38">Mandal et al., 2007</xref>) to target RNAi specifically to niche cells failed to identify any of the 21 lines as additional niche signaling components (not shown). Lastly, seven of the 21 RNAi lines did not cause a phenotype when expressed with any of the zone-specific Gal4 driver lines used. Taken together, our screen identified three genes, <italic>CTPsyn</italic>, <italic>bip1</italic>, and <italic>Nup98</italic>, which cause a loss of lymph gland progenitor cells upon RNAi knock down in differentiating cells, but not in progenitor cells or in niche cells. As described below, it was ultimately possible to connect two of these genes, <italic>bip1</italic> and <italic>Nup98</italic>, to the equilibrium signaling pathway through the control of Pvr expression.</p></sec><sec id="s2-4"><title>The bip1 gene functions in differentiating cells to regulate progenitor maintenance</title><p>The <italic>bip1</italic> gene was originally identified through a yeast two-hybrid screen that showed that its encoded protein binds the BTB/POZ domain of the transcription factor Bric à brac 1 (Bab1) (<xref ref-type="bibr" rid="bib54">Pointud et al., 2001</xref>), a protein that has several developmental roles including the formation of ovarian terminal filament cells that are required for germline stem cell maintenance (<xref ref-type="bibr" rid="bib34">Lin and Spradling, 1993</xref>; <xref ref-type="bibr" rid="bib57">Sahut-Barnola et al., 1995</xref>; <xref ref-type="bibr" rid="bib9">Couderc et al., 2002</xref>). Analysis of the predicted Bip1 amino acid sequence (InterPro) (<xref ref-type="bibr" rid="bib24">Hunter et al., 2012</xref>) identifies a THAP domain containing a C2CH-type zinc finger motif that is known to bind DNA (<xref ref-type="bibr" rid="bib56">Sabogal et al., 2010</xref>).</p><p>As no known <italic>bip1</italic> mutants exist, several different approaches were used to validate the <italic>bip1</italic> RNAi results and elucidate the function of the <italic>bip1</italic> gene in differentiating the blood cells. First, qRT-PCR confirmed that <italic>bip1</italic> is expressed in the lymph gland and demonstrated that the <italic>bip1</italic> RNAi line (NIG 7574R-2) actually targets <italic>bip1</italic> transcripts. Indeed, RNAi knock down of <italic>bip1</italic> using <italic>Hml-gal4</italic> (<xref ref-type="bibr" rid="bib62">Sinenko and Mathey-Prevot, 2004</xref>; <xref ref-type="bibr" rid="bib25">Jung et al., 2005</xref>) reduces <italic>bip1</italic> mRNA levels in the lymph gland to approximately ten percent of that observed in controls (<xref ref-type="fig" rid="fig5">Figure 5A</xref>). The <italic>bip1</italic> RNAi blood phenotype is also suppressible by the simultaneous overexpression of <italic>bip1</italic> (<italic>UAS-bip1</italic><sup><italic>LA645</italic></sup>; <xref ref-type="fig" rid="fig5">Figure 5B–B'</xref>), demonstrating the specific requirement for <italic>bip1</italic> in maintaining progenitors. Driving <italic>bip1</italic> RNAi with <italic>Pxn-gal4</italic>, an alternative differentiating- and mature-cell driver to <italic>Hml-gal4</italic>, also causes the loss of progenitor cells (<xref ref-type="fig" rid="fig5">Figure 5C–C′</xref>), thereby confirming that <italic>bip1</italic> knock-down in differentiating cells is key to its associated phenotype. Additionally, the progenitor cell marker <italic>dome-MESO-lacZ</italic> (<xref ref-type="bibr" rid="bib23">Hombria et al., 2005</xref>; <xref ref-type="bibr" rid="bib29">Krzemien et al., 2007</xref>) is strongly reduced relative to control lymph glands (<xref ref-type="fig" rid="fig5">Figure 5D–D′</xref>) in the <italic>bip1</italic> RNAi (<italic>Hml-gal4</italic>) background. This result confirms that progenitor cells fail to be maintained in <italic>bip1</italic> RNAi lymph glands, rather than ectopically upregulating Pxn and <italic>Hml-gal4</italic> expression.<fig id="fig5" position="float"><object-id pub-id-type="doi">10.7554/eLife.03626.010</object-id><label>Figure 5.</label><caption><title>Validation of the <italic>bip1</italic> RNAi phenotype.</title><p>(<bold>A</bold>) Quantitative RT-PCR demonstrates that <italic>bip1</italic> is expressed in the lymph gland and that the RNAi line NIG 7574R-1 targeting <italic>bip1</italic> indeed reduces <italic>bip1</italic> transcript level when expressed using <italic>Hml-gal4</italic>. <italic>Hml-gal4</italic> expresses GFP throughout the primary lobes in <italic>bip1</italic> RNAi lymph glands (<italic>Hml > bip1-i</italic>, <bold>B</bold>), and this phenotype is suppressed by overexpression of <italic>bip1</italic> (<bold>B′</bold>), restoring both the cortical and the medullary zones. (<bold>C</bold>–<bold>C′</bold>) Expression of <italic>bip1</italic> RNAi using <italic>Pxn-gal4</italic> phenocopies obtained with <italic>Hml-gal4</italic>, further supporting a cell-type-specific function of <italic>bip1</italic>. Expression of the progenitor cell marker <italic>dome-MESO-lacZ</italic> (<bold>D</bold>) is strongly reduced in <italic>bip1</italic> RNAi lymph glands (<bold>D′</bold>), demonstrating that the gain in differentiation markers is due to the loss of progenitor cells that normally express <italic>dome-MESO-lacZ</italic>. RNAi knock down of <italic>RpS8</italic>, encoding a putative Bip1-interacting protein, causes the expansion of Pxn and <italic>Hml-gal4</italic> expression throughout the lymph gland (<bold>E</bold>–<bold>E′</bold>), similar to that observed upon the loss of <italic>bip1</italic>.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03626.010">http://dx.doi.org/10.7554/eLife.03626.010</ext-link></p></caption><graphic xlink:href="elife03626f005"/></fig></p><p>Several ribosomal components, including Ribosomal protein S8 (RpS8), have been shown to associate with chromatin at active transcription sites and to associate with nascent transcripts to form ribonucleoprotein complexes (<xref ref-type="bibr" rid="bib5">Brogna et al., 2002</xref>). Interestingly, RpS8 has also been identified in genomic-scale yeast two-hybrid analyses as a Bip1-interacting protein (<xref ref-type="bibr" rid="bib21">Giot et al., 2003</xref>; <xref ref-type="bibr" rid="bib18">Formstecher et al., 2005</xref>; <xref ref-type="bibr" rid="bib66">Stark et al., 2006</xref>), which suggests that Bip1 and RpS8 may function together in vivo to regulate gene expression. Consistent with this idea, RNAi knockdown of <italic>RpS8</italic> also causes the expanded expression of both Pxn and <italic>Hml-gal4 UAS-GFP</italic> expression throughout the lymph gland primary lobes (<xref ref-type="fig" rid="fig5">Figure 5E–E′</xref>). This result reflects a specific function of RpS8 in these cells because knockdown directly in niche or progenitor cells (using <italic>Antp-gal4</italic> and <italic>dome-gal4</italic>, respectively) does not cause their loss to differentiation (not shown). Thus, <italic>RpS8</italic> RNAi effectively phenocopies <italic>bip1</italic> RNAi, as both cause a loss of progenitor cells when knocked down in differentiating cells. Collectively, these data support a model in which Bip1 functions along with RpS8 in a protein complex within differentiating cells to maintain multipotent lymph gland progenitors at later stages of development, consistent with a potential function in the equilibrium signaling pathway.</p></sec><sec id="s2-5"><title>bip1 functions genetically upstream of the equilibrium signaling pathway</title><p>The progenitor maintenance function of Pvr signaling in differentiated cells requires the downstream function of the STAT transcriptional activator and the secreted enzyme ADGF-A (<xref ref-type="bibr" rid="bib43">Mondal et al., 2011</xref>), and, consistent with this relationship, overexpression of either activated STAT (STAT<sup>ACT</sup>) (<xref ref-type="bibr" rid="bib16">Ekas et al., 2010</xref>) or ADGF-A in differentiating cells can suppress the <italic>Pvr</italic> loss-of-function phenotype (<xref ref-type="bibr" rid="bib43">Mondal et al., 2011</xref>). Likewise, we find that overexpression of STAT<sup>ACT</sup> or ADGF-A can suppress the <italic>bip1</italic> RNAi phenotype (<xref ref-type="fig" rid="fig6">Figure 6A–D′</xref>). Furthermore, overexpression of <italic>Pvr</italic> also strongly suppresses the <italic>bip1</italic> RNAi phenotype, returning lymph gland morphology and organization to essentially wild type (<xref ref-type="fig" rid="fig6">Figure 6E–E′</xref>). By contrast, overexpression of <italic>bip1</italic> does not suppress the <italic>Pvr</italic> RNAi phenotype (<italic>Hml-gal4 UAS-Pvr RNAi UAS-bip1</italic><sup><italic>LA645</italic></sup>; not shown). Collectively, these results place <italic>bip1</italic> function genetically upstream of <italic>Pvr</italic> and other equilibrium signaling components in lymph gland progenitor maintenance by differentiating cells.<fig-group><fig id="fig6" position="float"><object-id pub-id-type="doi">10.7554/eLife.03626.011</object-id><label>Figure 6.</label><caption><title><italic>bip1</italic>, <italic>RpS8</italic>, and <italic>Nup98</italic> control Pvr expression in the lymph gland.</title><p>Expression of <italic>bip1</italic> RNAi in differentiating cells (<italic>Hml-gal4 or Hml></italic>, <bold>B</bold>–<bold>B′</bold>) causes expansion of both Pxn and <italic>Hml-gal4 UAS-GFP</italic> throughout the lymph gland, as compared to controls (<bold>A</bold>–<bold>A′</bold>). Misexpression of either activated STAT (STAT<sup>ACT</sup>, <bold>C</bold>–<bold>C′</bold>), ADGF-A (<bold>D</bold>–<bold>D′</bold>), or Pvr (<bold>E</bold>–<bold>E′</bold>) partially (in the case of STAT activation or ADGF-A overexpression) or fully (in case of Pvr overexpression) suppresses this <italic>bip1</italic> phenotype, suggesting that <italic>bip1</italic> functions upstream of these genes. Expression of Pvr in control third-instar lymph glands (<bold>F</bold>–<bold>F′</bold>) and mid-second instar (40 hr post-hatching, <bold>G</bold>–<bold>G′</bold>). Reduced expression of Pvr is already apparent in <italic>bip1</italic> RNAi lymph glands by 40 hr (<bold>H</bold>–<bold>H′</bold>), and this loss is even stronger in homozygous animals expressing higher levels of RNAi (<bold>I</bold>–<bold>I′</bold>); increased differentiation, based upon <italic>Hml-gal4 UAS-GFP</italic> expression, is also apparent (<bold>I</bold>). Strong suppression of Pvr is also observed in homozygous <italic>bip1</italic> RNAi lymph glands (<bold>J</bold>–<bold>J′</bold>). RNAi knockdown of <italic>RpS8</italic> also causes differentiation and the loss of Pvr expression (<bold>K</bold>–<bold>K′</bold>). Likewise, RNAi knockdown of <italic>Nup98</italic> also causes differentiation and the loss of Pvr expression (<bold>L</bold>–<bold>L′</bold>). (<bold>M</bold>) Control background (<italic>Hml-gal4</italic>/<italic>+</italic>) showing normal expression of the differentiation marker Pxn in the cortical zone of the lymph gland. Progenitor cells in the MZ region are easily discerned by their lack of Pxn expression. By contrast, few progenitor cells (Pxn-negative cells) are observed in lymph glands when single-copy loss-of-function mutations of <italic>Pvr</italic> and <italic>Nup98</italic> (<italic>Pvr</italic><sup><italic>C2195</italic></sup>/<italic>+</italic>; <italic>Nup98</italic><sup><italic>Df(3R)mbc-R1</italic></sup>/<italic>+</italic>) are combined (<bold>M′</bold>), further indicating the close interaction between these genes. The middle-third (confocal z-stack) of the primary lobe is shown. Misexpression of <italic>bip1</italic> in this background is sufficient to suppress these phenotypes and restore Pvr expression to the lymph gland (<bold>N</bold>–<bold>N′</bold>).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03626.011">http://dx.doi.org/10.7554/eLife.03626.011</ext-link></p></caption><graphic xlink:href="elife03626f006"/></fig><fig id="fig6s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.03626.012</object-id><label>Figure 6—figure supplement 1.</label><caption><title><italic>bip1</italic> and <italic>RpS8</italic> are required for normal <italic>Pvr</italic> transcript levels.</title><p>Quantitative RT-PCR analysis demonstrating that RNAi knockdown of <italic>bip1</italic> in lymph glands (<bold>A</bold>) and circulating cells (<bold>B</bold>) using <italic>Hml-gal4</italic> causes a reduction in <italic>Pvr</italic> transcript levels to approximately 70% and 34% of the control (<italic>Hml-gal4</italic> only) level, respectively. <italic>RpS8</italic> RNAi using <italic>Hml-gal4</italic> reduces <italic>Pvr</italic> to approximately 32% of the control level (<bold>C</bold>, circulating cells). Note that <italic>Hml-gal4</italic> is only expressed in a subset of cells in the lymph gland but in the vast majority of cells in circulation.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03626.012">http://dx.doi.org/10.7554/eLife.03626.012</ext-link></p></caption><graphic xlink:href="elife03626fs003"/></fig><fig id="fig6s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.03626.013</object-id><label>Figure 6—figure supplement 2.</label><caption><title>Pvr expression is regulated autonomously by <italic>bip1</italic>, <italic>Nup98</italic>, and <italic>RpS8</italic> within the lymph gland.</title><p>Mock FLP-out Gal4-expressing clones (GFP-positive cells, green) show no reduction in Pvr expression (red, <bold>A</bold>–<bold>A′</bold>), whereas similar clones expressing Pvr RNAi show a very strong reduction in Pvr protein expression (<bold>B</bold>–<bold>B′</bold>). FLP-out Gal4-expressing clones expressing either <italic>bip1</italic> RNAi (<bold>C</bold>–<bold>C′</bold>) or <italic>Nup98</italic> RNAi (<bold>D</bold>–<bold>D′</bold>) also reduce Pvr protein levels, with <italic>bip1</italic> RNAi exhibiting somewhat stronger effects. FLP-out clones were made using <italic>Hand-gal4 UAS-FLP Ay-gal4 UAS-GFP</italic> at 25°C. Restricting the RNAi knockdown of <italic>bip1</italic> (<bold>F</bold>), <italic>Nup98</italic> (<bold>G</bold>), or <italic>RpS8</italic> (<bold>H</bold>) to circulating cells with <italic>srpHemo-gal4</italic> (<bold>E</bold>) shows no effect on lymph gland Pvr levels.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03626.013">http://dx.doi.org/10.7554/eLife.03626.013</ext-link></p></caption><graphic xlink:href="elife03626fs004"/></fig><fig id="fig6s3" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.03626.014</object-id><label>Figure 6—figure supplement 3.</label><caption><title>Loss of the nucleoporin Sec13 by RNAi neither causes a differentiation phenotype within the lymph gland nor the loss of Pvr expression.</title><p>(<bold>A</bold>) <italic>Hml-gal4</italic> control showing normal cortical zone expression (<italic>UAS-GFP</italic>). (<bold>A′</bold>) Pvr is expressed normally throughout the primary lobe in control lymph gland in (<bold>A</bold>). (<bold>B</bold>–<bold>B′</bold>) Loss of Sec13 by RNAi (<italic>Hml-gal4 UAS-Sec13-i</italic>) does not cause increased differentiation (GFP expression) or the loss of Pvr expression (red). DNA, blue.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03626.014">http://dx.doi.org/10.7554/eLife.03626.014</ext-link></p></caption><graphic xlink:href="elife03626fs005"/></fig><fig id="fig6s4" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.03626.015</object-id><label>Figure 6—figure supplement 4.</label><caption><title>Loss of Pvr expression is not a common feature of highly differentiated lymph glands.</title><p>The lymph gland marker P1 is normally restricted to the differentiated cells of the cortical zone. However, in <italic>collier</italic><sup><italic>1</italic></sup> (<italic>col</italic><sup><italic>1</italic></sup>) mutants that lack niche signaling, P1 expression (<bold>A</bold>, green) is observed throughout, indicating strong differentiation and a lack of progenitor cells. In this background, Pvr is expressed at normal levels (<bold>A′</bold>, compare with cortical zone levels in <xref ref-type="fig" rid="fig6">Figure 6F′</xref>), indicating that loss of Pvr is not a general feature of highly differentiated lymph glands.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03626.015">http://dx.doi.org/10.7554/eLife.03626.015</ext-link></p></caption><graphic xlink:href="elife03626fs006"/></fig><fig id="fig6s5" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.03626.016</object-id><label>Figure 6—figure supplement 5.</label><caption><title>Overexpression of <italic>bip1</italic>, <italic>Nup98</italic>, and <italic>RpS8</italic>, and RNAi knockdown of other nucleoporins does not affect Pvr levels in the lymph gland.</title><p>Compared with control levels (<bold>A</bold>, <italic>Hml-gal4</italic>), overexpression of <italic>bip1</italic> (<bold>B</bold>, <italic>UAS-bip1</italic><sup><italic>LA645</italic></sup>), <italic>Nup98</italic> (<bold>C</bold>, <italic>UAS-Nup98</italic> [<xref ref-type="bibr" rid="bib51">Parrott et al., 2011</xref>]), or <italic>RpS8</italic> (<bold>D</bold>, <italic>UAS-RpS8</italic><sup><italic>DP01446</italic></sup> [<xref ref-type="bibr" rid="bib67">Staudt t al., 2005</xref>]) does not significantly affect Pvr levels (red). Likewise, RNAi knockdown of <italic>Nup154</italic> (<bold>F</bold>), <italic>Nup214</italic> (<bold>G</bold>), or <italic>Nup358</italic> (<bold>H</bold>) (all with <italic>Hml-gal4</italic>) does not significantly alter Pvr level compared to controls (<bold>E</bold>), further supporting the specific function of Nup98 in Pvr regulation.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03626.016">http://dx.doi.org/10.7554/eLife.03626.016</ext-link></p></caption><graphic xlink:href="elife03626fs007"/></fig></fig-group></p></sec><sec id="s2-6"><title>Bip1 and RpS8 control the equilibrium signaling pathway by regulating Pvr expression</title><p>The suppression of the <italic>bip1</italic> RNAi phenotype by overexpression of <italic>Pvr</italic> suggested that <italic>bip1</italic> may positively control <italic>Pvr</italic> expression during normal development. Indeed, a reduction in Pvr protein expression within the lymph gland is observed in the <italic>bip1</italic> RNAi background by mid-second instar, soon after differentiation begins (∼40 hr post-hatching; <xref ref-type="fig" rid="fig6">Figure 6F–J′</xref>). This reduction in Pvr expression is even stronger (along with significantly increased differentiation, based upon <italic>Hml-gal4</italic> expression) at the same developmental time point in larvae having two copies of <italic>Hml-gal4 UAS-bip1 RNAi</italic> (compare <xref ref-type="fig" rid="fig6">Figure 6I′</xref> with <xref ref-type="fig" rid="fig6">Figure 6H′</xref>), further supporting the model that <italic>bip1</italic> RNAi causes the loss of Pvr expression. By the late third instar (when the <italic>bip1</italic> RNAi differentiation phenotype is most apparent), Pvr protein levels in the lymph gland remain strongly reduced (<xref ref-type="fig" rid="fig6">Figure 6J–J′</xref>).</p><p>Knockdown of <italic>bip1</italic> function in lymph glands by <italic>Hml-gal4</italic>-mediated RNAi reduces lymph gland <italic>Pvr</italic> transcript levels to approximately 70% of control levels (assessed by qRT-PCR; <xref ref-type="fig" rid="fig6s1">Figure 6—figure supplement 1A</xref>), consistent with the observed loss of Pvr protein (<xref ref-type="fig" rid="fig6">Figure 6H′–J′</xref>). However, because not all lymph gland cells express <italic>Hml</italic> (<italic>Hml-gal4</italic>), the actual reduction of <italic>Pvr</italic> transcript levels in cells expressing <italic>bip1</italic> RNAi is likely to be greater than the observed total reduction. In support of this idea, <italic>bip1</italic> RNAi in circulating blood cells (where greater than 90% express <italic>Hml-gal4</italic>) reduces <italic>Pvr</italic> transcript level to approximately 35% of the control level (<xref ref-type="fig" rid="fig6s1">Figure 6—figure supplement 1B</xref>). Expression of <italic>bip1</italic> RNAi in FLP-out Gal4-expressing cell clones made exclusively in the lymph gland strongly reduces Pvr levels compared to nearby cells not expressing RNAi as well as to mock clones (<xref ref-type="fig" rid="fig6s2">Figure 6—figure supplement 2A–C</xref>), consistent with the autonomous regulation of Pvr by <italic>bip1</italic> in the lymph gland. Furthermore, <italic>bip1</italic> RNAi expression with <italic>srpHemo-gal4</italic> (<xref ref-type="bibr" rid="bib6">Bruckner et al., 2004</xref>), which expresses in a large fraction of circulating cells but in few or no cells within the lymph gland, does not reduce lymph gland Pvr levels (<xref ref-type="fig" rid="fig6s2">Figure 6—figure supplement 2E–H</xref>). Collectively, these data indicate that <italic>bip1</italic> is required for proper Pvr protein expression, and therefore proper equilibrium signaling, within the developing lymph gland.</p><p>As described above, RpS8 is a putative Bip1-interacting protein in vivo and <italic>RpS8</italic> RNAi in differentiating lymph gland cells, like <italic>bip1</italic> RNAi, causes the loss of progenitor cells (<xref ref-type="fig" rid="fig5">Figure 5F–F′</xref>). This effect is likely due to the loss of equilibrium signaling during development since <italic>RpS8</italic> RNAi also reduces Pvr protein expression in the lymph gland (<xref ref-type="fig" rid="fig6">Figure 6K–K′</xref>). Knockdown of <italic>RpS8</italic> by RNAi, as with knockdown of <italic>bip1</italic>, also reduces <italic>Pvr</italic> transcript levels (<xref ref-type="fig" rid="fig6s1">Figure 6—figure supplement 1C</xref>). Interestingly, a <italic>Drosophila</italic> RNAi screen using the blood-related S2 cell line previously identified both Pvr and RpS8 as regulators of cell size and division (<xref ref-type="bibr" rid="bib61">Sims et al., 2009</xref>). Although the relationship between Pvr and RpS8 was not explored, their results as well as ours are consistent with RpS8 having a regulatory role in <italic>Pvr</italic> expression in blood cells.</p></sec><sec id="s2-7"><title>Nup98 also regulates Pvr expression</title><p>In addition to <italic>bip1</italic>, the screen described here identified <italic>Nup98</italic> as a potential equilibrium signaling component because its knockdown in differentiating cells specifically causes a loss of progenitors cells (<xref ref-type="fig" rid="fig3">Figure 3T</xref> and <xref ref-type="fig" rid="fig4">Figure 4F–F′′</xref>). Although Nup98 is widely known as a general component of the nuclear pore complex, recent work has demonstrated that Nup98 and other nuclear pore components such as Sec13 and Nup88, can regulate gene expression through the binding of target promoters (<xref ref-type="bibr" rid="bib7">Capelson et al., 2010</xref>; <xref ref-type="bibr" rid="bib28">Kalverda et al., 2010</xref>; <xref ref-type="bibr" rid="bib33">Liang et al., 2013</xref>). Moreover, chromatin immunoprecipitation experiments identified <italic>bip1</italic>, <italic>RpS8</italic>, and the equilibrium signaling genes <italic>Pvr</italic> and <italic>STAT</italic> (<italic>STAT92E</italic>) as in vivo Nup98 regulatory targets (<xref ref-type="bibr" rid="bib7">Capelson et al., 2010</xref>). Consistent with a function in regulation of equilibrium signaling genes, <italic>Nup98</italic> knockdown specifically in differentiating cells of lymph glands causes a strong reduction in Pvr expression (<xref ref-type="fig" rid="fig6">Figure 6L–L′</xref>). By contrast, RNAi knockdown of the nucleoporin Sec13 in differentiating cells has no effect on the maintenance of progenitor cells or Pvr expression (<xref ref-type="fig" rid="fig6s3">Figure 6—figure supplement 3</xref>) underscoring the specific role of <italic>Nup98</italic> in Pvr expression control. Furthermore, the close genetic relationship between <italic>Nup98</italic> and <italic>Pvr</italic> is illustrated by the fact that single-copy loss of these genes in combination causes extensive loss of progenitor cells to differentiation (<xref ref-type="fig" rid="fig6">Figure 6M–M′</xref>). Interestingly, overexpression of <italic>bip1</italic> in <italic>Nup98</italic> RNAi lymph glands (<italic>Hml-gal4 UAS-Nup98 RNAi UAS-bip1</italic><sup><italic>LA645</italic></sup>) is sufficient to restore Pvr protein expression and to suppress the loss of progenitors to differentiation (based upon lymph gland morphology and <italic>Hml-gal4</italic> expression; <xref ref-type="fig" rid="fig6">Figure 6N–N′</xref>).</p><p>As has been shown, knockdown of <italic>bip1</italic>, <italic>Nup98</italic>, or <italic>RpS8</italic> in differentiating cells each causes a strong reduction in Pvr expression in the lymph gland. Our interpretation of this common phenotype is that each gene works in the equilibrium signaling pathway to control Pvr expression, although an alternative hypothesis is that the loss of Pvr expression is a common feature of highly differentiated lymph glands and is not specifically related to the function of these genes. To test this, Pvr expression was examined in <italic>collier</italic> (<italic>col</italic>) mutant lymph glands, which lack niche signaling and are strongly differentiated by late larval stages (<xref ref-type="bibr" rid="bib11">Crozatier et al., 2004</xref>; <xref ref-type="bibr" rid="bib38">Mandal et al., 2007</xref>), and was found to be normal (<xref ref-type="fig" rid="fig6s4">Figure 6—figure supplement 4</xref>, compare with Pvr expression in wild-type cortical zone differentiating cells in <xref ref-type="fig" rid="fig6">Figure 6F′</xref>). Thus, <italic>Pvr</italic> requires <italic>bip1</italic>, <italic>RpS8</italic>, and <italic>Nup98</italic> for proper developmental expression in the lymph gland.</p><p>Several genetic screens, including overexpression and enhancer/suppressor screens of mutant or tumor phenotypes, have been conducted in the fly hematopoietic system (<xref ref-type="bibr" rid="bib42">Milchanowski et al., 2004</xref>; <xref ref-type="bibr" rid="bib77">Zettervall et al., 2004</xref>; <xref ref-type="bibr" rid="bib68">Stofanko et al., 2008</xref>; <xref ref-type="bibr" rid="bib3">Avet-Rochex et al., 2010</xref>; <xref ref-type="bibr" rid="bib70">Tan et al., 2012</xref>; <xref ref-type="bibr" rid="bib73">Tokusumi et al., 2012</xref>); however, the screen described here represents the first loss-of-function screen targeting normal developmental mechanisms throughout the lymph gland. This was accomplished with the development and use of the pan-lymph gland expression tool <italic>HHLT-gal4</italic> to drive <italic>UAS-</italic>mediated RNAi, which identified 20 different candidate genes that cause a loss of progenitor cells when knocked down within the lymph gland. From subsequent analyses using lymph gland zone-restricted Gal4 driver lines, we arrive at a model (<xref ref-type="fig" rid="fig7">Figure 7</xref>) in which Bip1, RpS8, and Nup98 are required in differentiating blood cells upstream of Pvr to control its expression and function in the equilibrium signaling pathway that maintains blood progenitors within the lymph gland. Future analyses will be required to identify additional components of this important signaling pathway and to provide more information about how equilibrium signaling interacts with other pathways in the control of blood cell progenitor maintenance, cell fate specification, and proliferation.<fig id="fig7" position="float"><object-id pub-id-type="doi">10.7554/eLife.03626.017</object-id><label>Figure 7.</label><caption><title>Schematic of the equilibrium signaling pathway demonstrating the proposed roles of Bip1, RpS8, and Nup98 in controlling Pvr.</title><p>Bip1, RpS8, and Nup98 are independently required for the expression of Pvr (direct arrows). Rescue of endogenous Pvr expression by misexpression of <italic>bip1</italic> in the <italic>Nup98</italic> RNAi background indicates that <italic>bip1</italic> functions genetically downstream of <italic>Nup98</italic> (dashed arrow) in the control of Pvr expression. Bip1 and RpS8 may work together in a complex (dashed line) to control Pvr expression in vivo. These components collectively comprise the known equilibrium signaling pathway working within the lymph gland to promote progenitor cell maintenance, along with the previously known Hh niche signaling mechanism.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03626.017">http://dx.doi.org/10.7554/eLife.03626.017</ext-link></p></caption><graphic xlink:href="elife03626f007"/></fig></p><p>The Pvr receptor, with its numerous developmental roles, is arguably one of the most important members of the <italic>Drosophila</italic> RTK family, yet most of what is known about Pvr stems from analyses of how it works in the context of intracellular signaling. Little is known about how <italic>Pvr</italic> gene or protein expression is regulated. Importantly, the work described here sheds new light upon this issue by demonstrating a role for <italic>bip1</italic>, <italic>RpS8</italic>, and <italic>Nup98</italic> in the regulation of <italic>Pvr</italic> expression. Our data and that of others suggest that this regulation of <italic>Pvr</italic> is likely taking place at the gene level, although other mechanisms are also possible. Ribosomes are required for protein translation, however specific ribosomal components or subunits may selectively stabilize transcripts and/or mediate preferential translation (<xref ref-type="bibr" rid="bib76">Xue and Barna, 2012</xref>), while nucleoporins control both nuclear entry of regulatory proteins and the exit of mRNAs to the cytoplasm, and specific subcomponents are known to exhibit differential functions in this regard (<xref ref-type="bibr" rid="bib69">Strambio-De-Castillia et al., 2010</xref>). Thus, RpS8 and Nup98 may selectively affect <italic>Pvr</italic> expression post-transcriptionally through transcript stabilization, transport, and translation. Although the specific mechanisms of molecular control of <italic>Pvr</italic> expression by <italic>bip1</italic>, <italic>RpS8</italic>, and <italic>Nup98</italic> remain to be determined, their function is clearly critical in mediating proper equilibrium signaling and, therefore, proper blood progenitor maintenance within the lymph gland. The finding that <italic>bip1</italic> regulates Pvr expression in the context of hematopoietic equilibrium signaling represents the first functional association for <italic>bip1</italic> in <italic>Drosophila</italic>. The predicted Bip1 protein exhibits only one recognizable structural sequence, namely a THAP domain that contains a putative DNA-binding zinc finger motif. Our results suggest that Bip1 behaves as a positive regulator of <italic>Pvr</italic> transcription, but whether this occurs directly through Bip1 interaction with the <italic>Pvr</italic> locus will require further investigation.</p><p>Understanding how progenitor cell maintenance and homeostasis is controlled over developmental time is crucial for understanding normal cellular and tissue dynamics, especially in the context of ageing or disease. The identification of Bip1 and Nup98 as regulators of hematopoietic progenitors in <italic>Drosophila</italic> may be indicative of important conserved functions of related proteins within the vertebrate blood lineages similar to what has been shown previously for GATA, FOG, and RUNX factors (<xref ref-type="bibr" rid="bib75">Waltzer et al., 2010</xref>). THAP-domain proteins are conserved across species and have been reported to have a variety of important functions in mammalian systems, including maintenance of murine embryonic stem cell pluripotency (<xref ref-type="bibr" rid="bib8">Cayrol et al., 2007</xref>; <xref ref-type="bibr" rid="bib12">Dejosez et al., 2008</xref>, <xref ref-type="bibr" rid="bib13">2010</xref>). What role, if any, THAP-domain proteins have in vertebrate blood progenitor maintenance (or hematopoiesis in general) remains to be established. Likewise, Nup98 has not been implicated in any normal hematopoietic role despite being a well-studied protein in other contexts.</p><p>With regard to the diseased state, mutations in the human <italic>THAP1</italic> gene have been associated with dystonia (<xref ref-type="bibr" rid="bib20">Fuchs et al., 2009</xref>; <xref ref-type="bibr" rid="bib50">Paisan-Ruiz et al., 2009</xref>; <xref ref-type="bibr" rid="bib26">Kaiser et al., 2010</xref>; <xref ref-type="bibr" rid="bib41">Mazars et al., 2010</xref>), a neuromuscular disorder that causes repetitive, involuntary muscular contraction, and THAP1/Par4 protein complexes have been shown to promote apoptosis in leukemic blood cells in various experimental contexts in vitro (<xref ref-type="bibr" rid="bib36">Lu et al., 2013</xref>; <xref ref-type="bibr" rid="bib78">Zhang et al., 2014</xref>). Chromosomal translocations that generate Nup98 fusion proteins have been implicated in numerous human myelodysplastic syndromes and leukemias (<xref ref-type="bibr" rid="bib48">Nishiyama et al., 1999</xref>; <xref ref-type="bibr" rid="bib1">Ahuja et al., 2001</xref>; <xref ref-type="bibr" rid="bib35">Lin et al., 2005</xref>; <xref ref-type="bibr" rid="bib46">Nakamura, 2005</xref>; <xref ref-type="bibr" rid="bib74">van Zutven et al., 2006</xref>; <xref ref-type="bibr" rid="bib65">Slape et al., 2008</xref>; <xref ref-type="bibr" rid="bib27">Kaltenbach et al., 2010</xref>; <xref ref-type="bibr" rid="bib45">Murayama et al., 2013</xref>), further underscoring the need to explore Nup98 function in the hematopoietic system. Therefore, the study of <italic>bip1</italic> and <italic>Nup98</italic> in <italic>Drosophila</italic>, a powerful molecular genetic system, will likely be of benefit to understand the function of related vertebrate genes in normal and disease contexts.</p></sec></sec><sec id="s3" sec-type="materials|methods"><title>Materials and methods</title><sec id="s3-1"><title>Fly stocks</title><p>Misexpression <italic>P{Mae-UAS.6.11}</italic> inserts (LA lines) were obtained from John Merriam, UCLA (Los Angeles, California). <italic>UAS-RNAi</italic> lines were obtained from the Vienna <italic>Drosophila</italic> RNAi Center (VDRC, Vienna, Austria), the National Institute of Genetics (NIG, Kyoto, Japan), and the Bloomington <italic>Drosophila</italic> Stock Center (TRiP lines, BDSC, Bloomington, Indiana). The lines <italic>UAS-FLP.JD1, UAS-2XEGFP, P{GAL4-Act5C(FRT.</italic>CD2<italic>).P}S</italic>, <italic>UAS-human Raf</italic><sup><italic>ACT</italic></sup>, <italic>Df(3R)mbc-R1</italic>, <italic>UAS-RpS8</italic><sup><italic>PD01446</italic></sup>, and <italic>w</italic><sup><italic>1118</italic></sup> (BDSC 5905) were from the BDSC. <italic>Pvr</italic><sup><italic>c02195</italic></sup> was from Exelixis (available from BDSC, obtained from D Montell). <italic>Hml</italic><sup><italic>Δ</italic></sup><italic>-gal4 UAS-2XEGFP</italic> (S Sinenko)<italic>, Antp-gal4/TM6B Tb</italic> (S Cohen)<italic>, P{ubi-gal80 ts}10; Antp-gal4/TM6B Tb</italic> (this lab)<italic>, domeless-gal4 UAS-2XEYFP/FM7i</italic> (this lab), <italic>UAS-DAlk</italic><sup><italic>ACT</italic></sup> (R Palmer), <italic>dome-MESO-lacZ</italic> (S Brown), <italic>Pxn-gal4</italic> (M Galko), <italic>UAS-STAT</italic><sup><italic>ACT</italic></sup> (E Bach), <italic>UAS-ADGF-A</italic> (T Dolezal), <italic>collier</italic><sup><italic>1</italic></sup><italic>; P(col5-cDNA)/CyO-TM6B, Tb</italic> (M Crozatier), <italic>srpHemo-gal4</italic> (K Brückner), and <italic>Hand-gal4</italic> (Z Han) have been previously described.</p></sec><sec id="s3-2"><title>HHLT-gal4 construction and whole animal screening</title><p>Second chromosome inserts of <italic>Hand-gal4</italic>, <italic>HmlΔ-gal4</italic>, <italic>UAS-FLP.JD1</italic>, and <italic>UAS-2XEGFP</italic> were recombined onto a single chromosome and placed with <italic>P{GAL4-Act5C(FRT.</italic>CD2<italic>).P}S</italic> on Chromosome 3. Because Gal4 reporter lines with specific, pan-lymph gland expression are unknown, we took advantage of a FLP-out lineage tracing approach that we have used previously to perpetually mark lymph gland cells (<xref ref-type="bibr" rid="bib25">Jung et al., 2005</xref>; <xref ref-type="bibr" rid="bib17">Evans et al., 2009</xref>). The <italic>Hand-gal4</italic> reporter reflects the expression of the <italic>Hand</italic> gene, which is expressed in the cardiogenic mesoderm, from which the lymph gland is derived. Within the lymph gland, <italic>Hand-gal4</italic> is expressed from the late embryo through the first larval instar but then is downregulated (<xref ref-type="bibr" rid="bib22">Han and Olson, 2005</xref>). Using <italic>Hand-gal4</italic> in conjunction with <italic>UAS-FLP</italic> and a FLP-out Gal4-expressing line (<italic>P{GAL4-Act5C(FRT.</italic>CD2<italic>).P}S</italic>) (<xref ref-type="bibr" rid="bib53">Pignoni and Zipursky, 1997</xref>), lymph gland cells are perpetually with EGFP throughout all subsequent developmental stages. To express EGFP in circulating cells, we used <italic>Hemolectin-gal4</italic> (<italic>HmlΔ-gal4</italic>) (<xref ref-type="bibr" rid="bib62">Sinenko and Mathey-Prevot, 2004</xref>), which is specific to mature blood cells both in circulation and in the lymph gland cortical zone (<xref ref-type="bibr" rid="bib25">Jung et al., 2005</xref>). <italic>HHLT-gal4</italic> expression is easily detectable in lymph glands and circulating cells of whole animals throughout larval development. Due to the embryonic activity of <italic>Hand-gal4</italic>, <italic>HHLT-gal4</italic> also labels dorsal vessel cardioblasts and pericardial cells, although by late larval stages the expression of EGFP in the former is almost undetectable.</p><p><italic>HHLT-gal4</italic> virgins were crossed to males from individual LA lines, RNAi lines, or <italic>w</italic><sup><italic>1118</italic></sup> as a control. All crosses were reared at 29°C to maximize Gal4 activity. Wandering third-instar larvae from control and experimental crosses were collected, washed with water, and placed in glass spot wells (Fisher) on ice to minimize movement. Animals were scored visually using a Zeiss Axioskop 2 compound fluorescence microscope. Non-screen images of <italic>HHLT > GFP</italic> larvae were collected with a Zeiss SteREO Lumar fluorescence microscope. Images were collected using either an AxioCam HRc or HRm camera with AxioVision software.</p></sec><sec id="s3-3"><title>Tissue dissection and antibody staining and analysis</title><p>Lymph glands were dissected and processed as previously described (<xref ref-type="bibr" rid="bib25">Jung et al., 2005</xref>). Briefly, lymph glands were dissecting from third-instar larvae in 1× PBS, fixed in 4% formaldehyde/1× PBS for 30 min, washed three times in 1×PBS with 0.4% Triton-X (1× PBST) for 15 min each, blocked in 10% normal goat serum/1× PBST for 30 min, followed by incubation with primary antibodies in block. Primary antibodies were incubated with tissue overnight at 4°C and then washed three times in 1× PBST for 15 min each, reblocked for 15 min, followed by incubation with secondary antibodies for 3 hr at room temperature. Samples were washed three times in 1× PBST, with TO-PRO-3 iodide (diluted 1:1000; Invitrogen, Carlsbad, California) added to the last wash to stain nuclei. Samples were washed briefly with 1× PBS to remove excess TO-PRO-3 and detergent prior to mounting on glass slides in VectaShield (Vector Laboratories, Burlingame, California). Mouse anti-Peroxidasin was a kind gift from John and Lisa Fessler (UCLA) and was used at 1:1500 dilution. Rat anti-Pvr was a kind gift from Benny Shilo and was used at 1:400 dilution. Secondary Cy3-labeled antibodies were obtained from Jackson ImmunoResearch Laboratories Inc. (West Grove, Pennsylvania) and used at 1:500 dilution.</p></sec><sec id="s3-4"><title>Quantitative real-time PCR analysis</title><p>Lymph glands from 50 third-instar larvae were isolated by dissection. For fat body analysis, ten third-instar larvae were used. RNA was extracted from these tissues with the RNeasy mini kit (Qiagen, Germantown, Maryland). Relative quantitative RT-PCR (comparative CT) was performed using Power SYBR Green RNA-to-CT 1-step kit (Applied Biosystems, Carlsbad, California) and a StepOne Real-Time PCR detection thermal cycler (Applied Biosystems) using primers specific for <italic>Pvr</italic>, <italic>bip1,</italic> and <italic>rp49</italic>. Primer sequences are: <italic>Pvr</italic>(forward), 5′-TTCGGATTTCGATGGTGAAT-3′; <italic>Pvr</italic>(reverse), 5′-CGGACACTAAGCTGGTCGAT-3′; <italic>bip1</italic>(forward), 5′-CGGAGTTTATGGACAGCACA-3′; <italic>bip1</italic>(reverse), 5′-CCTTAGCAGGAGGAGGAGGT-3′; <italic>rp49</italic>(forward), 5′-GCTAAGCTGTCGCACAAATG-3′; <italic>rp49</italic>(reverse), 5′-GTTCGATCCGTAACCGATGT-3′.</p></sec></sec></body><back><ack id="ack"><title>Acknowledgements</title><p>We thank Amir Yavari, Julia Manasson, Tanya Hioe, Sean Mofidi, Jesse Zaretsky, and Tina Muhkerjee for assistance generating the <italic>HHLT-gal4</italic> line and with various aspects of the screen. We thank John Merriam (UCLA) for use of the LA lines and John and Lisa Fessler (UCLA) for the kind gift of anti-Pxn antibodies. Lastly, we thank Ira Clark, John Merriam, and members of the Banerjee Lab for discussion of the project and comments on the manuscript.</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>UB: Reviewing editor, <italic>eLife</italic>.</p></fn><fn fn-type="conflict" id="conf2"><p>The other 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>BCM, Acquisition of data, Analysis and interpretation of data</p></fn><fn fn-type="con" id="con2"><p>JS, Acquisition of data, Analysis and interpretation of data</p></fn><fn fn-type="con" id="con3"><p>CJE, 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>UB, Conception and design, Analysis and interpretation of data, Drafting or revising the article</p></fn></fn-group></sec><sec sec-type="supplementary-material"><title>Additional files</title><supplementary-material id="SD1-data"><object-id pub-id-type="doi">10.7554/eLife.03626.018</object-id><label>Supplementary file 1.</label><caption><p>Genes and phenotypes associated with <italic>P{Mae-UAS.6.11}</italic> LA insertion lines expressed with <italic>HHLT-gal4</italic>. Screen and line identifiers are shown along with the predicted misexpressed gene and the associated whole-animal lymph gland and circulating cell phenotype.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03626.018">http://dx.doi.org/10.7554/eLife.03626.018</ext-link></p></caption><media mime-subtype="xlsx" mimetype="application" xlink:href="elife03626s001.xlsx"/></supplementary-material><supplementary-material id="SD2-data"><object-id pub-id-type="doi">10.7554/eLife.03626.019</object-id><label>Supplementary file 2.</label><caption><p>Genes and phenotypes associated with <italic>UAS-RNAi</italic> lines expressed with <italic>HHLT-gal4</italic>. Screen and line identifiers are shown along with the targeted gene for knockdown by RNAi, the associated whole-animal lymph gland phenotype based upon GFP expression, and the Peroxidasin (Pxn) expression phenotype of dissected lymph glands.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03626.019">http://dx.doi.org/10.7554/eLife.03626.019</ext-link></p></caption><media mime-subtype="xlsx" mimetype="application" xlink:href="elife03626s002.xlsx"/></supplementary-material></sec><ref-list><title>References</title><ref id="bib1"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Ahuja</surname><given-names>HG</given-names></name><name><surname>Popplewell</surname><given-names>L</given-names></name><name><surname>Tcheurekdjian</surname><given-names>L</given-names></name><name><surname>Slovak</surname><given-names>ML</given-names></name></person-group><year>2001</year><article-title>NUP98 gene rearrangements 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pub-id-type="doi">10.7554/eLife.03626.020</article-id><title-group><article-title>Decision letter</article-title></title-group><contrib-group content-type="section"><contrib contrib-type="editor"><name><surname>Ohlstein</surname><given-names>Benjamin</given-names></name><role>Reviewing editor</role><aff><institution>Columbia University Medical Center</institution>, <country>United States</country></aff></contrib></contrib-group></front-stub><body><boxed-text><p>eLife posts the editorial decision letter and author response on a selection of the published articles (subject to the approval of the authors). 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://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 “Maintenance of <italic>Drosophila</italic> blood progenitors by equilibrium signal regulators functioning in differentiating blood cells” for consideration at <italic>eLife</italic>. Your article has been favorably evaluated by K (Vijay) VijayRaghavan (Senior editor) and 3 reviewers, one of whom served as a guest Reviewing editor.</p><p>The Reviewing editor and the other reviewers discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission.</p><p>This paper from Mondal and colleagues addresses how blood cell progenitors within the Drosophila larval lymph gland are maintained. The work follows on from an earlier Cell paper that showed the existence of an 'equilibrium signal' from differentiating blood cells centered around PVR. In this study the authors have carried out a well-designed screen to identify novel genes required for blood progenitor maintenance and successfully identify 20 novel regulators of this process. The study then focuses on those that regulate the 'equilibrium signal' in the differentiating cells, namely bip1 and Nup98. The upstream regulation of PVR is an understudied and very important question that has wide-reaching implications, which will likely benefit our understanding of the regulation of this receptor family across species.</p><p>Technically this paper is excellent, the screen is beautifully designed and the HHLT-gal4 line is likely to be a great resource to the research community. However, there is little follow up work to the screen. Whilst the screen must have been very labour intensive the paper does feel more like a 'technique paper' rather than a ground-breaking research paper. Therefore, the storyline of the manuscript would benefit from re-shaping and focusing, placing less emphasis on the early steps of screening and more on PVR regulation. The authors may even want the title to reflect this. Some experimental clarifications and amendments of the text would be required to make the manuscript suitable for publication.</p><p>Substantive concerns:</p><p>1) The authors should address whether regulation of PVR by Nup98, bip1 and RpS8 is cell autonomous, or related to non-autonomous events, which could even originate from the population of tissue hemocytes/macrophages (by the authors called 'circulating hemocytes', see comment below), which express the same drivers as differentiating/-ed hemocytes of the LG (ie Hml-GAL4 and Pxn-GAL4). This overlap in expression is in fact a major caveat of most LG papers, an issue that receives increasing attention now that the biology of larval tissue hemocytes, the active hemocyte population of the larva, has been revealed in more detail (see below). The LG field would greatly benefit from an expression system for differentiated LG-hemocytes only (e.g. by inducible elimination of GAL4 in the earlier differentiated population of tissue hemocytes). However, in the absence of such a tool the authors need to retreat to other methods to demonstrate whether or not the presented regulation of PVR is cell autonomous. These methods should comprise:</p><p>1a) Use of flipout-GAL4 to generate <italic>in vivo</italic> RNAi clones of the genes in question, assessing PVR expression and the GAL4 expressing clones (e.g. by UAS-GFP) in the LG. Side-by-side comparison of kd and control tissue should allow to evaluate cell autonomous effects.</p><p>1b) Use of Hand-GAL4 with UAS-GAL4, or HHLT without Hml-GAL4, as driver to determine the effects of RNAi of the genes in question on LG PVR expression, in the absence of expression in tissue hemocytes.</p><p>2) The authors show all genes of the RNAi screen that led to increased Pxn expression. Do all or just some of these kds affect PVR expression? Was this a selection criterion by the authors to go forward with specific genes?</p><p>3) What is the overexpression phenotype of Nup98, bip1 and RpS8? From the phenotypes listed in <xref ref-type="supplementary-material" rid="SD1-data">Supplementary File 1</xref> it looks as if they may not be fully complementary to the RNAi phenotypes. This is not a big issue as complex regulatory effects are possible, but nevertheless the authors should mention phenotypes and expression levels of PVR.</p><p>4) The discussion mainly focuses on specific hypotheses, i.e. a functional transcription complex consisting of bip1 and RpL8, suggested by third party interaction reports, and Nup98 targeting the promoter of PVR and other genes by third party ChIP data. While these are possible mechanisms, the authors should also discuss alternative and more traditional scenarios, such as general roles of nuclear pore components and consequences of their loss-of-function, and a role of ribosomal subunits either as part of the general translation machinery or in the translation regulation of specific transcripts (see e.g. review by Xue and Barna Nat Rev Mol Cell Biol 2012). Do the authors know whether other nup genes or other ribosomal proteins also have an effect on PVR expression in their system? In this context it would be very interesting to find out whether the identified PVR upstream regulators, and maybe related genes of the same functional families, may have been found by other approaches such as genome-wide RNAi screening for PVR modifiers. Any information from complementary systems would greatly synergize with the observations made by the authors.</p><p>5) Some phrasing of the manuscript is misleading and should be corrected.</p><p>5a) In the introduction, the LG is presented as 'the larval hematopoietic organ'. This may mislead to think that all hemocyte tools have high LG specificity and no crosstalk with another blood cell population may take place. To provide a more balanced view, the population of larval self-renewing tissue hemocytes/macrophages needs to be mentioned, and appropriate literature should be cited (Markus PNAS 2009; Makhijani et al. Development 2011; Makhijani and Brückner Fly 2012; Gold and Brückner Exp Hematology 2014).</p><p>5b) Related to this, when using Hml-GAL4 or another driver active in both the LG and tissue hemocyte populations, the authors should stay away from overstating specificity such as “overexpression of bip1 in Nup98 RNAi lymph glands...” (main text discussing <xref ref-type="fig" rid="fig6">Figure 6</xref>).</p><p>5c) Hml-GAL4 does not only express in 'differentiating' hemocytes, but mainly in fully 'differentiated' hemocytes of both the tissue hemocyte population and differentiated LG hemocytes. The term 'differentiating' should therefore be used with caution.</p><p>5d) 'Circulating hemocytes' should be changed to 'tissue hemocytes', which are also known as 'larval hemocytes'. Recent research has shown that this independent population of hemocytes is largely resident in inductive tissue microenvironments (Hematopoietic Pockets), and under unchallenged conditions enters circulation only gradually and rather late in larval life (see references above).</p></body></sub-article><sub-article article-type="reply" id="SA2"><front-stub><article-id pub-id-type="doi">10.7554/eLife.03626.021</article-id><title-group><article-title>Author response</article-title></title-group></front-stub><body><p><italic>Technically this paper is excellent, the screen is beautifully designed and the HHLT-gal4 line is likely to be a great resource to the research community. However, there is little follow up work to the screen. Whilst the screen must have been very labour intensive the paper does feel more like a 'technique paper' rather than a ground-breaking research paper. Therefore, the storyline of the manuscript would benefit from re-shaping and focusing, placing less emphasis on the early steps of screening and more on PVR regulation. The authors may even want the title to reflect this. Some experimental clarifications and amendments of the text would be required to make the manuscript suitable for publication</italic>.</p><p>Thank you very much to the reviewers for the positive comments and constructive criticism. As suggested by the reviewers, the text of the manuscript has now been reshaped to focus less on the early screening steps. In response to specific reviewer concerns about the description of the screen (below), we have removed the proof-of-principle details of the screen from <xref ref-type="fig" rid="fig2">Figure 2</xref> (to a child figure and legend for those that would like to see this part of the screen) and have streamlined the text accordingly. Additionally, we have changed the title to reflect the central role of PVR and its regulation.</p><p><italic>Substantive concerns</italic>:</p><p><italic>1) The authors should address whether regulation of PVR by Nup98, bip1 and RpS8 is cell autonomous, or related to non-autonomous events, which could even originate from the population of tissue hemocytes/macrophages (by the authors called 'circulating hemocytes', see comment below), which express the same drivers as differentiating/-ed hemocytes of the LG (ie Hml-GAL4 and Pxn-GAL4). This overlap in expression is in fact a major caveat of most LG papers, an issue that receives increasing attention now that the biology of larval tissue hemocytes, the active hemocyte population of the larva, has been revealed in more detail (see below). The LG field would greatly benefit from an expression system for differentiated LG-hemocytes only (e.g. by inducible elimination of GAL4 in the earlier differentiated population of tissue hemocytes). However, in the absence of such a tool the authors need to retreat to other methods to demonstrate whether or not the presented regulation of PVR is cell autonomous. These methods should comprise</italic>:</p><p><italic>1a) Use of flipout-GAL4 to generate in vivo RNAi clones of the genes in question, assessing PVR expression and the GAL4 expressing clones (e.g. by UAS-GFP) in the LG. Side-by-side comparison of kd and control tissue should allow to evaluate cell autonomous effects</italic>.</p><p><italic>1b) Use of Hand-GAL4 with UAS-GAL4, or HHLT without Hml-GAL4, as driver to determine the effects of RNAi of the genes in question on LG PVR expression, in the absence of expression in tissue hemocytes</italic>.</p><p>For 1a and 1b, the major issue is whether the observed PVR phenotype in the lymph gland is due to autonomous RNAi effects or due to non-autonomous, secondary effects of RNAi in circulating/sessile, non-lymph gland hemocytes. We have addressed both 1a and 1b by expressing RNAi using Hand-gal4, which is expressed in the lymph gland but not in circulating or sessile hemocytes. Small flipout-gal4 clones (UAS-FLP Ay-gal4 UAS-GFP) constitutively driving RNAi were made at low temperature (room temp and 25 °C), while larger clones covering the most or all of the lymph gland were generated at 29 °C. In both cases, PVR expression was assessed in RNAi backgrounds. In both cases, the evidence indicates a lymph gland-autonomous role in the regulation of PVR. Clones expressing RNAi to PVR show a robust knockdown of PVR protein expression, whereas mock clones do not. RNAi knockdown of bip1 and Nup98 strongly and moderately reduces PVR protein expression, respectively (<xref ref-type="fig" rid="fig6s2">Figure 6–figure supplement 2</xref>). By contrast, clones expressing RNAi for RpS8 show little if any effect on PVR expression (not shown). Why RpS8 RNAi fails to give a PVR phenotype is unclear, however it is likely that the regulation of PVR by these factors is complex and that the generation of small clones is not equivalent to expressing RNAi throughout a defined cell population (e.g., the Hml-gal4-expressing cells of the CZ). More importantly, to address whether bip1, Nup98, or RpS8 knockdown in the circulating/sessile cell population can affect lymph gland PVR expression, we used srpHemo-gal4 (Bruckner et al., 2004) as an RNAi driver, which expresses in a large fraction of third-instar circulating cells but with essentially no expression in lymph glands. RNAi for bip1, Nup98, and RpS8 were used and for each PVR expression in the lymph gland was unaffected (<xref ref-type="fig" rid="fig6s2">Figure 6–figure supplement 2</xref>).</p><p><italic>2) The authors show all genes of the RNAi screen that led to increased Pxn expression. Do all or just some of these kds affect PVR expression? Was this a selection criterion by the authors to go forward with specific genes</italic>?</p><p>PVR expression was not a primary screening criterion, so the RNAi lines were not assessed as a group. However, we do know that not all RNAi knockdowns cause a reduction or loss of PVR expression, because CTPsynthase RNAi exhibits normal PVR levels (see Results and Discussion section). PVR expression was only assessed after three RNAi lines (targeting bip1, Nup98, and CTPsynthase) were identified that cause a loss of progenitors when expressed with Hml-gal4 (<xref ref-type="fig" rid="fig4">Figure 4</xref>, similar to RNAi of PVR/loss of equilibrium signaling; Mondal et al., 2011) in differentiating/mature cells, and after the bip1RNAi phenotype could be suppressed by expressing PVR (<xref ref-type="fig" rid="fig6">Figure 6</xref>).</p><p><italic>3) What is the overexpression phenotype of Nup98, bip1 and RpS8? From the phenotypes listed in</italic> <xref ref-type="supplementary-material" rid="SD1-data"><italic>Supplementary File 1</italic></xref> <italic>it looks as if they may not be fully complementary to the RNAi phenotypes. This is not a big issue as complex regulatory effects are possible, but nevertheless the authors should mention phenotypes and expression levels of PVR</italic>.</p><p>Overexpression of bip1, Nup98, or RpS8 has no significant effect on PVR levels (<xref ref-type="fig" rid="fig6s5">Figure 6–figure supplement 5</xref>).</p><p><italic>4) The discussion mainly focuses on specific hypotheses, i.e. a functional transcription complex consisting of bip1 and RpL8, suggested by third party interaction reports, and Nup98 targeting the promoter of PVR and other genes by third party ChIP data. While these are possible mechanisms, the authors should also discuss alternative and more traditional scenarios, such as general roles of nuclear pore components and consequences of their loss-of-function, and a role of ribosomal subunits either as part of the general translation machinery or in the translation regulation of specific transcripts (see e.g. review by Xue and Barna Nat Rev Mol Cell Biol 2012)</italic>.</p><p>We agree with the reviewers’ comments and have now revised and expanded our discussion of this issue in the text and have included the appropriate references.</p><p>Do the authors know whether other nup genes or other ribosomal proteins also have an effect on PVR expression in their system? In this context it would be very interesting to find out whether the identified PVR upstream regulators, and maybe related genes of the same functional families, may have been found by other approaches such as genome-wide RNAi screening for PVR modifiers. Any information from complementary systems would greatly synergize with the observations made by the authors.</p><p>This is an important question. We found that unlike Nup98, loss of the nucleoporin Sec13 does not cause a loss of Pvr (<xref ref-type="fig" rid="fig6s3">Figure 6–figure supplement 3</xref>). In response to the reviewer’s question, we have now used RNAi to knock down 3 additional nucleoporins (Nup154, 214, and 358; using Hml-gal4) and found that they do not affect PVR levels (<xref ref-type="fig" rid="fig6s5">Figure 6–figure supplement 5</xref>). Thus, the function of Nup98 is specific. We have not analyzed additional ribosomal protein genes since there are a large number of genes that belong to this family.</p><p>During our analysis we looked at the literature for possible connections between nucleoporins or ribosomal protein and PVR, but did not find any reports connecting them beyond what we cite in the manuscript already. We are not aware of any previous screen to identify regulators of PVR; however, as mentioned in the manuscript, a genome-wide RNAi screen in S2 cells that identified PVR as a major regulator of proliferation also identified RpS8 (Sims et al., 2009), although no attempt was made by the authors to link it to PVR regulation.</p><p><italic>5) Some phrasing of the manuscript is misleading and should be corrected</italic>.</p><p><italic>5a) In the introduction, the LG is presented as 'the larval hematopoietic organ'. This may mislead to think that all hemocyte tools have high LG specificity and no crosstalk with another blood cell population may take place. To provide a more balanced view, the population of larval self-renewing tissue hemocytes/macrophages needs to be mentioned, and appropriate literature should be cited (Markus PNAS 2009; Makhijani et al. Development 2011; Makhijani and Brückner Fly 2012; Gold and Brückner Exp Hematology 2014)</italic>.</p><p>We have added text to state that multiple hematopoietic populations are evident in Drosophila. We have also cited the appropriate literature, however we do want to emphasize that this manuscript deals only with the hematopoietic compartments found within the lymph gland.</p><p><italic>5b) Related to this, when using Hml-GAL4 or another driver active in both the LG and tissue hemocyte populations, the authors should stay away from overstating specificity such as “overexpression of bip1 in Nup98 RNAi lymph glands...” (main text discussing</italic> <xref ref-type="fig" rid="fig6"><italic>Figure 6</italic></xref><italic>)</italic>.</p><p>To say that Hml-gal4 is used to overexpress UAS constructs in the lymph gland is not incorrect; however we have clarified that Hml-gal4 expression is not limited to the lymph gland (see 5c below) at its first description in the manuscript. For the sake of simplicity, and, given our experimental results (including those conducted here to demonstrate autonomy), we have kept the original language elsewhere since Hml-gal4-expressing cells in the lymph gland are the relevant cells.</p><p><italic>5c) Hml-GAL4 does not only express in 'differentiating' hemocytes, but mainly in fully 'differentiated' hemocytes of both the tissue hemocyte population and differentiated LG hemocytes. The term 'differentiating' should therefore be used with caution</italic>.</p><p>This point is correct. Hml-gal4 activity is a definitive marker for the earliest stages of differentiation in the lymph gland (<xref ref-type="fig" rid="fig6">Figure 6</xref> and Jung et al., 2005) but it is also expressed by more mature cells in the lymph gland (such as those expressing the P1 marker) and those cells in circulation and sessile populations. We have rechecked the manuscript and have clarified this point where appropriate.</p><p><italic>5d) 'Circulating hemocytes' should be changed to 'tissue hemocytes', which are also known as 'larval hemocytes'. Recent research has shown that this independent population of hemocytes is largely resident in inductive tissue microenvironments (Hematopoietic Pockets), and under unchallenged conditions enters circulation only gradually and rather late in larval life (see references above)</italic>.</p><p>In consideration of established and newer contributions to the literature (Rizki, 1978; Zetterval et al., 2004; Markus et al., 2009; Honti et al., 2010; Makhijani et al., 2011, among others) we have chosen to use “circulating and sessile cells” in the manuscript to refer to non-lymph gland larval hemocytes. These terms are well established and widely used, including the view that circulating/sessile cells and lymph gland cells represent distinct hemocyte populations arising from their different developmental origins. Perhaps there is need for new nomenclature that combines the sessile and circulating pool. However neither “tissue hemocyte” nor “larval hemocyte” is appropriate for them as the hemocytes within the lymph gland are contained within a larval tissue and released into the hemocoel both upon infection and at the end of the larval period.</p></body></sub-article></article> |