Sumoylation is tumor-suppressive and confers proliferative quiescence to hematopoietic progenitors in Drosophila melanogaster larvae

Summary How cell-intrinsic regulation of the cell cycle and the extrinsic influence of the niche converge to provide proliferative quiescence, safeguard tissue integrity, and provide avenues to stop stem cells from giving rise to tumors is a major challenge in gene therapy and tissue engineering. We explore this question in sumoylation-deficient mutants of Drosophila. In wild type third instar larval lymph glands, a group of hematopoietic stem/progenitor cells acquires quiescence; a multicellular niche supports their undifferentiated state. However, how proliferative quiescence is instilled in this population is not understood. We show that Ubc9 protein is nuclear in this population. Loss of the SUMO-activating E1 enzyme, Aos1/Uba2, the conjugating E2 enzyme, Ubc9, or the E3 SUMO ligase, PIAS, results in a failure of progenitors to quiesce; progenitors become hyperplastic, misdifferentiate, and develop into microtumors that eventually detach from the dorsal vessel. Significantly, dysplasia and lethality of Ubc9 mutants are rescued when Ubc9wt is provided specifically in the progenitor populations, but not when it is provided in the niche or in the differentiated cortex. While normal progenitors express high levels of the Drosophila cyclin-dependent kinase inhibitor p21 homolog, Dacapo, the corresponding overgrown mutant population exhibits a marked reduction in Dacapo. Forced expression of either Dacapo or human p21 in progenitors shrinks this population. The selective expression of either protein in mutant progenitor cells, but not in other hematopoietic populations, limits overgrowth, blocks tumorogenesis, and restores organ integrity. We discuss an essential and complex role for sumoylation in preserving the hematopoietic progenitor states for stress response and in the context of normal development of the fly

Sumoylation is tumor-suppressive and confers proliferative quiescence to hematopoietic progenitors in Drosophila melanogaster larvae

Summary How cell-intrinsic regulation of the cell cycle and the extrinsic influence of the niche converge to provide proliferative quiescence, safeguard tissue integrity, and provide avenues to stop stem cells from giving rise to tumors is a major challenge in gene therapy and tissue engineering. We explore this question in sumoylation-deficient mutants of Drosophila. In wild type third instar larval lymph glands, a group of hematopoietic stem/progenitor cells acquires quiescence; a multicellular niche supports their undifferentiated state. However, how proliferative quiescence is instilled in this population is not understood. We show that Ubc9 protein is nuclear in this population. Loss of the SUMO-activating E1 enzyme, Aos1/Uba2, the conjugating E2 enzyme, Ubc9, or the E3 SUMO ligase, PIAS, results in a failure of progenitors to quiesce; progenitors become hyperplastic, misdifferentiate, and develop into microtumors that eventually detach from the dorsal vessel. Significantly, dysplasia and lethality of Ubc9 mutants are rescued when Ubc9wt is provided specifically in the progenitor populations, but not when it is provided in the niche or in the differentiated cortex. While normal progenitors express high levels of the Drosophila cyclin-dependent kinase inhibitor p21 homolog, Dacapo, the corresponding overgrown mutant population exhibits a marked reduction in Dacapo. Forced expression of either Dacapo or human p21 in progenitors shrinks this population. The selective expression of either protein in mutant progenitor cells, but not in other hematopoietic populations, limits overgrowth, blocks tumorogenesis, and restores organ integrity. We discuss an essential and complex role for sumoylation in preserving the hematopoietic progenitor states for stress response and in the context of normal development of the fly

Role for Sumoylation in Systemic Inflammation and Immune Homeostasis in Drosophila Larvae

To counter systemic risk of infection by parasitic wasps, Drosophila larvae activate humoral immunity in the fat body and mount a robust cellular response resulting in encapsulation of the wasp egg. Innate immune reactions are tightly regulated and are resolved within hours. To understand the mechanisms underlying activation and resolution of the egg encapsulation response and examine if failure of the latter develops into systemic inflammatory disease, we correlated parasitic wasp-induced changes in the Drosophila larva with systemic chronic conditions in sumoylation-deficient mutants. We have previously reported that loss of either Cactus, the Drosophila (IκB) protein or Ubc9, the SUMO-conjugating enzyme, leads to constitutive activation of the humoral and cellular pathways, hematopoietic overproliferation and tumorogenesis. Here we report that parasite infection simultaneously activates NF-κB-dependent transcription of Spätzle processing enzyme (SPE) and cactus. Endogenous Spätzle protein (the Toll ligand) is expressed in immune cells and excessive SPE or Spätzle is pro-inflammatory. Consistent with this function, loss of Spz suppresses Ubc9− defects. In contrast to the pro-inflammatory roles of SPE and Spätzle, Cactus and Ubc9 exert an anti-inflammatory effect. We show that Ubc9 maintains steady state levels of Cactus protein. In a series of immuno-genetic experiments, we demonstrate the existence of a robust bidirectional interaction between blood cells and the fat body and propose that wasp infection activates Toll signaling in both compartments via extracellular activation of Spätzle. Within each organ, the IκB/Ubc9-dependent inhibitory feedback resolves immune signaling and restores homeostasis. The loss of this feedback leads to chronic inflammation. Our studies not only provide an integrated framework for understanding the molecular basis of the evolutionary arms race between insect hosts and their parasites, but also offer insights into developing novel strategies for medical and agricultural pest control

Polydnaviral Ankyrin Proteins Aid Parasitic Wasp Survival by Coordinate and Selective Inhibition of Hematopoietic and Immune NF-kappa B Signaling in Insect Hosts

Polydnaviruses are mutualists of their parasitoid wasps and express genes in immune cells of their Lepidopteran hosts. Polydnaviral genomes carry multiple copies of viral ankyrins or vankyrins. Vankyrin proteins are homologous to IκB proteins, but lack sequences for regulated degradation. We tested if Ichnoviral Vankyrins differentially impede Toll-NF-κB-dependent hematopoietic and immune signaling in a heterologous in vivo Drosophila, system. We first show that hematopoiesis and the cellular encapsulation response against parasitoid wasps are tightly-linked via NF-κB signaling. The niche, which neighbors the larval hematopoietic progenitors, responds to parasite infection. Drosophila NF-κB proteins are expressed in the niche, and non cell-autonomously influence fate choice in basal and parasite-activated hematopoiesis. These effects are blocked by the Vankyrin I2-vank-3, but not by P-vank-1, as is the expression of a NF-κB target transgene. I2-vank-3 and P-vank-1 differentially obstruct cellular and humoral inflammation. Additionally, their maternal expression weakens ventral embryonic patterning. We propose that selective perturbation of NF-κB-IκB interactions in natural hosts of parasitic wasps negatively impacts the outcome of hematopoietic and immune signaling and this immune deficit contributes to parasite survival and species success in nature

Vankyrins and GFP-Dorsal localization in blood cells.

<p><b>A–A″</b>. Uninfected <i>Cg>GFP-Dorsal</i> shows speckled distribution (arrowhead). <b>B–B″</b>. Infected <i>Cg>GFP-Dorsal</i>. Infection relocalizes some GFP-Dorsal to nucleus (arrow). <b>C–C″</b>. Uninfected <i>Srp>GFP, I3</i>. Both I3 (C, arrowhead) and GFP-Dorsal (C′) are mostly cytoplasmic in blood cells of uninfected animals and do not show much co-localization. <b>D–D″</b> Infected <i>Srp>GFP, I3</i>. In infected animals, I3 is strongly nuclear (D, arrow) but most of the GFP-Dorsal colocalizes with the remaining cytoplasmic I3 (D″ – yellow). <b>E–E″</b>. P1 is also mostly cytoplasmic in cells from uninfected animals (E, E″, arrowhead). <b>F–F″</b>. Upon infection, both GFP-Dorsal and P1 colocalize in the nucleus (F″, white). All images are presented at the same magnification and scale bars represent 20 µm. <i>Cg>GFP-dl, I3</i> cells from uninfected animals show cytoplasmic localization of both proteins similar to <i>Srp>GFP-dl I3</i> (data not shown).</p

Effect of Vankyrins on immune gene expression.

<p><b>A</b>. Both Vankyrins strongly reduce the expression of <i>Drosomycin</i> in manually-poked larvae (t = 4.1, df = 6, p = 0.006 for <i>Cg>GFP, P1</i> and t = 4, df = 5, p = 0.009 for <i>Cg>GFP, I3</i>). <b>B–B′</b>. Levels of ProPO transcripts are increased in <i>Cg>GFP, Toll^10b</i> animals compared to controls (t = 14.2, df = 5, p<0.001 for ProPO59 and t = 11.2, df = 3, p = 0.001 for <i>ProPO54</i>). <b>B</b>. <i>ProPO59</i> expression level is reduced to control levels by expression of I3 (<i>Cg>GFP, Toll^10b, I3</i>) (t = 3.4, df = 6, p = 0.01 compared to <i>Cg>GFP, Toll^10b</i> and t = 0.4, df = 5, p = 0.7 compared to <i>Cg>GFP</i>). Expression of P1 (<i>Cg>GFP, Toll^10b, P1</i>) also decreases (t = 6.9, df = 6, p<0.001 compared to <i>Cg>GFP, Toll^10b</i> and t = 1.96, df = 5, p = 0.1 compared to controls) the expression of <i>ProPO59</i> to control levels. <b>B′</b>. The levels of <i>ProPO54</i> transcripts are only affected by I3 expression (<i>Cg>GFP, Toll^10b, I3</i>) (t = 5.1, df = 5, p = 0.003) but not by P1 expression (<i>Cg>GFP, Toll^10b, P1</i>) (t = 0.5, df = 5, p = 0.6). Stars indicate conditions that are different from controls (* for 0.05</p

Vankyrin localization and their effects on niche properties and on crystal cell development.

<p><b>A–A′</b>. Niche without any Vankyrin expression stained with anti-FLAG antibody. <b>B–B′</b>. Antp-Gal4 simultaneously drives the expression of GFP and P1 from their respective UAS sequences. P1 localizes to nuclei and in the cytoplasm (yellow) where it is relatively uniformly distributed. <b>C–C′</b>. Antp-Gal4 simultaneously drives the expression of GFP and I3 from their respective UAS sequences. I3 is expressed mostly in the cytoplasm; it colocalizes with <i>Antp>GFP</i> expression in some cells (yellow), and its distribution is speckled. <b>D–F1</b>. Effect of Vankyrins on crystal cells development. <b>D–F</b>. Crystal cells in the anterior lobes of the lymph gland. Crystal cell number is not significantly different from the control when either Vankyrin is expressed. <b>D1–F1</b>. However, their number is increased in the three posterior larval segments when I3 is expressed (for quantification, see Panel I.) <b>G</b>. Expression of I3 reduces the number of <i>Antp>GFP</i>-positive cells in the niche compared to controls (W = 438, p = 0.03 while P1 does not (W = 229, p = 0.09). N = 16 animals for control; N = 10 animals for I3 and P1-expressing glands. Cell counts represent an average per niche. <b>H</b>. Expression of I3 decreases the intensity (measurement done on more than 15 cells – see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003580#s4" target="_blank">Methods</a>) of <i>Antp>GFP</i> signal. Pixel intensity is reduced in <i>Antp>GFP, I3</i> (t = 3.3, df = 30.9, p = 0.002) but not in <i>Antp>GFP, P1</i> (t = 1.9, df = 47.6, p = 0.07) compared to controls (N = 15 animals for <i>Antp>GFP</i> controls, N = 9 animals for I3 expressing animals and N = 10 for P1 expressing animals). <b>I</b>. Quantification of crystal cell changes in panels E1 and F1, relative to D1. Crystal cell number in the three posterior larval segments is increased by <i>Antp>I3</i> (t = −3.7, df = 65, p<0.001; control, N = 33; I3 expressing animals, N = 34) but not with <i>Antp>P1</i> (t = −1.7, df = 59.197, p = 0.09 - P1 expressing animals, N = 29). Scale bars represent 20 µm. Bars indicate standard deviation. Stars indicate conditions that are different from controls (* for 0.05</p

Polydnaviral Ankyrin Proteins Aid Parasitic Wasp Survival by Coordinate and Selective Inhibition of Hematopoietic and Immune NF-kappa B Signaling in Insect Hosts

<div><p>Polydnaviruses are mutualists of their parasitoid wasps and express genes in immune cells of their Lepidopteran hosts. Polydnaviral genomes carry multiple copies of viral <i>ankyrins</i> or <i>vankyrins</i>. Vankyrin proteins are homologous to IκB proteins, but lack sequences for regulated degradation. We tested if Ichnoviral Vankyrins differentially impede Toll-NF-κB-dependent hematopoietic and immune signaling in a heterologous <i>in vivo Drosophila</i>, system. We first show that hematopoiesis and the cellular encapsulation response against parasitoid wasps are tightly-linked via NF-κB signaling. The niche, which neighbors the larval hematopoietic progenitors, responds to parasite infection. <i>Drosophila</i> NF-κB proteins are expressed in the niche, and non cell-autonomously influence fate choice in basal and parasite-activated hematopoiesis. These effects are blocked by the Vankyrin I²-vank-3, but not by P-vank-1, as is the expression of a NF-κB target transgene. I²-vank-3 and P-vank-1 differentially obstruct cellular and humoral inflammation. Additionally, their maternal expression weakens ventral embryonic patterning. We propose that selective perturbation of NF-κB-IκB interactions in natural hosts of parasitic wasps negatively impacts the outcome of hematopoietic and immune signaling and this immune deficit contributes to parasite survival and species success in nature.</p></div

Effect of wasp infection on <i>D4-lacZ</i> expression.

<p><b>A–A′</b>. Uninfected <i>Antp>GFP</i> animals. <i>D4-lacZ</i> is expressed mostly in the niche (A′, arrowhead) where it colocalizes with <i>Antp>GFP</i> (A, yellow). <b>B–B′</b>. <i>L. boulardi</i> infection triggers four-fold increase in the expression of <i>D4-lacZ</i> (23.06±4.22 versus 89.65±41.4; t = −6.37, df = 14.7, p<0.001; N = 4 glands for uninfected and 8 for infected). <i>D4-lacZ</i> is also activated in cells of the anterior lobes. <b>C–D′</b>. Niche expression of <i>D4-lacZ</i> is abolished in glands lacking a functional <i>dl</i> gene (D, D′), but is observed in controls (<b>C–C′</b>). The reporter is expressed ectopically in the mutant but not control lobe cortex.</p