The Endo-SiRNA Pathway is Essential for Robust Development of The

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The Endo-siRNA Pathway Is Essential for Robust Development of the Drosophila Embryo Elena M. Lucchetta1*, Richard W. Carthew2, Rustem F. Ismagilov1 1 Department of Chemistry and Institute for Biophysical Dynamics, The University of Chicago, Chicago, Illinois, United States of America, 2 Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, Illinois, United States of America Abstract Background: Robustness to natural temperature fluctuations is criti
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  The Endo-siRNA Pathway Is Essential for RobustDevelopment of the Drosophila  Embryo Elena M. Lucchetta 1 * , Richard W. Carthew 2 , Rustem F. Ismagilov 1 1 Department of Chemistry and Institute for Biophysical Dynamics, The University of Chicago, Chicago, Illinois, United States of America, 2 Department of Biochemistry,Molecular Biology, and Cell Biology, Northwestern University, Evanston, Illinois, United States of America Abstract Background:  Robustness to natural temperature fluctuations is critical to proper development in embryos and to cellularfunctions in adult organisms. However, mechanisms and pathways which govern temperature compensation remain largelyunknown beyond circadian rhythms. Pathways which ensure robustness against temperature fluctuations may appear to benonessential under favorable, uniform environmental conditions used in conventional laboratory experiments where thereis little variation for which to compensate. The endo-siRNA pathway, which produces small double-stranded RNAs in Drosophila , appears to be nonessential for robust development of the embryo under ambient uniform temperature and tobe necessary only for viral defense. Embryos lacking a functional endo-siRNA pathway develop into phenotypically normaladults. However, we hypothesized that small RNAs may regulate the embryo’s response to temperature, as aribonucleoprotein complex has been previously shown to mediate mammalian cell response to heat shock. Principal Findings:  Here, we show that the genes DICER-2 and ARGONAUTE2, which code for integral protein componentsof the endo-siRNA pathway, are essential for robust development and temperature compensation in the Drosophila embryowhen exposed to temperature perturbations. The regulatory functions of DICER-2 and ARGONAUTE2 were uncovered byusing microfluidics to expose developing Drosophila embryos to a temperature step, in which each half of the embryodevelops at a different temperature through developmental cycle 14. Under this temperature perturbation, dicer-2 or argonaute2 embryos displayed abnormal segmentation. The abnormalities in segmentation are presumably due to theinability of the embryo to compensate for temperature-induced differences in rate of development and to coordinatedevelopmental timing in the anterior and posterior halves. A deregulation of the length of nuclear division cycles 10–14 isalso observed in dicer-2 embryos at high temperatures. Conclusions:  Results presented herein uncover a novel function of the endo-siRNA pathway in temperature compensationand cell cycle regulation, and we hypothesize that the endo-siRNA pathway may regulate the degradation of maternal cellcycle regulators. Endo-siRNAs may have a more general role buffering against environmental perturbations in otherorganisms. Citation: Lucchetta EM, Carthew RW, Ismagilov RF (2009) The Endo-siRNA Pathway Is Essential for Robust Development of the Drosophila Embryo. PLoSONE 4(10): e7576. doi:10.1371/journal.pone.0007576 Editor: Patrick Callaerts, Katholieke Universiteit Leuven, Belgium Received February 27, 2009; Accepted August 3, 2009; Published October 23, 2009 Copyright: ß 2009 Lucchetta et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the srcinal author and source are credited. Funding: This work was supported by the NIH (1R01GM077331 to R.F.I. and 5R01GM068743-05 to R.W.C.), by the Yen Postdoctoral Fellowship (E.M.L) and wasperformed in part at the Chicago MRSEC microfluidic facility funded by the NSF. The funders had no role in study design, data collection and analysis, decision topublish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist.* E-mail: elucchetta@gmail.com Introduction ‘‘There is no greater biological question of greater moment thanthe means by which the individual cell activities are coordinated,and the organic unity of the body maintained’’ [1]. In thedeveloping embryo, the coordination of individual cell activities, orrobustness, under environmental fluctuations such as temperatureis critical in forming the adult organism. Although robustness totemperature fluctuations is critical to proper development, thepathways and molecular mechanisms that govern robustness havelong remained perplexing [2–4]. Mechanisms of robustness againsttemperature fluctuations have been largely unexplored outside of circadian rhythms [5,6].In developing embryos such as those of  Drosophila melanogaster  , ahierarchy of precise protein expression guides the growth of theembryo in space and time at a large range of temperatures. Thesepatterns separate the embryo into well-defined segments that formthe adult body plan. In Drosophila  , the earliest segmented patternsof protein expression appear in the embryo while it is a syncytium,i.e. concurrent with rapid nuclear divisions in a commoncytoplasm. Later, the syncitial blastoderm cellularizes intothousands of cells, each cell inheriting the pattern of geneexpression expressed by its nucleus during cellularization. Thelack of cellular compartmentalization in developing  Drosophila  embryos makes the maintenance of robustness and coordination of cellular processes at a large range of temperatures during earlydevelopment particularly puzzling.The pathways involved in maintaining robustness to tempera-ture fluctuations may appear to be nonessential under favorable,uniform environmental conditions such as those used in PLoS ONE | www.plosone.org 1 October 2009 | Volume 4 | Issue 10 | e7576  conventional laboratory experiments. However, the importance of these pathways can be revealed under controlled environmentalperturbations [7–9]. We previously developed a microfluidicplatform that can be used to precisely control and perturb theenvironment around a syncitial Drosophila  embryo by maintaining each half of the embryo at a specific and different temperature[10,11], i.e. creating a temperature step across the body of theembryo. Early embryonic events, such as nuclear divisions, occurasynchronously in such temperature perturbations, and earlysegmentation events are also asynchronous. Remarkably, both thedensity of nuclei across the embryo [12] and segmented geneexpression [10] become stabilized by cycle 14, and embryosdevelop into normal animals. Although this paradigm reveals thedegree of robustness to temperature inherent in embryonicsegmentation and development, the pathways responsible fortemperature compensation have remained unclear.It has been speculated that microRNAs (miRNAs) might beused to build robustness within biological networks [13]. There areexamples of individual miRNAs that impart robustness todevelopmental decisions in various organisms [14–17]. Moreover,bioinformatic analysis suggests that 45–70% of mammalianmiRNAs act in feedback and feedforward network motifs [18],which build robustness into networks. miRNAs are related to smallinterfering RNAs (siRNAs) in many respects. Both types of RNAsare produced from double-stranded RNA (dsRNA) precursors bypost-transcriptional processing by the Dicer (Dcr) ribonuclease[19]. Both types of RNAs are associated with Argonaute (Ago)proteins to form ribonucleoprotein effector complexes. Both typesof RNAs repress gene expression by base-pairing with messengerRNAs of complementary sequence. Despite these similarities,there are several differences between miRNAs and siRNAs. WhilemiRNAs are processed from short imperfect hairpin RNAs, thedsRNA precursors for siRNAs are generally perfect duplexes orextended hairpins. Animal miRNAs typically repress mRNAtranslation by imperfect pairing to messages, while siRNAs causemRNA degradation by a perfect pairing mechanism. Moreover, insome species different Dcr and Ago proteins are specific forparticular miRNAs or siRNAs. For example, Drosophila  Dicer-1(Dcr-1) processes miRNAs, whereas Dicer-2 (Dcr-2) processessiRNAs [20] (Figure 1). Ago1 loads miRNAs, whereas Ago2 loadssiRNAs. Thus, the two types of RNAs can be distinguished by anumber of features. Although miRNAs have been implicated in building robustness,they are also important for major developmental events affecting cell differentiation, division, morphogenesis, and apoptosis [21].This broad range of functions for miRNAs might be due to theirsheer numbers (at least in the hundreds) and their broadspecificity. In contrast, siRNAs appear dispensable for majordevelopmental events under uniform laboratory conditions. This ismost simply evident in Drosophila  , where mutations in DCR-2 and AGO2 specifically impair the siRNA pathway but have negligibleeffect on fly development–mutants are viable and fertile [20,22]. Afew weak defects have been described for ago2 embryos but thesedefects do not impair the outcome of embryogenesis [23]. Instead,the Drosophila  siRNA pathway appears to be critical only for innateimmunity against viral infection. Viral RNAs are processed byDcr-2 into siRNAs that repress viral gene expression, and this hostmechanism attenuates virus replication and pathogenicity [24–26].These findings might suggest that siRNAs are derived only fromexogenous RNA sources as a defensive response against foreignagents. However, it is becoming clear that siRNAs are also derivedfrom endogenous RNA sources. These endo-siRNAs were firstdescribed in the nematode Caenorhabditis elegans  , and wereimplicated in silencing expression of transposable elements withinits genome [27,28]. More recently, endo-siRNAs have beendiscovered to be prevalent in Drosophila  at many stages of development and in distinct tissues [29–32]. Genetic experimentsdemonstrate that Dcr-2 and Ago2 are required for activity of  Figure 1. siRNA silencing pathway initiated by Dicer-2 (Dcr-2). Dcr-2 and R2D2 process dsRNAs into small interfering (siRNAs) in anATP-dependent mechanism. The double-stranded siRNA is then assembled in a pre-RISC complex containing Argonaute2 (Ago2) prior to formingthe holo-RISC complex, which contains only the guide siRNA strand and facilitates cleavage of target mRNA.doi:10.1371/journal.pone.0007576.g001Endo-siRNAs and RobustnessPLoS ONE | www.plosone.org 2 October 2009 | Volume 4 | Issue 10 | e7576  endo-siRNAs. As in C. elegans, Drosophila  endo-siRNAs correspond-ing to transposable elements have been found, and they appear torepress these elements. However, many endo-siRNAs have alsobeen found corresponding to various endogenous fly genes,prompting the question as to their functions in Drosophila  .Several lines of evidence prompted us to hypothesize that theendo-siRNA pathway may have a role in generating robustnessduring  Drosophila  embryonic development. Previously, we deter-mined a period of time critical to robust development of embryosexposed to a temperature step, the orientation of which waschanged for a 35 minute window [10]. The critical time identifiedwas from 65 to 100 minutes of development, during which nucleardivision cycles are very rapid and may only suppsort the synthesisof short zygotic transcripts [33]. Additionally, staufen HL  mutantembryos displayed a decrease in robust development [34], whichcould be due to another double-stranded RNA binding function of Staufen apart from bicoid  or oskar  .To test our hypothesis that the endo-siRNA pathway is essentialfor maintaining robustness to temperature fluctuations, we usedmicrofluidics to expose developing  Drosophila  embryos to atemperature step. We uncover a novel function of the endo-siRNA pathway in maintaining robustness to temperaturefluctuations in the developing embryo, and show that the endo-siRNA pathway is essential to development under environmentalperturbations. This result could shed light on the role of endo-siRNA silencing in robust development among other organisms. Inaddition, this work demonstrates the usefulness of controlledenvironmental perturbations to uncover pathways essential forgenerating robustness. Results Segmentation is Normal in dcr-2 and ago2 MutantEmbryos in Uniform Temperature Environments Core components of the endo-siRNA pathway are producedand are potentially active in the Drosophila  germline [35].Moreover, these components are maternally contributed to theearly zygotic embryo [20,22]. Therefore, to perform ourexperiments studying early embryogenesis, we generated mutantembryos that are missing both maternal and zygotic expression of endo-siRNA pathway components. This strategy was complicatedby the fact that homozygous null dcr-2 adults show a considerablyenhanced susceptibility to viral infection [24,25]. We wanted toensure that any effects on embryogenesis that we observed werenot due to stresses imparted through viral infection, either to themother or to the embryo. To obviate this possibility, we preparednull dcr-2 homozygous embryos from heterozygous wild type (WT)mothers by using the FLP/FRT system to generate homozygousmutant germ cells within these mothers [20,36]. Such heterozy-gous females exhibit normal levels of viral infection compared toWT females, as tested by qPCR assays for a variety of  Drosophila   viruses (Marques, J. and Carthew, R.W., unpublished data). The dcr-2 heterozygous females were crossed to heterozygous mutantmales to generate embryos that were missing both maternal andzygotic contributions of the DCR-2 gene. qPCR assays confirmedthat there was no increase of the viral load in these embryosrelative to controls (Marques, J. and Carthew, R.W., unpublisheddata).We wished to determine if the loss of DCR-2 results in defects inearly segmentation under uniform temperature conditions. Weexamined this by visualizing the expression profile of the Even-skipped (Eve) protein [37], an early marker of embryonicsegmentation (Figure S1). Normal Eve expression is manifestedby seven precisely positioned stripes along the antero-posterior axisof the embryo [37]. We found that dcr-2 R416x  (Figure 2A) and dcr-2 L811fsX  (Figure 2C) embryos displayed normal Eve expressionpatterns at a uniform temperature of 24 u C, as well as at uniformtemperatures of 20 u C and 27 u C (Figure S1A, C–D, F). Thesemutant embryos subsequently developed into properly segmentedlarvae having eight abdominal segments (Figure 2E, H and FigureS2A, C). Additionally, dcr-2 R416x  embryos displayed normal Eveexpression when developing at alternating uniform temperatures,where the temperature was alternated between 20 u C and 27 u Cevery 15 or every 30 minutes (data not shown). Similar results wereobserved in homozygous ago2 embryos and larvae (Figure S3A).We conclude from these experiments that DCR-2 and AGO2appear to be nonessential for early embryonic segmentation underuniform temperature conditions. Segmentation is Abnormal in dcr-2 and ago2 EmbryosExposed to a Temperature Step Next, we wanted to determine if mutation of the DCR-2 generesults in defects in early segmentation when embryos are exposedto the environmental perturbation of a temperature step. We useda microfluidic device that we had previously developed andcharacterized [10,11]. In this device, each half of a live, developing embryo is maintained at a different controlled temperature by twostreams of fluid flowing laminarly side by side. In the experimentsdescribed below, embryos were allowed to develop in atemperature step with their anterior half at 27 u C and theirposterior half at 20 u C for the first 200 minutes of development,until the cellular blastoderm stage. During the course of these 200minutes, an anterior and a posterior organizing center, establishedin the oocyte, normally interact through a cascade of segmentationpatterning genes to form the fourteen segments that comprise thefly. Under these temperature step conditions, the anterior and theposterior organizing centers trigger the cascade under verydifferent conditions. The Bicoid gradient is highly abnormal inWT embryos exposed to a temperature step [12], and nucleidivide faster in the warm half of the embryo, creating a differencein nuclear density between the two halves of the embryo [10,12].However, the segmentation gene network is highly robust andcompensates for any variation that is exerted by the temperaturestep such that WT embryos correct for differences in nucleardensities and become normally segmented [10] during cycle 14.We initially tested that the genetic background was not acomplicating factor, exposing both y w eyFLP; FRT 42D  embryos,and dcr-2 mutant embryos carrying a rescue DCR-2 transgene tothe temperature step. y w eyFLP; FRT 42D  embryos developnormally in a temperature step–embryos expressed all seven Evestripes normaly (Figure 3A) and develop into normally segmentedlarvae after being removed from the temperature step (Figure 3C).Likewise, transgenically rescued embryos compensated for thetemperature step and expressed all seven Eve stripes normally(Figure 3B) and developed into normally segmented larvae afterbeing removed from the temperature step (Figure 3D and FigureS4).We next tested dcr-2 mutant embryos that did not carry therescue DCR-2 transgene. Surprisingly, these embryos were unableto compensate for the perturbation of a temperature step(Figure 2B, D). In dcr-2 R416x  embryos, between three and fiveEve stripes were expressed. In dcr-2 L811fsX  embryos, between fourand six Eve stripes were expressed. These results indicate that the dcr-2 embryos lost robust expression and positioning of Eveexpression domains.Embryos with extra copies of maternal factors have been shownto compensate for early segmentation defects prior to reaching larval stage [38]. To determine if  dcr-2 mutant embryos exposed to Endo-siRNAs and RobustnessPLoS ONE | www.plosone.org 3 October 2009 | Volume 4 | Issue 10 | e7576  a temperature step could nevertheless correct for the earlyabnormalities in segmentation, exoskeletal cuticles were preparedfrom larvae that had developed in a temperature step for the first200 minutes of embryogenesis, and then further developed at auniform temperature of 24 u C. Larval cuticles from both dcr-2 R416x  (Figure 2F) and dcr-2 L811fsX  (Figure 2H) displayed abnormalsegmentation. Four out of five dcr-2 R416x  larvae had between fourand six abdominal segments bearing denticle belts rather than thenormal eight segments (Figure 2F and Figure S2B). All three dcr-2 L811fsX  larvae had only one to four segments (Figure 2H, andFigure S2D). The loss of segments seen in the mutant larvae isconsistent with loss of Eve expression domains seen in dcr-2 embryos. Thus, the segmentation gene network in dcr-2 embryos issensitive to environmental perturbation, resulting in partial loss of segmentation gene expression.The effect of the loss of DCR-2 in the temperature step is quitespecific for segmentation. Segmentation occurs independently of establishment of the body axes [39]. Other gene networks arecritical for forming the anteroposterior and dorsoventral axes,though the anteroposterior determinants profoundly influenceaction of the segmentation network. The dcr-2 embryos exhibitednormal or near-normal body axis organization, as seen by thelarval cuticle patterns. Head and tail structures were formedcorrectly, although tail structures were underdeveloped. More-over, the dcr-2 embryos exhibited normal dorsal and ventralidentities. These observations argue that null dcr-2 mutations donot have a general and catastrophic effect on embryonicpatterning.To confirm that the endo-siRNA pathway is critical for robustsegmentation and development, we determined the effect of thetemperature step on null ago2 414  embryos. ago2 414  embryosdeveloped normally under uniform temperature conditions(Figure 4A, C), but they displayed abnormal Eve expressionwhen developing in a temperature step with the anterior half at27 u C and the posterior half at 20 u C for the first 200 minutes of development (Figure 4B, D). Between four and six Eve stripeswere present, with only one out of four embryos showing allseven Eve stripes. Larval cuticles prepared from ago2 414  embryosthat had developed in a temperature step for the first 200minutes of embryogenesis were also abnormal (Figure 4D andFigure S3B). Most ago2 414  larvae had between four and sixabdominal segments, rather than the normal eight segments(Figure S3B). Most mutant larvae exhibited normal head andtail formation, with underdeveloped tail structures, as well asnormal dorsoventral patterning, consistent with the dcr-2 results.Since the ago2 414  embryos displayed similar patterning defects to dcr-2 embryos, we conclude that the endo-siRNA pathway iscritical for robustness to temperature during development of theembryo and, therefore, that the mechanism responsible formaintaining robustness of early development is dependent uponendogenous siRNAs. Since virus levels in embryos are notaffected by loss of the endo-siRNA pathway under ourexperimental conditions (Marques, J. and Carthew, R.W.,unpublished data), it suggests that the poor robustness of  dcr-2 embryos is not due to their lack of ability to get rid of exogenous viral siRNAs. Figure 2. dcr-2  is required for robust development of segmental patterning in Drosophila  . (A–D) Eve expression. (A,C) dcr-2 R416x  and dcr-2 L811fsX  embryos developed at uniform temperature (24 u C) have normal Eve stripe positions and express all seven Eve stripes. One of four embryos isshown for dcr-2 R416x  and one of three embryos is shown for dcr-2 L811fsX  mutants. (B,D) dcr-2 R416x  and dcr-2 L811fsX  embryos developed in a temperaturestep have an abnormal number of Eve stripes and abnormal stripe positions. One of seven embryos is shown for dcr-2 R416x  and one of four embryos isshown for dcr-2 L811fsX  mutants. (E–H) Larval cuticle preparations. (E,G) dcr-2 R416x  and dcr-2 L811fsX  larvae developed at uniform 24 u C have a normalsegmentation pattern of denticle belts (arrows). One of five larvae is shown for both dcr-2 R416x  and dcr-2 L811fsX  mutants. (F,H) dcr-2 R416x  and dcr-2 L811fsX  larvae that were exposed to the temperature step for the first 200 minutes of development and then allowed to reach larval stage at uniform 24 u Chave missing or abnormally positioned denticle belts (arrows). One of five larvae is shown for dcr-2 R416x  and one of three larvae is shown for dcr-2 L811fsX  mutants.doi:10.1371/journal.pone.0007576.g002Endo-siRNAs and RobustnessPLoS ONE | www.plosone.org 4 October 2009 | Volume 4 | Issue 10 | e7576
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