Two way controls of apoptotic regulators consign DmArgonaute-1 a better clasp on it

Argonaute family proteins are well conserved among all organisms. Its role in mitotic cell cycle progression and apoptotic cell elimination is poorly understood. Earlier we have established the contribution of Ago-1 in cell cycle control related to G2/M cyclin in Drosophila. Here we have extended our study in understanding the relationship of Ago-1 in regulating apoptosis during Drosophila development. Apoptosis play a critical role in controlling organ shape and size during development of multi cellular organism. Multifarious regulatory pathways control apoptosis during development among which highly conserved JNK (c-Jun N-terminal kinase) pathway play a crucial role. Here we have over expressed Ago-1 in Drosophila eye and brain by employing UAS (upstream activation sequence)-GAL4 system under the expression of eye and brain specific driver. Over expression of Ago-1 resulted in reduced number of ommatidia in the eye and produced smaller size brain in adult and larval Drosophila. A drastic reversal of the phenotype towards normal was observed upon introduction of a single copy of the dominant negative mutation of basket (bsk, Drosophila homolog of JNK) indicating an active and physical involvement of the bsk with Ago-1 in inducing developmental apoptotic process. Further study showed that Ago-1 stimulates phosphorylation of JNK through transforming growth factor-β activated kinase 1- hemipterous (Tak1-hep) axis of JNK pathway. JNK phosphorylation results in up regulation of pro-apoptotic genes head involution defective (hid), grim & reaper (rpr) and induces activation of Drosophila caspases (cysteinyl aspartate proteinases);DRONC (Death regulator Nedd2-like caspase), ICE (alternatively Drice, Death related ICE-like caspase) and DCP1 (Death caspase-1) by inhibiting apoptotic inhibitor protein DIAP1 (Death-associated inhibitor of apoptosis 1). Further, Ago-1 also inhibits miR-14 expression to trigger apoptosis. Our findings propose that Ago-1 acts as a key regulator in controlling cell death, tumor regression and stress response in metazoan providing a constructive bridge between RNAi machinery and cell death.

Introduction Apoptosis is collection of multiple perturbations of the cellular architecture and play an important role in tissue homeostasis and pattern formation of an organism. This process allows apoptotic cells to be dismantled in a manner that minimizes damage and disruption of adjacent cells [1]. These events are orchestrated by a series of cysteine proteases family members referred as caspases. Developmental apoptotic death response of cells is mediated by JNK (c-Jun N-terminal kinase), a member of the MAPK (mitogen-activated protein kinase) family [2]. JNK activity is under tight regulation of reversible phosphorylation process stimulated by protein kinases wherein JNK-specific phosphatases are responsible for dephosphorylation and inactivation of JNK. Activation of JNK pathway releases cytochrome-c from mitochondria and activates caspase cascade to induce apoptosis [3]. Abolition of JNK signaling causes a series of defects in response to stress induced apoptotic response during developmental processes, enabling the tumor cells to bypass apoptotic cell death and achieve metastatic activity [4]. JNK1 and JNK2 are essential for suppressing the oncogenic changes and tumorigenesis. Loss of function of JNK3 is associated with development of brain tumors [5]. An upstream JNK kinase, encoded by mkk4 gene has been identified as suppressor of tumorigenesis and metastasis [6]. In Drosophila, TNF ortholog egr (eiger) induces cell death through dTAK1 (transforming growth factor-β activated kinase 1, Fly JNK kinase kinase), hep (hemipterous, the Drosophila JNK kinase) and Drosophila JNK bsk (basket) [7][8][9][10]. Other bsk-mediated signaling pathways that modulate developmental apoptosis have not been elucidated fully.
AGO (Argonaute) family proteins are highly conserved across both animal and plant kingdom. In mammals both AGO and PIWI (P-element Induced WImpy testis) includes four members in their subfamily [11]. Structurally AGO proteins contain four domains-an amino terminal domain, a highly conserved mid domain, PAZ (Piwi/Argonaute/Zwille) domain & PIWI domain. AGO proteins are direct binding partners of existing small RNA. Both AGO1 and 2 are well studied, because of its role in miRNA (microRNA) biogenesis and as a major representative of miRNA effector complex (RISC complex). By interacting with AGO proteins miRNA guide the RISC (RNA-induced silencing complex) to its mRNA (messenger RNA) targets to induce translational repression or degradation. As a post-transcriptional regulator for a large number of vital genes, miRNAs play crucial role in controlling a wide range of cellular processes and their mis expression lead to development of various diseases [12]. miRNAs can act as a tumor inducer or inhibitor by altering the expression of tumor suppressor genes and oncogenes. Altered expression of miRNA biogenesis associated proteins such as DROSHA and DICER is frequently observed during tumorigenesis and cancer progression [13]. Drosophila Ago-1 is broadly expressed in the embryo; central nervous system and imaginal discs of larvae. Maternal and zygotic Ago-1 mutant embryos show different developmental defects. Earlier studies have also shown that Drosophila AGO1 is localized to the cytoplasmic region of the cell [14]. In our earlier study we have shown the regulatory role of Ago-1 in association with the G2/M cyclin in controlling cell cycle during development. We also, identified the involvement of two embryonic miRs (miRNAs)-miR-981 and miR-317 as spatiotemporal regulator of cyclin B [15]. However, till date, little is known about the role of Ago on apoptotic process.
In this study, we report a connection between AGO1 and the JNK signaling pathway that opens a crosstalk with different signaling pathways and gene silencing mechanism. We demonstrate that subtle changes in the expression of Ago-1 lead to chronic effect on gene regulation that promotes different diseases including cancer. Our results demonstrate that AGO1 activates JNK signaling pathway through JNK phosphorylation in vivo and mediate Ago-1 induced apoptosis in developing fly organs and thereby control organ size and structure. Apart from that, Ago-1 also negatively regulates the expression of apoptotic inhibitor micro RNA, miR-14. Regulation of JNK pathway in one hand and miR-14 expression in other hand allows Ago-1 to take a better control with more perfection on developmental apoptosis. This understanding of the new role of Ago-1 unravels the pathway dictating the principle and cause for various diseases including cancer and neuro degenerative diseases.

Fly stocks and genetic crosses
All flies were obtained from Bloomington Drosophila Stock Centre and maintained at standard condition, at 24˚±1˚C temperature and reared on yeast-agar standard medium. All crosses were carried out at 18˚C (except GMR GAL4 and CyO marker related crosses). SEM (Scanning Electron Microscope) images of newly immerged flies were taken after maintaining them at 24˚C for 3 days. All details of genetic crosses are mentioned in S2 File.

Microarray: Labeling and hybridization
The miRNA labeling was performed using miRNA Complete Labeling and Hyb Kit (Agilent Technologies, Part Number: 5190-0456). About 200ng of total RNA was hybridized on the miRNA 8x60K Arrays. The hybridization was carried out at 55˚C for 20 hours followed by washes using Gene Expression Wash Buffer1 (Agilent Technologies, Part Number 5188-5325) at room temperature for 5 minutes and Gene Expression Wash Buffer 2 (Agilent Technologies, Part Number 5188-5326) at 37˚C for 5 minutes. Slide was scanned on a G2600D scanner (Agilent Technologies).

Chromatin immunoprecipitation (ChIP)
Chromatin immunoprecipitation (ChIP) assay was performed after isolating chromatin from 10,000 UAS Ago-1/GMR GAL4 adult fly heads followed by crosslinking DNA-protein with formaldehyde. Target amplification was carried out using region-specific primer sets (Table-B in S1 File).

Immuno Precipitation
Immuno Precipitation (IP) was carried out using RNA pol II antibody (detailed method has mentioned in supplemental section). The eluted proteins were run on SDS-PAGE along with 5% input sample and western blot was carried out using RNA pol II (Abcam# ab817) and AGO1 (Abcam# ab5070)antibody. during cell cycle is extremely essential in maintaining a check for proper development. We therefore generated Drosophila lines that ectopically overexpress Ago-1 gene in different organs of Drosophila. UAS Ago-1 flies were crossed with GMR-GAL4 driver carrying flies, thereby ectopically expressing Ago-1 in the developing eye. Ectopic expression of Ago-1 at both transcriptional and translational level was confirmed by performing quantitative real time PCR and western blot analysis. Results showed an increase in the intensity of expression of both Ago-1 transcripts and proteins (S1 Fig and Fig 1IA and 1IB). Interestingly, ectopic overexpression of Ago-1 in the adult eyes lead to phenotypic changes that resembled apoptotic cell death compared to the control flies that contained endogenous copy of Ago-1. Observation of adult eyes under SEM (Scanning Electron Microscopy) showed reduced and deformed omatidia in Ago-1 overexpressed flies compared to control flies (Fig 1IIA'-1IIC'). Number of fully formed omatidia from each individual eye was counted and average number from twenty individual flies presented as a graphical format (S2 Fig). To understand whether apoptotic cell death is responsible, we performed AO (Acridine Orange) staining in eye imaginal discs of third instar larvae arising from stocks that contained overexpressed Ago-1 (UAS Ago-1/GMR GAL4) as well as control stocks (+/GMR GAL4) and Ago-1 loss of function allele mutant fly stock (P{hs-Ago-1}; Ago-1 k08121 / Ago-1 k08121 ). Due to absence of Ago-1 functionality, homozygous mutants arising from this stock do not complete their embryonic development [14,16,17], resulting in embryonic lethality. However, these homozygous mutants are capable of surviving to adulthood with the insertion of transgene P{hs-Ago-1} along with homozygous mutant genotype and upon receiving daily heat shock treatment consequently that supplies the amount of AGO1 needed for completion of its embryonic development. To revive a minimum number of survivals till the larval stage for this study we performed a series of heat shock experiments by subjecting embryos to heat shock for different time periods. Proteins were isolated from emerging larvae for performing western blot analysis. Result showed that a minimum expression of AGO1 was seen when the embryos were subjected to one hour heat shock per day to achieve the basal level of AGO1 for completion of embryonic development and reach to larval stage. Acridine Orange staining (AO) in the eye imaginal discs of the third instar larvae showed a large number of positively stained apoptotic cells in the discs arising from larvae in which Ago-1 is overexpressed (UAS Ago-1/GMR GAL4) compared to discs isolated from wild type stock as well as discs isolated from stock in which Ago-1 is down regulated (Fig 1IIA1-1IIC2) confirming that apoptosis was indeed induced due to over expression of Ago-1 (S2 Fig). For further confirmation of apoptosis, we co-expressed p35 protein, a well known inhibitor of apoptosis in Ago-1 over expressed eye disc. A clear reduction of Ago-1 induced apoptosis was observed clearly indicating that over expression of Ago-1 indeed lead to induction of apoptosis in developing fly eye (Fig 1IID1, 1IID2 and 1IID'). To check whether the observed apoptosis upon over expression of Ago-1 is clearly due to its effect and not related to any stress response, we over expressed Green Fluorescence Protein (GFP) in developing fly eye. Interestingly, no significant apoptotic cell death was observed indicating clearly that the observed apoptotic cell death in the fly eye was due to the over expression of Ago-1 (Fig 1IIE1, 1IIE2 and 1IIE'). We also performed a similar experiment to see the generalized effect of inducing apoptosis by over expression of Ago-1 using ptc GAL4 that shows its expression in the developing wing discs as well as in the adult wings. AO staining was performed in the wing discs of 3 rd instar larvae. Similar positively stained cells were seen in the discs arising from Ago-1 overexpressed stocks compared to controls. Moreover, over expression of Ago-1 produced by ptc GAL4 also resulted in the loss of anterior cross vein and led to narrowing of the space between longitudinal vein3 and longitudinal vein4 in adult fly wings demonstrating induction of apoptosis (S3 Fig). Over expression of Ago-1 stimulates Drosophila JNK pathway. Argonaute family proteins play an important role in RNAi pathway. As overexpression of Ago-1 resulted in increased cell death, we wanted to see its effect on the players of cell death inducers NF-κB and JNK pathway proteins. JNK cascade comprises of several members among whom JNKs are MAPK superfamily signal-transduction protein involved in apoptotic signaling [2,18]. Also MAPKKK superfamily protein TAK1 known as D-tak, DmTak1 in Drosophila is an important component of JNK signaling pathway and play a critical role in JNK induced cell death in the development of fly [19]. Flies were generated that carried a copy of Tak1 (Tak1 ds ) along with over expressed Ago-1(Tak1 ds ;UAS Ago-1/GMR GAL4). Number of omatidia from adult eyes of these heterozygous mutant flies was compared to the control and results indicated a significant level of recovery towards normal ( Fig 1IIIA, 1IIIA', 1IIIB and 1IIIB'). Quantitative real time PCR analysis of Tak1 expression under same genetic background showed an increase in transcript level (S1 Fig) in the eye of adult flies confirming that indeed Tak1 interacted with Ago-1 in recovering the eye phenotype. Drosophila homolog of mammalian JNK also known as DJNK is encoded by bsk [20] and acts downstream of Tak1 [19]. bsk mutants lead to defects in dorsal closure [21]. It is a stress activated protein kinase involved in upstream activator functions required for morphogenetic movements in embryos and in maintaining cell and tissue polarity in adults [20]. JNK-mediated apoptosis triggers apoptosis in intrinsic tumor suppression process, wherein activation of JNK functions as a cell editor by removing epithelial aberrant cells [22]. As bsk is a major component of the JNK signaling cascade in Drosophila, we generated flies carrying one copy of over expressed Ago-1 along with one copy of mutant bsk gene (UAS Ago-1/GMR GAL4; UAS bsk ds /+). We generated two heterozygous stocks; one carrying single copy of Ago-1 over expression under the GMR driven GAL4 driver along with one copy of P element inserted RNAi construct against bsk (UAS Ago-1/GMR GAL4; UAS bsk ds /+), and another one containing same single copy of Ago-1 over expression along with a dominant negative form of bsk allele (UAS bsk DN ; UAS Ago-1/GMR GAL4). Adult fly eyes visualized under SEM showed an significant increase in the number of omatidia with the introduction of one copy of RNAi mutant of bsk (bsk ds ) ( Fig 1IIID and 1IIID') in comparison to control ( Fig  1IIIA and 1IIIA'), whereas introduction of one copy of the dominant negative form of bsk (bsk DN ) resulted in complete rescue of eye phenotype ( Fig 1IIIC and 1IIIC').
Hid, a member of the Drosophila pro-apoptotic protein family is a known inducer of apoptosis [23][24][25]. Drosophila HID is a member of the RHG (REAPER, HID and GRIM) group of proteins that act by binding to the IAPs (Inhibitor of Apoptosis Proteins). We combined a loss of function mutation of hid (hid H109 ) with the stock having an over expression of Ago-1 (UAS Ago-1/GMR GAL4; hid H109 /+). Introduction of a single copy of loss of function mutation of hid was capable of reverting the eye phenotype towards normal with a strong increase in the number of ommatidia ( Fig 1IIIF and 1IIIF').
Ago-1 induces apoptosis by regulating JNK phosphorylation. JNKs are members of MAP kinase superfamily that regulate cell proliferation, differentiation and apoptosis [26,27]. JNK functions downstream of the TNF homologue egr (eiger) and its receptor Wengen via a conserved kinase signaling cascade that includes fly JNK kinase kinase Tak1, JNK kinase, hep and Drosophila Jun kinase bsk. Following the same route, downstream to JNK, the core apoptotic machinery that includes Hid and caspases get activated to cause an apoptotic response.
To inspect whether the JNK phosphorylation is induced by Ago-1, we produced transient expression of Drosophila Ago-1 in adult eyes using eye specific GMR-GAL4 driver and checked the expression of phospho-JNK (active form of JNK) by performing western blot analysis with proteins isolated from the adult eyes of the overexpressed Ago-1 flies. Significant increase in the expression of phospho JNK was seen in flies carrying overexpressed Ago-1 compared to control ( Fig 2I) providing strong evidence that phospho JNK is a physiological target of Ago-1. Furthermore, to see the association of JNK with AGO1 we performed immunofluorescent staining (co-localization) using anti AGO1 and anti phospho-JNK antibodies in the eye imaginal discs isolated from late third instar larvae arising from flies carrying over expressed Drosophila Ago-1 (UAS Ago-1; P{w[+mW.hs] = GawB}32B). These larvae were collected from fly stock produced by crossing UAS Ago-1 lines to imaginal disc specific GAL4 [P{w[+mW.hs] = GawB}32B] lines that produced a specific expression only in the 3 rd instar larval imaginal disc. A clear activation of JNK in the area of AGO1 overexpression in the eye-antennal imaginal discs of the larvae, confirmed that AGO1 is an activator of JNK ( Fig 2IIA-2IIE'"). To check the phosphorylation status of JNK in developing eye we further performed immuno staining studies using p-JNK antibodies in the eye disc of UAS Ago-1/GMR GAL4 larvae. Simultaneously the same eye discs were double stained with anti AGO1 antibody to visualize and confirm the level of co-expression of AGO1 and p-JNK protein in eye discs isolated from Ago-1 over expressed lines ( Fig 2IIIA-2IIIE"). Staining performed with the same antibodies in eye discs isolated from larvae produced by crossing UAS Ago-1 with GMR GAL4 to induce over expression line and Ago-1 mutant (UAS Ago-1/GMR GAL4 and P{hs-Ago-1}; Ago-1 k08121 /Ago-1 k08121 ) along with GMR GAL4 stocks, served as control. There was a significant increase in the expression and association of p-JNK antibody in eye discs from Ago-1 overexpressed lines compared to control and mutant lines. As expected, AGO1 anti body showed an increased level of expression of AGO1 in the discs isolated from Ago-1 over expressed lines (Fig 3 and S4  Fig). Taken together the results clearly confirmed that phosphorylation of JNK is indeed induced by Ago-1.
Hep activation and JNK phosphorylation by Ago-1 is related to Tak1 expression. Our next question was to see whether Ago-1 dependent apoptosis is directly regulated via activation of JNK or is dependent on its upstream players that in turn activate JNK. We generated flies that carry hep mutation (hep 1 ), homologue of mammalian MKK7 (also known as JNKK) that TAK1 an upstream player of JNKK (hep), activates a number of intracellular kinases (p38 MAPK, JNK and IKK) by inducing or inhibiting apoptotic cell death in a number of cells depending upon cell types [28,29]. Role of TAK1 in the regulation of autophagic cell death has been reported [30]. To explore whether there is any direct involvement of TAK1 we performed both Immuno Precipitation (IP) and ChIP assay using chromatin extracted from Ago-1 over expressed head & eye. PCR amplification was performed using three different primer sets, designed against predicted (by Neural Network Promoter Prediction tool, http:// www.fruitfly.org/seq_tools/promoter.html) promoter sequences. Results demonstrated a significant protein-DNA interaction at upstream sequence of Tak1 (Fig 5A). Interestingly, IP study with nuclear extract (extracted from Ago-1 over expressed head along with eye) showed   We next introduced puc a known Drosophila MAPK phosphatase, that inhibits apoptosis by preventing JNK phosphorylation in Ago-1 over expressed flies (UAS Ago-1/GMR GAL4; UAS puc). Relatively less Ago-1 induced apoptosis under puc over expressed background in fly eye was observed (Fig 4IA and 4IA') indicating the involvement of puc in Ago-1 induced JNK signaling. Collectively, Ago-1 activates JNK by the phosphorylation process, which is dependent on JNK kinase hep and JNK phosphatase puc and controls apoptotic process in Drosophila melanogaster.
expression induced small eye phenotype not changed significantly in over expression of those genes along with Ago-1 heteroallelic mutation background (Ago-1 72 /Ago-1 45 ).
https://doi.org/10.1371/journal.pone.0190548.g004 Chromatin Immuno Precipitation (ChIP) using AGO1 antibody, 5% crosslinked fragmented DNA was used as an input and the sample from "no antibody beads (IgG+beads)" was used as negative control, (B) Blot image showing the interaction between nuclear AGO1 and RNA Pol II. Immuno Precipitation (IP) of nuclear extract, extracted from Ago-1 over expressed eye was carried out using RNA Pol II antibody, followed by detected with RNA Pol II and AGO1 antibody (upper two). Same experiment was performed using nuclear extract, extracted from Ago-1 over expressed as well as Ago-1 mutant fly eye (lower two) (C) Model figure showing interaction of AGO1 with transcription machinery at the 5' upstream region of Tak1 gene.
Pro apoptotic gene hid, grim and reaper are downstream to Ago-1. Although apoptotic defects found in Ago-1 mutants do not show exactly similar phenotypes as seen in other Drosophila deficient mutants of major pro apoptotic genes, such as hid, grim and rpr but activation of apoptotic initiator also induced extensive activation of caspases. Therefore we speculated probability of pro apoptotic genes involvement in Ago-1 induced apoptosis. We therefore introduced one mutant copy of hid gene (hid H109 ) into Ago-1 over expressed flies and observed the eye phenotype under SEM and also checked the expression of RNA and proteins. Interestingly, reducing the dose of hid (hid H109 ) prevented Ago-1 over expressed induced apoptosis significantly (Fig 1IIIF & 1IIIF'). However, the trans-heterozygotic mutation of Ago-1 (Ago 72 / Ago 45 ) produced minimum effect on hid induced apoptosis. Further, this trans-heterozygotic mutation was not capable of reversing the hid, grim and rpr induced apoptosis significantly (Fig 4IIA-4IIF'), implicating that Ago-1 exists upstream of these pro apoptotic genes. A similar pattern of expression was observed at both the transcript (Fig 6I) and protein level. Ago-1 over expression activates an IAP-sensitive cell death pathway. In Drosophila, initiation of apoptosis is mainly controlled by hid, reaper and grim who share a short region of homology at the N terminus which interacts with DIAP-1 to regulate its function negatively [31]. thread (th) gene encodes Drosophila Inhibitory apoptotic protein, DIAP1 which functions similar to the inhibitor of apoptosis protein. Evolutionarily conserved DIAP1 is homologous to baculovirus IAPs and also share common domain with mammalian IAPs and prevents apoptosis [32]. To investigate the role of DIAP-1 in inducing cell death under Ago-1 over expression, we inserted UAS carrying th allele (UAS th) in flies having over expression of Ago-1 (UAS Ago-1/GMR GAL4; UAS th). Over expression of th rescued several ommatidia that were lost as a result of Ago-1 over expression induced cell death in developing eye (Fig 6IIIC, 6IIIC' and S8  Fig). Low level of expression of th transcript was observed in Ago-1 over expressed background (Fig 6I), whereas with the introduction of UAS th in the same Ago-1 over expressed flies recovered the expression of th transcript as well as the expression level of DIAP protein.
Ago-1 induced apoptosis is caspase dependent. CARD-containing Drosophila caspase DRONC or Nedd2-like caspase are protein factors that regulate programmed cell death during Drosophila development. DRONC which is under regulation of DIAP1 [33] act as ICE interacting caspases to activate downstream apoptotic processes [34,35]. To understand the role of Dronc in Ago-1 induced cell death, trans-heterozygous Dronc mutant flies were generated (Dronc I24 /Dronc I29 ) along with Ago-1 over expression {(UAS Ago-1/GMR GAL4; Dronc I24 / Dronc I29 ; homozygous double mutants (Dronc I24 / Dronc I24 or Dronc I29 /Dronc I29 ) are lethal}. Introduction of trans-heterozygous Dronc mutant produced almost near to full recovery of rough eye phenotype (Fig 6IIID and 6IIID') indicating a clear involvement of Dronc in Ago-1 over expression induced cell death. To observe the involvement of Ark (Dark), a homolog of mammalian Apaf-1 and C. elegans CED-4 cell death protein, we introduced RNAi induced silenced Dark gene in Ago-1 over expressed flies (UAS Ago-1/GMR GAL4; Ark ds ). Heterozygous flies carrying silenced Ark partly recovered the eye phenotype towards normal (Fig 6IIIE  and 6IIIE'), whereas, introduction of a single copy of RNAi silenced Drice (ice), a Drosophila effector caspase in Ago-1 over expressed flies failed to recover Ago-1 induced apoptotic cell death phenotype in fly eye (Fig 6IIIF and 6IIIF'). An increased level of both ice RNA and ICE protein in Ago-1 over expressed line indicated clear involvement of ice in Ago-1 induced apoptosis. But, failure to suppress the Ago-1 induced apoptosis by ice mutant suggests the involvement of other effector caspase. Expression studies of Dronc transcript ( Fig 6I) and protein ( Fig  6IIA and 6IIB) showed an increased level of expression in Ago-1 over expressed flies. Immuno On other hand, eye discs isolated from larvae carrying heterozygous mutation of CARD domain containing Drosophila caspase, Dronc (Dronc I24 /Dronc I29 ) along with Ago-1 over expression showed reduced level of expression indicating it's incapability of inducing apoptosis significantly. These observations clearly demonstrate the involvement of caspases in Ago-1 induced apoptosis. To identify involvement of other caspases in Ago-1 induced apoptosis, we studied the expression of effector caspase Drice both at transcript and protein level. Although Drice level was slightly increased in Ago-1 over expressed flies both at transcript (Fig 6I) and protein level (Fig 6II), the genetic data showed that single copy RNAi silenced Drice with Ago-1 over expression was unable to recover Ago-1 induced apoptotic cell death (Fig 6IIIF and 6IIIF') indicating the involvement of other effector caspases in this process. We therefore checked the transcript and protein expression of Dcp-1, another effector caspase known to be actively involved in apoptotic process [36,37]. Ago-1 over expressed flies showed more expression of Dcp-1 mRNA ( Fig 6I) along with high cleavage level of Dcp-1 protein (Fig 6II). To confirm the elevated level of cleaved Dcp-1 under Ago-1 overexpression, we performed immunostaining experiments in wing discs of larvae arising from ptc GAL4 driven Ago-1 over expressed stocks. These wing discs expressed high level of cleaved Dcp-1 at AGO1 over expressing areas of the disc (S10 Fig). Interestingly the adult flies arising from this stock also showed loss of one anterior cross vein between 3 rd and 4 th longitudinal vein (S3 Fig). Similar result of elevated accumulation of active Dcp-1 was also observed in the eye imaginal discs (driven by GMR GAL4) isolated from 3 rd instar larvae arising from Ago-1 over expressed lines. A clear decrease in the accumulation of the active Dcp-1 in mutant line (P{hs-Ago-1}; Ago-1 k08121 / Ago-1 k08121 ), confirmed that elevated expression was indeed due to over expression of Ago-1 (Fig 3E-3Q'  and S4 Fig). Interestingly, increased level of JNK phosphorylation was observed at the same area of eye imaginal discs of Ago-1 over expression and Dcp-1 activation (Fig 3M-3M', 3O-3O', 3Q-3Q'and graphics S, T and U). Together results suggest active involvement of Ago-1 in JNK phosphorylation and association of JNK phosphorylation with the activation of Dcp-1 in the Ago-1 over expression induced apoptotic process. Moreover, immune staining results showed few DCP-1 positive cells in eye discs arising from UAS Ago-1/GMR GAL4; Dronc I24 / Dronc I29 larvae (Fig 7). These observations confirm that Ago-1 induced apoptosis is caspase dependent.
eiger is not essential for Ago-1 induced apoptosis. egr (eiger), TNF (Tumor necrosis factor) ligand of Drosophila and TRAF1 (TNF-receptor-associated factor1) have been reported as a critical inducer of JNK-dependent apoptotic process [7,38,39]. To uncover the role of Ago-1 in egr-induced apoptosis, flies co-expressing inverted repeat induced silencing of egr with Ago-1 over expression (UAS Ago-1/ GMR GAL4; UAS egr-IR) were generated. No phenotypic changes were observed upon introduction of UAS egr-IR in the eye phenotypes (Panel A and B in S11 Fig other component of egr signaling pathway (Panel E in S11 Fig). Collectively, results demonstrate that there is no direct involvement of egr in Ago-1 induced apoptosis and Ago-1 is not essential for egr/TRAF1 induced apoptosis.
Endogenous Ago-1 is necessary for developmental apoptotic process. As apoptosis is a crucial process throughout development in maintaining organ size and shape [40], our next question was to see whether endogenous Ago-1 is required to regulate the developmental apoptosis and thereby control organ size in Drosophila. We therefore generated an RNAi mutant Ago-1 stock that suppresses Ago-1 expression in larval brain by crossing P{TRiP.HM04006}attP2 (BSC#31700) flies with larval brain and fat body specific GAL4-P{GawB}c754 (BSC#6984) flies. Larval developing brain, eye and wing imaginal discs from growing 3 rd instar larvae arising from the Ago-1 over expressed stock, mutant stock as well as from control stocks were isolated and AO staining was performed. Larval brain from RNAi silenced Ago-1 mutants showed brain hyperplasia compared to wild type controls; similar to a brain condition as seen in the Apaf-1 (Apoptotic protease activating factor-1) null mutants which is a key regulator of apoptosis. Similar bigger sized brain was also seen in Drosophila 3 rd instar larvae, carrying mutation in its initiator caspase, Dronc (S12 Fig). In depth view of third instar larval brain lobes along with ventral ganglion from wild-type Ago-1 over expressed and Ago-1 RNAi mutants showed significantly enlarged brain hemispheres and ventral ganglion in Ago-1 mutant larvae, compared to those in wild type whereas in Ago-1 over expressed line the size was significantly smaller (Fig 8IA-8IC'  and 8IE). Moreover, results from AO staining and flow cytometry showed cells from brain hemispheres and ventral ganglion of Ago-1 over expressed line with more apoptotic population as compared to cells from mutant brain (Fig 8IA'-8IC' and 8IE and S13 Fig). Ectopic expression of Ago-1 was capable of suppressing the Ago-1 deficient big brain phenotype to normal size suggesting that the brain hyperplasia produced by Ago-1 silencing was indeed due to lack of proper Ago-1 activity. Larval brain, dissected from P{GawB}c754 (served as control), P{GawB}c754; P{TRiP. HM04006}attP2 (Ago-1 RNAi mutant) and P{GawB}c754; UAS Ago-1 (Ago-1 over expressed) larve were immunostained with human cleaved caspase-3 antibody (which indicates Dronc activity in fly [41]) and fly specific effector caspase, Dcp-1 antibody. High level of both initiator (Dronc) and effector (Dcp-1) caspase activation was observed in Ago-1 over expressed developing brain (Fig 8IIA-8IIC" and 8IIIA-8IIIC") providing a clear evidence that endogenous Ago-1 is highly essential for controlling organ size of the CNS by inducing developmental apoptosis at the time of organogenesis.
Ago-1 regulates miR-14 expression to sensitize cells for apoptosis and control organ development. As miRs are one of the crucial regulators of various cellular processes [42][43][44][45][46], our interest was to see whether any change in their expression occurs under Ago-1 overexpression. miRNA microarray analysis was carried out using total RNA extracted from control (GMR-GAL4) and Ago-1 overexpressed (UAS Ago-1/GMR GAL4) and Ago-1 mutant (Ago-1 72 / Ago-1 45 ) adult eye. miRNAs which has significant difference in their expression are presented in the form of heat map after statistical analysis of the array data (P < 0.05). Several miRNAs were upregulated and several showed down regulation. Interestingly a huge down regulation of mir-14 which is related to apoptosis was observed (Fig 9I and S3 File). miR-14 is expressed throughout development of Drosophila. It has already been established as a suppressor of cell death, induced by hid, grim and rpr [47] Further, miR-14 has also been reported as a negative regulator of Dronc-dependent cell death and a regulator of Drice expression either directly or indirectly [47]. Several gene transcripts including Drice, Dcp-1 and grim possesses potential target sites for miR-14 binding (S14 Fig). Drice is essential for cell death events and the apical caspase, Dronc acts as an activator of it, that inflate rpr, hid and grim induced cell death [48,49]. For further validation of the involvement of miR-14 with Ago-1 regulated apoptotic cell death quantitative PCR analysis was also performed. A little up regulation of miR-14 expression in Ago-1 mutant line and high down regulation in Ago-1 over expressed group was noticed (Fig 9II). Over expression of Ago-1 produced flies with deformed organ development. Since miR-14 expression was extremely down regulated in Ago-1 overexpressed lines, we wanted to see whether it plays any role in reverting the organ shape and size to normal when it is over expressed under Ago-1 over expressed condition. We therefore introduced over  14) successfully recovered Ago-1 over expression induced eye phenotype to normal (Fig 9III and S15 Fig). Further, flies carrying over expressed Ago-1 under sd-GAL4 (scalloped GAL4) resulted in flies having wings with reduced width compared to normal were also recovered with the introduction of over expressed miR 14 (sd-GAL4; UAS Ago-1; UAS miR-14) when driven with scalloped GAL4 in the wings of adult Drosophila. This ectopic expression of miR-14 by sd-GAL4 successfully rescued Ago-1 over expression induced wing phenotype (S16 Fig) to nearly normal phenotype. These observations positively suggest that Ago-1 regulates miR-14 expression to take a better hold in the control of developmental apoptosis in fruit fly.

Discussion
Programmed cell death is very crucial to maintain proper shape and size of organs by removing excess number of cells. It is also important to eliminate defective and abnormal cells from living system to maintain proper development and activity of organism. JNK pathway is one of the conserved pathways that play a lead role to maintain this activity. Activation of kinase cascade by any intrinsic or extrinsic signal turns on the pathway switch to induce apoptosis.
Ago-1, a well conserved protein factor and documented for its role in RISC and RITC complex serve as a RNAi effector complex in RNAi pathway. Apart from this, Ago-1 plays a role in maintaining proper cellular activity. Here, for the first time we report the role of Ago-1 in controlling apoptosis during organ development. A cross talk between core RNAi component Ago-1 and apoptosis via JNK circuit elucidate an in depth mechanism for RNAi role in apoptosis and progammed cell death in Drosophila. In continuity of earlier findings these results provide an explicit explanation of how RNAi factor Ago-1 controls caspase-dependent apoptosis in developing organs to maintain the proper size and shape of organs. Over expression of Ago-1 produce less number of ommatidia in adult eye (Fig 1IIC') and suppression of this phenotype (Fig 1IIIC and 1IIIC') by dominant negative form of bsk, a Drosophila JNK (bsk DN ) indicates a clear involvement of JNK pathway with developmental apoptotic process. Our study revealed an interesting finding that Ago-1 promotes cell death and controls JNK phosphorylation by regulating Drosophila JNK kinase hep through its upstream activator, Tak1 to trigger the pathway in one hand and down regulate onco-miR miR-14 expression in another hand. In flies, the pro apoptotic genes reaper, grim and hid promotes caspase activation by performing their antagonistic activity against DIAP (Drosophila inhibitor of apoptosis proteins). Our findings confirm the involvement of these pro apoptotic genes with Ago-1 induced apoptosis; as Ago-1 triggered cell death was suppressed by the co-expression of mutant alleles of these pro-apoptotic genes (hid H109 ; Fig 1IIIE and 1IIIE') and co-expression of elevated Diap1 as well (Fig 6IIIC  and 6IIIC'). Further, significant recovery of eye phenotype upon introduction of trans-heterozygous mutant of dronc (dronc i24 /dronc i29 ) in Ago-1 over expressed background (Fig 6IIID  and 6IIID') confirms Ago-1 induced cell death is caspase dependent. Moreover, a clear overexpression of cleaved DRONC, ICE and Dcp-1 level at Ago-1 over expressed condition further supports the involvement of caspases with Ago-1 induced cell death. Our findings clearly demonstrate the need of endogenous Ago-1 expression for proper regulation of apoptosis during organ development. Though Ago-1 is not a strong inducer of apoptosis like hid, grim, reaper or Dronc; it plays a crucial role in fine tuning of apoptosis during development. Ago-1 mutants accumulate cells due to low level of apoptosis (Fig 8 and S13 Fig) during development. Ago-1 is required for physiological elimination of cells during development to maintain the cell death process in a well controlled manner.
Development and organogenesis are vital processes wherein nature maintains various control mechanism to eliminate developmental errors. Micro RNAs are also known to play key role in controlling apoptosis. Here, we've identified miR-14 as one of the key regulators of Ago-1 mediated developmental apoptotic process. In Drosophila, miR-14 is expressed throughout development and has already been demonstrated as a negative cell death regulator at different stages of insect development by targeting pro apoptotic genes and caspases [47,50]. Therefore, miR-14 expression equips Ago-1 to apprehend developmental apoptosis in more disciplined manner. This study sheds a new beam of light on the crosstalk and involvement of Ago-1 in the induction of cell death through JNK signaling in one hand and through regulating miR-14 in other hand (Fig 10) during organ development. As JNK is implicated in apoptotic process induced by variety of death stimuli including oncogenic transformation and defective transformation in a number of cell types therefore it is clear that JNK signaling plays a pivotal role on tumor formation and metastasis also. Deregulation of this well orchestrated process result in abnormal organ development or progression of oncogenesis. Based on the present study in Drosophila it is resolved that, Ago-1 acts as a regulator of cell death, which is crucial for proper development of organs and might act as a tumour suppressor by inhibiting onco-miR-14 expression as well as by promoting JNK-dependent apoptosis. DmAgo-1 controls apoptosis

Conclusions
In this study we have uncovered Ago-1 as a critical regulator of JNK mediated cell death process in Drosophila melanogaster. Our genetic and biochemical analysis established Ago-1 as a potent cell death modulator downstream of egr. Moreover, we have shown that Ago-1 is independent of egr to activate JNK and induce JNK dependent apoptotic process through hep and puc. Furthermore, Ago-1 acts as a regulator of puc through Tak1 to control JNK phosphorylation and an inducer of JNK dependent cell death. It also regulates miR-14 expression to control the cell death with better control. Evolutionarily conserved nature of Ago-1 gives a hope that similar process might have a major contribution in modulating JNK-mediated cell death in other animals also.