Jumu is required for circulating hemocyte differentiation and phagocytosis in Drosophila
The regulatory mechanisms of hematopoiesis and cellular immunity show a high degree of similarity between insects and mammals, and Drosophila has become a good model for investigating cellular immune responses. Jumeau (Jumu) is a member of the winged-helix/forkhead (FKH) transcription factor family and is required for Drosophila development. Adult jumu mutant flies show defective hemocyte phagocytosis and a weaker defense capability against pathogen infection. Here, we further investigated the role of jumu in the regulation of larval hemocyte development and phagocytosis.
In vivo phagocytosis assays, immunohistochemistry, Real-time quantitative PCR and immunoblotting were performed to investigate the effect of Jumu on hemocyte phagocytosis. 5-Bromo-2-deoxyUridine (BrdU) labeling, phospho-histone H3 (PH3) and TdT-mediated dUTP Nick-End Labeling (TUNEL) staining were performed to analyze the proliferation and apoptosis of hemocyte; immunohistochemistry and Mosaic analysis with a repressible cell marker (MARCM) clone analysis were performed to investigate the role of Jumu in the activation of Toll pathway.
Jumu indirectly controls hemocyte phagocytosis by regulating the expression of NimC1 and cytoskeleton reorganization. The loss of jumu also causes abnormal proliferation and differentiation in circulating hemocytes. Our results suggest that a severe deficiency of jumu leads to the generation of enlarged multinucleate hemocytes by affecting the normal cell mitosis process and induces numerous lamellocytes by activating the Toll pathway.
Jumu regulates circulating hemocyte differentiation and phagocytosis in Drosophila. Our findings provide new insight into the mechanistic roles of cytoskeleton regulatory proteins in phagocytosis and establish a basis for further analyses of the regulatory mechanism of the mammalian ortholog of Jumu in mammalian innate immunity.
KeywordsDrosophila Hemocytes Phagocytosis Cytoskeleton reorganization Jumu
Janus Kinase/ signal transducer and activator of transcription
c-Jun N-terminal kinase
Mosaic analysis with a repressible cell marker
Phosphate Buffered Saline
TdT-mediated dUTP Nick-End Labeling
Drosophila lacks adaptive immunity and relies on multiple innate immune responses, such as humoral and cellular immunity, to defend against invading pathogens . The humoral response mainly depends on systematic secretion of antimicrobial peptides (AMPs) by the fat body, and AMP synthesis is triggered and regulated by the Toll and Imd pathways [2, 3]. The cellular response is provided by the hemocyte lineage. The Drosophila hemocyte population consists of three broad subtypes of cells: plasmatocytes, crystal cells and lamellocytes [4, 5]. Plasmatocytes represent 90–95% of all mature larval circulating hemocytes and are involved in the phagocytosis of microbial pathogens, encapsulation of parasites and production of AMPs [1, 6, 7]. Crystal cells constitute 5% of larval hemocytes and participate in the melanization process during the encapsulation of invading organisms, wound repair and coagulation [8, 9]. Lamellocytes are rarely observed in healthy larvae but appear after parasitization and are involved in the encapsulation of foreign pathogens that are too large to undergo phagocytosis [7, 9].
In recent years, the mechanisms underlying the humoral innate immune response have been intensively investigated particularly in insects and mammals. However, many of the mechanisms and roles of the cellular immune response have yet to be determined. Phagocytosis is an important defense mechanism in cellular immunity involved in both innate and adaptive immunity and has been conserved throughout evolution. The first step in phagocytosis is microbial recognition. In Drosophila, several proteins have been identified as phagocytic recognition receptors, such as Scavenger receptor class C, type I (Sr-CI) , Down syndrome cell adhesion molecule (Dscam) , peptidoglycan recognition protein LC (PGRP-LC)  and the EGF-like repeat-containing proteins Nimrod C1 (NimC1)  and Eater . These phagocytosis receptors can recognize various pathogens by binding to phagocytosis markers present on the surface of target pathogenic organisms. After binding target cells, the intracellular portion of phagocytosis receptors activates a signaling pathway that leads to rearrangement of the actin cytoskeleton. The plasma membrane of phagocytes then extends and surrounds their targets. Finally, target cells are incorporated into phagocytes as phagosomes and then ingested . The dynamics of the actin network are required for phagocytosis, cell migration and adhesion, and many proteins have been suggested to be involved in the rearrangement of the actin cytoskeleton. Arp2/3 controls filament polymerization and depolymerization through interactions with regulatory proteins . The Rho-family GTPases Rho, Rac and Cdc42 direct the formation of different cellular protrusions, such as filopodia, membrane ruffles or large lamellipodial extensions [17, 18]. Rho1 can promote hemocyte cell spreading and the formation of filopodia [19, 20]. Drosophila Profilin, which is encoded by the chickadee gene, is required for the formation of normal filopodial and lamellipodial extensions during wound repair . Enabled (Ena)/VASP family proteins can protect the growing barbed ends from capping by binding to them, thereby allowing continuous filament elongation and positively regulating the number and length of filopodia [22, 23, 24]. Fascin (Drosophila Singed, Sn) is a conserved actin-binding protein that cross-links clustered actin filaments and converts them into stable, bundled filopodia [25, 26, 27]. However, although several phagocytic receptors and a number of components of the cytoskeletal regulatory networks have been identified, the signaling pathways involved in phagocytosis and the reorganization of the actin network have yet to be determined.
Jumeau (Jumu) is a member of the winged-helix/forkhead (Fkh) transcription factor family in Drosophila and contains a conserved winged-helix/forkhead domain (WH/FKH) for DNA binding. The gene is widely expressed in most organizations throughout development, such as brain lobes, imaginal discs, the CNS, salivary gland and the hindgut [28, 29, 30]. Jumu is required for neurogenesis as well as eye, wing, and bristle development [28, 29]. Homozygous null mutants of jumu die as embryos or young larvae, and fitness and fertility are impaired in heterozygote null mutants . Jumu regulates nucleolar morphology and function as well as chromatin organization . A recent study indicated that Jumu regulates cardiac progenitor specification by controlling the expression of receptors of the fibroblast growth factor and Wnt signaling pathways . Additionally, our previous studies have shown that Jumu is also expressed in hemocytes and fat body, involved in proper bacterial phagocytosis and resistance in adult flies, and overexpression of jumu both in the fat body and hemocytes induces melanotic nodules by activating Toll signaling [32, 33]. Our recent study showed that Jumu plays crucial roles during Drosophila lymph gland hematopoiesis .
In this study, we show that Jumu is required for larval circulating hemocyte development as well as phagocytosis and filopodium formation. The loss of one jumu copy induces an increase in the number of hemocytes. The loss of two jumu copies can inhibit normal hemocyte mitosis by affecting spindle formation and cytokinesis, resulting in enlarged multinucleated hemocytes. The severe deficiency of jumu also induces the generation of lamellocytes through activation of the Toll signal pathway. Furthermore, we found that Jumu regulates hemocyte phagocytosis by affecting the expression of NimC1 and cytoskeletal reorganization and controls the formation of lamellipodia and filopodia by regulating the expression of Ena and Fascin.
The following fly stocks were used: jumuGE27806 was purchased from GenExel (Daejeon, South Korea). Df(3R)Exel6157 and UAS-jumu were gifts from Alan M. Michelson . Hml-delta-Gal4 UAS-2xEGFP was a gift from Utpal Banerjee . eaterGFP was a gift from Mika Rämet . Dif1 was a gift from Bruno Lemaitre. UAS-jumu RNAi (jumuGD4099), UAS-ena RNAi, UAS-fascin RNAi, UAS-eater RNAi and UAS-NimC1 RNAi were obtained from the Vienna Drosophila RNAi Center (VDRC). Hml-Gal4 were obtained from the Tsinghua Fly Center. UAS-Rho1N19, UAS-Rho1v14, UAS-Rac1 DN, UAS-Rac1 CA, and da-Gal4 were obtained from the Bloomington Stock Center. ppl-Gal4 was obtained from Xun Huang . The w1118 and jumu mutants were reared at 25 °C and, except for the hemocyte number comparison shown in Fig. 6b, the offspring of crosses involving RNAi lines or the UAS-jumu line and all controls of these crosses were reared at 29 °C.
To generate the UAS-NimC1 strain, the full-length NimC1-RA coding sequence was PCR amplified and cloned into pUAST between the underlined restriction sites. Then, the transgenic flies were generated using standard methods.
Circulating hemocyte counts
Larvae were staged according to procedures described previously . Precisely staged late-wandering third-instar larvae were used to obtain hemocyte counts. Circulating hemocyte counts were performed as described previously [32, 38]. Briefly, 5–6 wandering larvae were opened via an incision at both the posterior and anterior ends in 20 μl Phosphate Buffered Saline (PBS), and their hemolymph was allowed to leak out. The hemocytes were then transferred to a Neubauer improved hemocytometer (Marienfeld) for counting of the cells. Counts were conducted in at least 50 larvae in each experiment. All counting assays were performed for at least three independent experiments.
In vivo phagocytosis of hemocytes
In vivo phagocytosis assays of larvae were performed as described previously . Briefly, the ventral side of third-instar larvae was injected with fluorescent latex beads (Thermo Fisher Scientific, F8821), Alexa Fluor 488-labeled heat killed spores of B. bassiana, dead fluorescein-conjugated E. coli (K-12), S. aureus and pHRodo-E. coli (1 mg/ml, 180–200 nl) (Thermo Fisher Scientific) using a Picospritzer III injector. After incubation for 1 h, the circulating hemocytes were collected from the injected larvae by ripping the larval cuticle near the posterior end in PBS solution containing 0.4% trypan blue (trypan blue is used to quench fluorescence of nonphagocytosed bacteria), and the hemocytes were then transferred and attached to a glass slide for 30 min. The cells were subsequently fixed at room temperature with 3.7% formaldehyde in PBS for 10 min and then washed three times for 5 min with PBS. The phagocytosis of latex beads and bacteria by the circulating blood cells was observed using a Zeiss Axioplan 2 microscope equipped with fluorescence optics. For each genotype, 500–1000 cells were counted using ImageJ software. All phagocytosis assays were performed in at least three independent experiments.
For antibody staining, hemocytes were bled from third-instar larva and allowed to attach to a glass slide for 30 min. The cells were then fixed at room temperature with 3.7% formaldehyde in PBS for 10 min, pre-incubated in blocking solution (PBS with 0.1% Tween-20 and 5% goat serum) and incubated with primary antiserum diluted in blocking solution. The following primary antibodies were used: mouse anti-NimC1, mouse anti-L1 and mouse anti-H2 (gifts from I. Ando); rat anti-Jumu (made in our lab); mouse anti-α-tubulin (sigma); rabbit anti-PH3 (Millipore); mouse anti-Dorsal, mouse anti-Ena, mouse anti-Fascin, mouse anti-Rho1 and mouse anti-Profilin (Developmental Studies Hybridoma Bank); rabbit anti-Dif (gift from Dominique Ferrandon). Alexa Fluor 488-, Alexa Fluor 568- and Alexa Fluor 594-conjugated secondary antibodies (Thermo Fisher Scientific) were employed. For phalloidin staining, hemocytes were preincubated with PBST (PBS with 0.1% TritonX-100) for 5 min and then incubated with Alexa Fluor 488-labeled phalloidin (Thermo Fisher Scientific) diluted in PBS for 30 min. Images were obtained using a Zeiss Axioplan 2 microscope equipped with fluorescence optics. All staining was performed in at least three independent experiments.
BrdU labeling and TUNEL staining
The BrdU labeling was performed according to previously described procedures . Briefly, BrdU was diluted in standard fly food at 0.5 mg/ml (Sigma), and trypan blue was added to the fly food to identify whether the larvae eat the fly food. The third-instar larvae were maintained on this media for 4 h, and then, the dissected hemocytes from the third-instar larvae were fixed with 3.7% formaldehyde in PBS for 30 min and washed three times for 10 min each in PBS with 0.1% Tween-20. The samples were then treated with 3 M HCl for 30 min and washed three times in PBS with 0.1% Tween-20. After blocking the samples with blocking solution (PBS with 0.1% Tween-20 and 5% goat serum), the samples were stained with a mouse anti-BrdU antibody (Developmental Studies Hybridoma Bank), followed by mouse-TRITC secondary antibody staining and mounting. TUNEL staining was conducted using TUNEL solution according to the manufacturer’s instructions (Roche Biochemicals). At least three independent experiments were performed.
MARCM clone analysis
The genotype of the strain used for the MARCM clone generation was as follows: hsFLP, UAS-GFP, actGal4; FRT42D, tubGal80. We crossed this strain with UAS-jumu RNAi. The embryo collections (1 h) were incubated for 10 h at 25 °C and subsequently shifted to 37 °C for 1 h to induce the MARCM clones.
Transfection and immunoblotting
S2 cells (CVCL_Z232, Invitrogen, Cat#R690–07) were transfected with pMK33-Flag or the pMK33-Flag-jumu full CDS construct using the Effectene Transfection kit (Qiagen). Whole-cell extracts were prepared in lysis buffer containing 20 mM Tris (pH 7.6), 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM dithiothreitol (DTT), 2 mM EDTA, and protease inhibitors. Then, 30 μg of the lysate was loaded into a 12% SDS-PAGE gel, followed by electroblotting onto nitrocellulose membranes and probing with mouse anti-α-tubulin (1:500, Sigma), mouse anti-Ena, mouse anti-Fascin, mouse anti-Rho1 and mouse anti-Profilin (1:300, Developmental Studies Hybridoma Bank) for 2 h. The blot was subsequently probed with anti-mouse HRP-conjugated secondary antibodies for 1.5 h and detected using the ECL Plus detection system (Program). ImageJ was employed to measure the intensity values of the blots. Representative blots obtained from at least three independent experiments with similar results are presented.
Image analysis and quantification
All images used for quantification were captured with a Zeiss Axioplan 2 microscope, and all analyses were performed using ImageJ. The mitotic index and BrdU index of the circulating hemocytes were determined by dividing the number of PH3-positive cells and BrdU-positive cells, respectively, by the total number of cells, and at least 500 cells were counted. The signal intensities of NimC1, Ena, Fascin, Roh1 and Profilin were defined as the average pixel intensity values in each cell (integrated intensity in one cell/area of the cell), and at least 200 cells were measured. For quantification of the fluorescence signal intensity, the fluorescent images were first converted to 8-bit images, and the total intensity value with an identical threshold was captured and measured with ImageJ. The freehand selection tool in ImageJ was used to capture and measure the area of the circulating hemocytes and lamellipodia. Filopodial length was quantified using the line tool of ImageJ with protrusions > 1 μm long being classified as filopodia. At least 50 cells were measured for the quantification of the filopodial length, and at least 100 cells were measured for the quantification of the lamellipodial area.
Real-time quantitative PCR
The total RNA obtained from 6 to 8 dissected third-instar larvae or circulating hemocytes (from 600 to 800 third-instar larvae) was prepared using TRIzol (Invitrogen). The obtained total RNA was used to generate cDNA with M-MLV Reverse Transcriptase (Promega). Real-time PCR amplification was performed using SYBR Green I Master Mix (Roche, LightCycler480) on a Roche 480 real-time PCR system. The results were normalized to the level of RpL32 mRNA in each sample. Three experiments per genotype were averaged. Two biological replicates were performed. The primer sequences used are shown in Additional file 1: Table S1.
Statistical analyses were performed with two-tailed unpaired Student’s t-tests or one-way ANOVAs using GraphPad Prism software. The thresholds for statistical significance were established as *P < 0.05, **P < 0.01 and ***P < 0.001.
Jumu is required for hemocyte phagocytosis and development
Jumu regulates phagocytosis by modulating the expression of NimC1
Loss of jumu and NimC1 in hemocytes cause defects of filopodia
Jumu affects the expression of the proteins associated with the formation of actin filopodia
To further identify the relationship between cell spreading and phagocytosis, we evaluated the phagocytosis of latex beads after knockdown of ena and fascin. The phagocytosis ability was reduced in the ena knockdown hemocytes; however, compared with that in the control, the knockdown of fascin in the hemocytes increased the phagocytosis of latex beads (Fig. 4h-l). Similar to Fascin, a previous study showed that loss of profilin suppresses the formation of filipodia but also causes increased phagocytosis, and the authors suggested that the loss of profilin may change the balance between elongation and filament branching and lead to greater membrane ruffling, thereby increasing phagocytosis indirectly . These results suggest that the proteins associated with the formation of actin filopodia regulate phagocytosis by different manners.
Overexpression of jumu induces enhanced cell spreading and large numbers of filopodia
Jumu maintains proper hemocyte division by regulating the cell cycle and cytokinesis
It has been suggested that a defect in cytokinesis or DNA overreplication can lead to enlarged cells [19, 45]. Moreover, the above results showed that the loss of jumu affects actin-dependent cytoskeletal remodeling; therefore, we speculate that the loss of jumu might also cause a defect in microtubule cytoskeleton rearrangement during mitosis. To investigate whether the enlarged hemocytes caused by the loss of jumu are due to these causes, third-instar larvae hemocytes were stained with antibodies against Tubulin and phospho-histone H3 (PH3) to visualize microtubules and cell mitosis. The hemocytes that were not undergoing mitosis showed a similar microtubule cytoskeletal organization in w1118 and jumuGE27806/Df(3R)Exel6157 (Fig. 6c and d). We found that less than 1% of circulating w1118 hemocytes were undergoing mitosis (PH3+ cells), most of which displayed clear spindles, especially during metaphase, and nuclear division accompanied cytokinesis during anaphase and telophase (Fig. 6c1-c3 and e). However, more than 3% of circulating jumuGE27806/Df(3R)Exel6157 hemocytes were PH3+ cells, nearly half of which did not display spindles or show signs of nuclear division associated with cytokinesis, and most defective cells were larger and multinucleated (Fig. 6d1-e). Next, we examined whether the phagocytic deficit observed in jumu lacking hemocytes is a secondary consequence of the defects in mitosis. We found that compared with the PH3-negative cells in w1118, the PH3-positive cells have an obviously reduced phagocytosis ability (Fig. 6f, h). However, the phagocytosis ability of the PH3-positive cells is not reduced in jumuGE27806/Df(3R)Exel6157, although some normally sized PH3-positive cells showed a reduced phagocytosis of latex beads, and the enlarged PH3-positive cells have a stronger phagocytosis ability (Fig. 6g, h). This result suggests that the phagocytic deficit of the hemocytes in jumuGE27806/Df(3R)Exel6157 is not attributed to a mitotic deficit. Moreover, the Hml > GFP > jumu RNAi hemocytes displayed a mitotic phenotype similar to that observed in jumuGE27806/Df(3R)Exel6157 (Fig. 6i-j2). We investigated whether the loss of jumu could also cause DNA overreplication in hemocytes. To investigate this possibility, we detected cells in the S phase through BrdU incorporation assays. However, the incorporation of BrdU was not increased in the jumuGE27806/Df(3R)Exel6157 third-instar larvae hemocytes (Additional file 2: Figure S5a-c), and we found that the BrdU-positive cells and BrdU-negative cells in w1118 and jumuGE27806/Df(3R)Exel6157 hemocytes had a similar phagocytosis ability (Additional file 2: Figure S5d-f’). Moreover, the TUNEL staining showed that the jumuGE27806/Df(3R)Exel6157 circulating hemocytes did not display an increase in apoptotic cells compared with the number in w1118 (Additional file 2: Figure S5 g and h). Taken together, these results suggest that compared with jumu heterozygotes, a severe deficiency in Jumu levels can induce hemocyte mitosis but inhibit the formation of spindles and cytokinesis, leading to the generation of enlarged hemocytes with multiple nuclei and a reduced number of circulating hemocytes in jumuGE27806/Df(3R)Exel6157.
Similar to cells lacking jumu, hemocytes expressing dominant-negative Rho1 (Rho1N19) or Rac1 (Rac1 DN) were enlarged and multinucleated (Fig. 6l and m) . We investigated whether the enlargement of hemocytes resulting from the loss of jumu was related to the inactivation of Rho1 and Rac1. However, the expression of constitutively active Rho1 (Rho1V14) and Rac1 did not rescue the enlarged cell size of the Hml > jumu RNAi hemocytes (Fig. 6n and o), suggesting that jumu regulates hemocyte size in a Rho1- and Rac1-independent manner.
The above result shows that the loss of jumu causes an increased number of circulating hemocytes in the M phase and accelerates the cell cycle process. Thus, we investigated whether Jumu deficiency affects the expression of Cyclins. We detected the mRNA levels of CycA, CycB, CycD and CycE using real-time PCR and found that the knockdown of jumu reduces the CycA level and increases the CycB and CycD levels (Fig. 6p). Previously, we analyzed the gene expression profiles of larval circulating hemocytes with overexpression of jumu using the GeneChip Drosophila Genome 2.0 Array and found four genes, pav, png, bam and piwi, which were significantly upregulated (> 5-fold), participated in cell cycle and cell division according to gene ontology analysis (unpublished data). Moreover, a previous study showed that the RNAi of pav in the S2R or Kc cell could result in enlarged and multinucleate cells . Therefore, we next detected the expression of pav, png, bam and piwi in jumu-deficient hemocytes. Quantitative RT-PCR indicated that the expression of pav and bam were not changed, but the transcription levels of png and piwi were significantly downregulated (Fig. 6p). Moreover, a similar change in the mRNA level of these genes was observed in the jumu mutant (Additional file 2: Figure S5i). These findings suggest that Jumu may control the cell cycle and mitosis process by affecting the expression of Cyclin genes, png and piwi.
Knockdown of jumu induces the generation of lamellocytes via activation of the toll pathway in hemocytes
Jumu regulates hemocyte phagocytosis by affecting NimC1 expression and cytoskeletal reorganization
In addition to microbial recognition by several receptors, the phagocytosis process requires actin filament rearrangement to engulf invading microbes. Some cytoskeletal regulatory proteins have been shown to regulate phagocytosis in Drosophila; for example, positive regulation occurs through SCAR and Arp2/3, whereas Profilin negatively regulates the phagocytosis of E. coli and S. aureus . Additionally, Rho GTPases, such as Cdc42, Rac1 and Rac2, regulate phagocytosis by altering hemocyte cell shape after infection [42, 50, 51]. In the present study, we found that proteins associated with filopodium formation (Ena, Fascin and Rho1) also affect phagocytosis in hemocytes. Knockdown of ena led to a defect in the phagocytosis of latex beads, whereas loss of fascin increased the ability of the cells to engulf latex beads. Ena-deficient hemocytes rarely exhibited filopodia or extended lamellipodia under normal conditions or after latex bead infection, suggesting that Ena is involved in the regulation of plasma membrane protrusion during phagocytosis. Although the number of filopodia was reduced, numerous irregular lamellipodia were observed in fascin-deficient hemocytes after latex bead infection, which suggested that irregular lamellipodia may favor the dynamic rearrangement of the plasma membrane and that loss of fascin may promote cytoskeletal reorganization during phagocytosis. Additionally, a previous study indicated that the small GTPase Rho1 also positively controls the formation of cell protrusions . However, we found that the knockdown of Rho1 did not impair the phagocytosis ability of hemocytes (data not shown), the numbers of engulfing cells were not reduced, and the PIs of the latex beads and pHRodo-E. coli phagocytosis were increased. In fact, most Rho1-defective hemocytes showed similar phagocytosis to the controls, but the enlarged Rho1-defective hemocytes exhibited strong phagocytosis, which led to increased phagocytosis indexes (data not shown). Taken together, these results suggest that the proteins regulating the actin cytoskeleton affect phagocytosis in different manners, and the essential element for phagocytosis is dynamic actin filament rearrangement rather than simple filopodium elongation. Thus, the various regulations of cytoskeletal regulatory proteins on phagocytosis might explain why the increased lamellipodia area and filopodia number obtained with the overexpression of jumu did not sufficiently enhance the phagocytosis ability of hemocytes. Additionally, we also revealed that the loss of NimC1 affects the recruitment of Ena at the tips of filopodia and lamellipodia, and Jumu likely regulates the subcellular localization of Ena by altering the expression of NimC1 (Fig. 9a and b). The role of phagocytic receptors in the regulation of plasma membrane protrusion during phagocytosis is not well understood and has yet to be investigated.
Loss of Jumu changes hemocyte differentiation
Here, we showed that Jumu affects the differentiation and development of circulating hemocytes. The activation of several signaling pathways, such as the JNK, JAK/STAT and Toll pathways, in circulating hemocytes can promote lamellocyte formation [47, 48]. In a previous study, we showed that overexpression of jumu in both the fat body and hemocytes induces melanotic nodules and lamellocytes by activating Toll signaling, but activated Dorsal/Dif were only found in hemocytes deposited on the fat body and aggregated circulating hemocytes, and not within the fat body or scattered circulating hemocytes . In the present study, we further indicated that severe deficiency of jumu can autonomously induce activation of the Toll signal pathway in the fat body and circulating hemocytes, but lamellocyte formation caused by loss of jumu only depends on Toll signaling from circulating hemocytes (Fig. 9c). Moreover, in contrast to overexpression of jumu, loss of jumu in both the fat body and hemocytes does not induce melanotic nodules and deposited hemocytes. These results suggest that overexpression of jumu or a lack of jumu affects the activation of Toll signaling in a different manner, and the correct genetic dose of jumu balances the activation of immune signaling and prevents chronic inflammation.
We found that some lamellocytes induced by jumu deficiency can efficiently engulf latex beads after infection. Lamellocytes are formed in response to wasp infection and are mainly involved in the encapsulation and melanization of foreign pathogens that are too large to undergo phagocytosis [7, 52], although their role in phagocytosis has not been extensively investigated. Previous studies have shown that wasp infection induces a two-lineage model of lamellocyte hematopoiesis: one lineage of lamellocytes is derived from the direct transdifferentiation of plasmatocytes, while the other is a designated lamellocyte lineage, referred to as lamelloblasts [53, 54]. Moreover, the lamellocytes generated from plasmatocytes, expressing the plasmatocyte-specific marker NimC1, exhibit phagocytosis, but the terminally differentiated large lamellocytes without NimC1 expression do not engulf any bacteria . Therefore, the lamellocytes exhibiting phagocytosis among jumu-defective mutants may be derived from plasmatocytes. Furthermore, jumu knockdown in plasmatocytes (Hml > GFP > jumu RNAi) can induce the expression of the lamellocyte marker L1, and the resultant round cells showing coexpression of Hml > GFP and L1 are similar to the activated plasmatocytes induced by wasp infection described in a previous study . These results further suggest that loss of jumu can lead to the transdifferentiation of plasmatocytes into lamellocytes in a manner that is similar to immune induction.
Severe deficiency of jumu affects cell mitosis
Our results indicate that Jumu maintains proper hemocyte proliferation and division by regulating the cell cycle, spindle formation and cytokinesis. The loss of jumu affects the transcription levels of the Cyclin genes CycA, CycB and CycD and the cell cycle- and division-associated genes png and piwi (Fig.9c). CycA is essential for the control of the cell cycle at the G2/M transition, and the CycA-Cdk1 complex can trigger entry into the S phase [55, 56]. CycB degradation is required for entry into anaphase, and the expression of a stable version of Drosophila CycB blocks cytokinesis along with numerous events of mitotic exit [55, 57]. Moreover, CycB is also required for normal spindle formation . The CycD/Cdk4 complex stimulates both cell cycle progression and cell growth, and the overexpression of CycD/Cdk4 leads to larger ommatidia and an enlargement and rough appearance of the eye [59, 60]. According to these results, we speculate that the reduced expression of CycA and the increased expression of CycB and CycD likely simultaneously contribute to the defect in the cell cycle and cytokinesis, consequently causing the generation of enlarged multinucleated hemocytes in jumu mutants. PNG is required to repress DNA replication and for proper coupling of the S and M phases in early embryos, and mutation of png leads to inappropriate DNA replication and results in large polyploidy nuclei [61, 62]. Thus, the multinucleated phenotype observed in the jumu-deficient hemocytes may also be associated with decreased png levels. Moreover, the PNG kinase complex regulates the translation of CycB, and the phosphorylation of GNU by CyclinB/CDK1 can also block the activation of the PNG [63, 64]. Moreover, PIWI plays a critical role in the maintenance of cell cycle progression during early embryogenesis; loss of piwi leads to severe mitotic defects, including abnormal nuclear morphology, cell cycle arrest and asynchronous nuclear division . The mechanism by which Jumu regulates the expression levels of CycA, CycB, CycD, png and piwi and the regulatory mechanism of these elements in the control of the cell cycle and division of hemocytes remain to be addressed.
Moreover, previous studies have suggested that activation or overexpression of Dorsal/Dif in hemocytes can promote proliferation . Thus, we speculate that the higher mitotic index observed in the jumu double heterozygotes may also be associated with the activation of the Toll signaling pathway. However, we found that the activation of Toll signaling in hemocytes did not result in enlarged hemocytes (data not shown), which suggests that the cell enlargement phenotype caused by the lack of jumu is not related to the Toll signaling pathway. The regulatory mechanisms of Jumu in hemocyte proliferation and development remain to be elucidated.
In this study, we also found that mitosis could severely impact phagocytosis (Fig.6f and h), this might be because most microtubule and actin filament participate in the formations of spindle and contractile ring during mitosis, and are not enough to involve phagocytosis. However, most enlarged PH3-positive cells in jumuGE27806/Df(3R)Exel6157 are mitotic defect, thus, they might have free microtubule and actin filament to participate in cytoskeletal reorganization during phagocytosis (Fig.6g, asterisk). How the cooperation between microtubule and actin filament mediate phagocytosis remain to be addressed.
Taken together, our findings in this study suggest that Jumu is required for larval circulating hemocyte development as well as phagocytosis and filopodium formation. The severe deficiency of jumu induces the generation of lamellocytes through activation of the Toll signal pathway. Furthermore, Jumu regulates hemocyte phagocytosis by affecting the expression of NimC1 and cytoskeletal reorganization.
We thank Alan M. Michelson, Utpal Banerjee, Mika Rämet and Bruno Lemaitre for providing the numerous fly strains used in this study. We acknowledge I. Ando and Dominique Ferrandon for providing the anti-NimC1, mouse anti-L1, mouse anti-H2, and anti-Dif antibodies. We thank the GenExel Drosophila Stock Center, Vienna Drosophila RNAi Center, and Tsinghua Fly Center for sharing the numerous fly stocks utilized in this research. This work was supported by the National Natural Science Foundation of China (31772521).
This work was supported by the National Natural Science Foundation of China (31772521).
Availability of data and materials
All data generated or analysed during this study are included in this published article [and its supplementary information files].
HY and JLH designed this research. HY carried out most of the experiments, analyzed the data and drafted this manuscript. YS and LF helped with in vivo phagocytosis assays and immunohistochemistry. All authors read and approved the final manuscript.
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