The Nuclear Receptor Seven Up Regulates Genes Involved in Immunity and Xenobiotic Response in the Adult Drosophila Female Fat Body
- Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD 21205
- 2Corresponding author. E-mail: dbarbosa{at}jhu.edu
Abstract
The physiology of organisms depends on inter-organ communication in response to changes in the environment. Nuclear receptors are broadly expressed transcription factors that respond to circulating molecules to control many biological processes, including immunity, detoxification, and reproduction. Although the tissue-intrinsic roles of nuclear receptors in reproduction have been extensively studied, there is increasing evidence that nuclear receptor signaling in peripheral tissues can also influence oogenesis. We previously showed that the Drosophila nuclear receptor Seven up (Svp) is required in the adult fat body to regulate distinct steps of oogenesis; however, the relevant downstream targets of Svp remain unknown. Here, we took an RNA sequencing approach to identify candidate Svp targets specifically in the adult female fat body that might mediate this response. svp knockdown in the adult female fat body significantly downregulated immune genes involved in the first line of pathogen defense, suggesting a role for Svp in stimulating early immunity. In addition, we found that Svp transcriptionally regulates genes involved in each step of the xenobiotic detoxification response. Based on these findings, we propose a testable model in which Svp functions in the adult female fat body to stimulate early defense against pathogens and facilitate detoxification as part of its mechanisms to promote oogenesis.
- Drosophila
- fat body
- Seven up
- oogenesis
- xenobiotics
- immunity
Nuclear receptors are evolutionarily conserved systemic physiology sensors that act as transcriptional regulators throughout the body to control a diverse array of biological processes, including female reproduction (Mouzat et al. 2013; Mazaira et al. 2018). For example, Estrogen Receptor global knockout results in elevated steroid synthesis, reducing the number of growing follicles and preventing meiotic oocyte progression (Lubahn et al. 1993; Liu et al. 2017). Global knockout of Liver X Receptors disrupts meiosis resumption and reduces mammalian fertility (Mouzat et al. 2013), whereas COUP-TFs are required in the mammalian uterine muscle for placenta formation and embryo implantation (Petit et al. 2007; Zheng et al. 2010). However, how nuclear receptor function in specific peripheral tissues influences oogenesis in adult females is understudied.
The Drosophila ovary is an ideal model to investigate how nuclear receptor action in peripheral tissues remotely influences oogenesis. The Drosophila ovary is composed of 16-20 ovarioles, and each ovariole has an anterior germarium followed by chronologically arranged developing follicles. Each germarium houses two to three germline stem cells (GSCs) in a specialized niche, and GSCs divide asymmetrically to self-renew and produce a cystoblast that undergoes four incomplete mitotic divisions to form a 16-cell cyst (one oocyte plus 15 supporting nurse cells). Follicle cells surround the 16-cell cyst to form a follicle that buds from the germarium and develops through 14 stages of oogenesis to form a mature oocyte (Figure 1A,B) (Greenspan et al. 2015; Laws and Drummond-Barbosa 2017). Many steps of oogenesis, including maintenance of GSCs, early germline cyst survival, vitellogenesis (i.e., yolk uptake), and ovulation, are highly sensitive to physiological inputs (Drummond-Barbosa 2019). Notably, each of these processes has been shown to be controlled by nuclear receptors [reviewed in (Ables and Drummond-Barbosa 2017; Laws and Drummond-Barbosa 2017)].
Svp functions through unknown downstream targets in the adult female fat body to regulate oogenesis. (A) Drosophila ovariole showing an anterior germarium followed by progressively older follicles. Each follicle represents a 16-cell germline cyst (one oocyte and 15 supporting nurse cells; light blue) surrounded by follicle cells (gray). (B) Germarium showing 2-3 germline stem cells (GSCs; dark blue) in a niche composed of somatic cells, including cap cells (yellow). GSCs give rise to cystoblasts that divide to form 16-cell cysts. Germline cysts are surrounded by follicle cells (gray) to bud from the germarium as a new follicle. (C) Svp is required specifically in adult female adipocytes to promote GSC maintenance and early germline cyst survival, and in oenocytes for survival of vitellogenic follicles.
Nuclear receptors have been shown to control Drosophila oogenesis through intrinsic and extrinsic mechanisms (Ables and Drummond-Barbosa 2017). The best characterized Drosophila nuclear receptor, EcR (FBgn0000546, FXR/LXR homolog), is required in ovarian cell types for regulation of GSC maintenance, early germline cyst survival and development, oocyte meiosis entry, and lipid uptake in later follicles (Ables and Drummond-Barbosa 2010; König et al. 2011; Morris and Spradling 2012; Sieber and Spradling 2015). E78 (FBgn0004865, potential PPAR homolog) is required for follicle development/survival (Ables and Drummond-Barbosa 2010), E75 (FBgn0000568, potential REV-ERB homolog) is cell autonomously required for vitellogenesis (Buszczak et al. 1999; Ables and Drummond-Barbosa 2010), and HR39 (FBgn0261239, LRH1 homolog) and Ftz-F1 (FBgn0001078, SF1 homolog) are required for ovulation (Sun and Spradling 2013; Knapp et al. 2020). In addition to having these ovary-intrinsic roles, nuclear receptors can also function in peripheral tissues to control oogenesis, as recent studies show (Sieber and Spradling 2015; Weaver and Drummond-Barbosa 2019). For example, EcR is required in the central nervous system to regulate female feeding behavior and thereby support egg production (Sieber and Spradling 2015). More recently, we showed that the nuclear receptor Svp is required in adult female adipocytes and hepatocyte-like oenocytes (collectively referred to as the fat body herein) to regulate distinct steps of oogenesis (Weaver and Drummond-Barbosa 2019). Specifically, Svp (FBgn0003651) is required in adipocytes to control GSC maintenance and early germline cyst survival, whereas Svp is required in oenocytes for survival of vitellogenic follicles (Figure 1C) (Weaver and Drummond-Barbosa 2019). However, the mechanisms underlying how nuclear receptor activity in peripheral tissues regulates oogenesis are not well understood.
The Drosophila fat body is a major endocrine organ with energy-intensive metabolic and immune roles (Arrese and Soulages 2010), raising many possibilities as to how Svp activity in adipocytes and oenocytes might remotely control oogenesis. Multiple studies have explored the nutrient sensing and metabolic roles of the fat body in regulating larval growth (Colombani et al. 2003; Hennig et al. 2006), in mobilizing lipids in response to starvation (Chatterjee et al. 2014), in controlling lifespan (Giannakou et al. 2004; Hwangbo et al. 2004), and in reproduction (Armstrong et al. 2014; Matsuoka et al. 2017; Armstrong and Drummond-Barbosa 2018). The function of the fat body as a major immune-responsive tissue has also been characterized (Lemaitre and Hoffmann 2007). In response to infection, the fat body activates nuclear factor-κB (NF-κB) signaling to produce and secrete antimicrobial peptides (AMPs) into the hemolymph (Lemaitre and Hoffmann 2007; Buchon et al. 2014; Roth et al. 2018; Suzawa et al. 2019). There is also evidence that the fat body acts as a detoxification tissue based on the expression of members of the Cytochrome P450 (Cyp450) superfamily of monooxygenases, which are enzymes involved in metabolizing foreign substances and drugs (Feyereisen 1999; Chung et al. 2009) and implicated in resistance to insecticides (Terhzaz et al. 2015). In this study, we used transcriptomics to identify candidate target genes downstream of Svp in the adult female fat body and generate hypotheses for future investigation into the molecular mechanisms underlying Svp control of oogenesis. Specifically, we took an RNA sequencing approach to compare the transcriptome of fat bodies from adult females with svp RNAi knockdown to that of control RNAi female fat bodies. We found that svp knockdown significantly reduces the expression levels of genes involved in the first line of defense against pathogens. In addition, Svp targets are significantly enriched for genes involved in xenobiotic detoxification responses. We propose a model according to which Svp normally functions in the adult female fat body to stimulate early immunity (and prevent later activation of the immune deficiency pathway) and to neutralize toxic compounds to facilitate their elimination from the body, thereby promoting optimal conditions for oogenesis.
Materials And Methods
Drosophila strains and culture conditions
Drosophila strains and cultures were maintained on medium containing 58 g/ml molasses, 46.5 g/ml yellow cornmeal, 4.7 g/ml agar, 17.4 g/ml active dry yeast, 0.1% tegosept, and 7.25 mM Propionic Acid at 22-25°. The previously described Gal4 lines, adipocyte-specific 3.1Lsp2-Gal4 (Lazareva et al. 2007; Armstrong et al. 2014) and PromE800-Gal4 (Billeter et al. 2009; Weaver and Drummond-Barbosa 2019), along with the previously described temperature-sensitive tub-Gal80ts transgene (Mcguire et al. 2003), were recombined by standard crosses to generate the PromE800-Gal4 tubGal80ts; 3.1Lsp2-Gal4 double driver specifically targeting both oenocytes and adipocytes with temporal control. UAS-LucJF01355 (Matsuoka et al. 2017) was obtained from the Bloomington Drosophila Stock Center and UAS-svpGD1546 was obtained from the Vienna Drosophila Resource Center (VDRC). Balancer chromosomes and other genetic elements are described in Flybase (www.flybase.org).
Tissue-specific RNAi
Females of genotypes y w; PromE800-Gal4 tubGal80ts; 3.1Lsp2-Gal4/UAS-hairpin (for adult fat body-specific RNAi) were raised at 18° [the permissive temperature for Gal80ts (Mcguire et al. 2003)] to prevent RNAi induction during development. Zero- to 2-day-old females were maintained at 18° for 3 days with y w males, and then switched to 29° (the restrictive temperature for Gal80ts) for 5 days to induce RNAi in the adult fat body (i.e., in both adipocytes and oenocytes). We chose this time point of svp RNAi in part because knockdown of svp specifically in adult oenocytes decreases egg production after 5 days (Weaver and Drummond-Barbosa 2019). We also note that, although reduced GSC numbers are only observed after 7-10 days of svp knockdown (Weaver and Drummond-Barbosa 2019), any changes in gene expression resulting from decreased Svp activity that could be causally involved in the increased rate of GSC loss would necessarily precede any observable decrease in GSC numbers. UAS-LucJF01355 was used as an RNAi control. For all conditions, medium was supplemented with wet yeast paste.
RNA isolation
Abdominal carcasses from 100 females of each genotype were dissected in Grace’s medium supplemented with 10% fetal bovine serum (FBS; Sigma). Fat body cells were dissociated from abdominal carcasses with 500 µl dissociation buffer [0.5% Trypsin (Sigma) plus 1 mg/ml collagenase (Sigma) in 1x PBS] per 50 carcasses for 30 min at room temperature. Samples were gently agitated every 5 min to facilitate separation of cells from the cuticle. 500 µl of Grace’s media plus 10% FBS was added to stop the enzymatic reaction and supernatants were placed in new tubes. Carcasses were rinsed with Grace’s medium plus 10% FBS and the two supernatants per genotype were combined. Dissociated cells were centrifuged at 3.3 rpm for 5 min at room temperature. Supernatants were removed and cells were immediately lysed in 250 µl lysis buffer from the RNAqueous-4PCR DNA-free RNA isolation for RT-PCR kit (Ambion). RNA was extracted from all samples following the manufacturer’s instructions. Three independent experiments were performed for RNA sequencing and RT-qPCR analysis.
RT-qPCR
RNA from abdominal carcasses was extracted as described above. cDNA was synthesized from 500 ng of total RNA for each sample using Supercript II Reverse Transcriptase (ThermoFisher) according to the manufacturer’s instructions. Table S1 lists all primers used in this study. PowerUp SYBR Green Master Mix (ThermoFisher) was used for quantitative PCR. The reactions for three independent biological replicates were performed in triplicate using LightCycler 96 (Roche). Amplification fluorescence threshold was determined by LightCycler 96 software, and ΔΔCT were calculated using Microsoft Excel. Rp49 transcript levels were used as a reference. Fold change of transcript levels were calculated using the equation 2-ΔΔCt (Microsoft Excel).
RNA sequencing and data analysis
cDNA library construction, Illumina sequencing, and differential expression analysis was performed by Novogene Bioinformatics Technology Co., Ltd (Beijing, China). The cDNA libraries were prepared using the NEBNext Ultra RNA Library Prep Kit for Illumina (New England Biolabs) according to the manufacturer’s instructions. The cDNA library for each sample was quality assessed using an Agilent Bioanalyzer 2100, and library preparations were sequenced on a NovaSeq6000 platform with PE150 read lengths.
Reads obtained from sequencing were aligned to the D. melanogaster reference genome using the TopHat read alignment tool (Trapnell et al. 2009) for each of the sequencing datasets. The reference sequences were downloaded from the Ensembl project website (useast.ensembl.org). TopHat alignments were used to generate read counts for each gene using HTSeq (Anders et al. 2015), which were subsequently used to generate the differential expression results using the DESeq2 R package (Anders et al. 2015). Gene ontology (GO) enrichment of differentially expressed genes was analyzed by the clusterProfiler R package (Yu et al. 2012). Enriched genes with a corrected P value of less than 0.05 were considered significant.
Data availability
Drosophila strains are available upon request. The data and analyses in this paper are described in the main figures. The raw data and processed data files are available through the NCBI GEO accession number GSE159703 and are also provided as supplemental tables. Supplemental material available at figshare: https://doi.org/10.25387/g3.13122728.
Results And Discussion
Expression levels of 132 transcripts are altered when svp is knocked down in the fat body
We previously showed that Svp is required in the adult female fat body to regulate distinct aspects of oogenesis (Weaver and Drummond-Barbosa 2019); however, the downstream factors mediating those effects are unknown. To identify downstream targets of Svp in the adult female fat body, we performed RNA sequencing analysis of fat bodies from fat body-specific Luc control RNAi and svp RNAi females (Figure 2A). We knocked down svp in the entire fat body of adult females for 5 days, using the combined adipocyte-specific 3.1Lsp2-Gal4 (Lazareva et al. 2007; Armstrong et al. 2014) and oenocyte-specific PromE800-Gal4 (Billeter et al. 2009; Weaver and Drummond-Barbosa 2019) drivers with tub-Gal80ts (Mcguire et al. 2003), prior to fat body dissections and RNA sequencing performed in triplicate. RT-qPCR analysis showed that svp transcript levels were significantly decreased by 21% in svp knockdown relative to control fat bodies (Figure 2B). [We note that this modest knockdown is sufficient to cause significant changes in oogenesis (Weaver and Drummond-Barbosa 2019), consistent with physiological regulators being highly sensitive to environmental and physiological fluctuations. In fact, we previously showed that similarly modest changes in the levels of single amino acid transporters in adipocytes also lead to increased GSC loss (Armstrong et al. 2014)]. RNA sequencing produced an average of 38,389,394 reads across the six sequencing libraries, ranging from 35.1 to 43.3 million reads per sample (Table 1), of which an average of 96% were mapped to the Drosophila melanogaster genome. Based on the analysis of those reads, over 16,000 transcripts were identified in the adult female fat body, with the majority (81%) representing protein-coding genes (Figure 2C; Table S2). In addition to protein-coding genes, our RNA sequencing analysis also identified transcripts for mitochondrial genes, long non-coding RNAs (lncRNAs), small nucleolar RNAs (snoRNAs), microRNAs (miRNAs), and additional RNA subtypes (Figure 2C). Our samples were enriched for mRNAs using oligo(dT) beads for poly(A) selection; however, this method does not completely eliminate non-coding RNAs, especially those containing poly(A) tails (Cai et al. 2004; Wilhelm et al. 2008; Cabili et al. 2011). Comparison of the identified protein-coding genes with our previous iTRAQ adult female fat body proteomic data (Matsuoka et al. 2017) revealed almost 2,000 common genes between the two datasets (Figure 2D; Table S3), representing ∼75% of the total proteins we identified by iTRAQ (Matsuoka et al. 2017). These results suggest that our transcriptomic and proteomic approaches reproducibly identified many of the genes expressed in the adult female fat body.
svp knockdown in the adult female fat body differentially regulates 132 genes. (A) Schematic of svp RNAi and RNA isolation in adult females. (B) RT-qPCR analysis of svp from fat bodies of females at five days of fat body Luc control or svp RNAi showing the mean±SEM. *P <0.05. (C) Pie chart showing the types of transcripts identified in the adult female fat body by RNA sequencing. (D) Venn diagram comparing the number of genes identified by RNA sequencing to the number of genes identified by iTRAQ proteomic analysis (Matsuoka et al. 2017) in adult female fat bodies. (E) Scatter plot of normalized transcript abundance in FPKM (fragments per kilobase of transcript per million mapped reads) of control vs svp knockdown fat bodies. Significantly upregulated genes, purple; significantly downregulated genes, green; unchanged, gray. (F) Volcano plot of differentially expressed genes graphing the statistical significance [-log10(padj)] against the magnitude of differential expression (log2 Fold Change). The horizontal dotted line represents P=0.05, such that all points above that line are considered statistically significant. (G) Classification of genes corresponding to significantly changed transcript levels in svp RNAi compared to control RNAi. (H) Pie charts of protein-coding gene classifications based on GLAD analysis for significantly downregulated (blue) and upregulated (purple) genes.
Differential expression analysis identified 132 genes with significantly altered transcript levels between the Luc control RNAi and svp RNAi groups (Figure 2E,F). Of those genes, 71 were downregulated (indicating genes normally positively regulated by Svp; Table S4), whereas 61 genes were significantly upregulated (indicating genes normally negatively regulated by Svp; Table S5). Of the upregulated genes, 92% were protein-coding genes and the remainder were identified as lncRNAs (Figure 2G). In addition, analysis using the gene group resource GLAD (Hu et al. 2015) classified most upregulated genes as encoding proteins that are either transmembrane, secreted, part of the matrisome, or do not belong to a specific category (referred to as “None,” Figure 2H). A small subset of genes were singly classified as encoding mitochondrial proteins, serine proteases, phosphatases, or methyltransferases (Figure 2H; listed as “Other”). Interestingly, a small subset of downregulated genes was identified as encoding proteins that are part of the “matrisome,” which form or remodel the extracellular matrix (Davis et al. 2019) (Figure 2H). The majority of downregulated genes were identified as protein-coding genes (Figure 2G), with most of those classified as encoding transmembrane or secreted proteins by GLAD analysis (Figure 2H). Similar to the case for upregulated genes, genes encoding serine proteases, kinases, and methyltransferases (referred to as “Other”) were also identified among downregulated genes (Figure 2H).
Multiple nuclear receptors have known roles in adipocytes in regulating lipid metabolism or controlling insulin secretion/sensitivity in mammals and Drosophila [reviewed in (King-Jones and Thummel 2005; Liu et al. 2015)]. For example, HNF4A-deficient mice are glucose intolerant and display impaired glucose-stimulated insulin secretion (Gupta et al. 2005), whereas mice heterozygous for the Svp homolog COUP-TFII have reduced adiposity and increased insulin sensitivity (Li et al. 2009). In Drosophila, Hnf4 (FBgn0004914) is required for lipid mobilization and β-oxidation in response to starvation in larvae (Palanker et al. 2009), whereas loss of svp in the larval fat body impairs lipid turnover and insulin signaling (Musselman et al. 2018). Surprisingly, however, the list of genes with significantly altered expression in the svp RNAi fat body was not enriched for genes encoding metabolic proteins based on GLAD or Gene Ontology (GO) analysis. In fact, genes encoding members of the insulin signaling, acetyl-CoA production, or β-oxidation pathways were not differentially expressed in svp knockdown fat bodies compared to RNAi controls (Table S2). These results suggest that, in stark contrast to known roles of nuclear receptors in regulating metabolic processes (e.g., HNF4, ERR, HR96) (Palanker et al. 2009; Sieber and Thummel 2009; Tennessen et al. 2011), Svp activity in the adult female fat body regulates genes that control other biological functions instead.
Svp induces genes involved in the early defense against microorganisms in the adult female fat body
To narrow down our list of candidate Svp targets, we first determined whether genes whose fat body expression levels were altered with a log2 fold change of at least ±1.5 when svp was knocked down in the adult female fat body belonged to common categories or pathways. Interestingly, we found that Svp positively regulates genes encoding proteins with roles in immunity, including those involved in particle recognition (e.g., PGRP-SC2, FBgn0043575), melanization and clotting (e.g., y, FBgn0004034), as well as anti-microbial peptides (e.g., Drsl4, FBgn0052282) (Figure 3A). For example, three microbial recognition proteins, which recognize bacteria through pattern recognition receptors and act upstream of the Toll and immune deficiency (IMD) pathways (Lemaitre and Hoffmann 2007), were significantly downregulated when svp was knocked down in the adult female fat body (Figure 3A-C). Peptidoglycan recognition protein (PGRP-SC2), whose transcript was downregulated with a log2 fold change of -3.2 (i.e., ∼9-fold) by svp RNAi (Figure 3A,B), is a secreted protein that scavenges bacteria in the hemolymph to prevent activation of the IMD pathway (Figure 3C) (Bischoff et al. 2006; Paredes et al. 2011). The immunoglobulin-superfamily receptor Down syndrome adhesion molecule 4 (Dscam4, FBgn0263219), whose transcript was downregulated with a log2 fold change of -4.4 by svp RNAi (Figure 3A,B), is a transmembrane protein required for phagocytic uptake of bacteria (Figure 3C) (Watson et al. 2005). CG7763 (FBgn0040503) was downregulated with a log2 fold change of -2.6 (Figure 3A,B) and encodes a predicted member of the C-type lectin family, which has been implicated in recognition of lipopolysaccharide [a component of gram negative bacterial cell walls (Zhang et al. 2013)] (Figure 3C) (Theopold et al. 1999). Drsl4, which encodes an antifungal peptide with homology to Drs (Jiggins and Kim 2005), was also significantly downregulated (log2 fold change of -1.9; Figure 3A,B) in the svp knockdown adult female fat body.
Knockdown of svp in adult female fat bodies reduces expression of genes involved in bacterial recognition, melanization, and clotting. (A) Classification of genes significantly downregulated with a log2 fold change of -1.5 or higher. (B) Quantification of normalized transcript expression (FPKM) for significantly downregulated recognition genes in control and svp RNAi fat bodies. Data shown as mean±SEM. *P <0.05; ***P <0.001, two-tailed Student’s t-test. (C) Cartoon of peptidoglycan recognition proteins (bound to peptidoglycan present in cell walls of most bacteria, yellow circles), whose transcripts are significantly downregulated by svp RNAi. (D) Quantification of normalized transcript expression (FPKM) for significantly downregulated melanization and clotting genes in control and svp RNAi fat bodies. Data shown as mean±SEM. *P <0.05; **P <0.01; ***P <0.001, two-tailed Student’s t-test. (E) Simplified cartoon of the melanization and clotting cascade in response injury. Proteins encoded by significantly downregulated genes in the absence of svp are included.
In addition to the responses involved in recognizing foreign pathogens upon injury and infection, another important immune defense is proper wound healing (Theopold et al. 2004). At the site of injury, a melanization cascade is initiated; melanization requires recognition of a pathogen (through pattern recognition receptors) followed by activation of phenoloxidase by serine proteases to ultimately produce melanin, which crosslinks and encapsulates microbes (Bidla et al. 2005; Binggeli et al. 2014). In addition to melanization, clotting is critical for limiting hemolymph loss as well as for physically immobilizing bacteria (Goto et al. 2003; Theopold et al. 2004). Our RNA sequencing analysis identified eight genes with roles in promoting melanization and clotting that are positively regulated by Svp in the adult female fat body (Figure 3D,F). For example, the y gene, downregulated with a log2 fold change of -4 (i.e., 16-fold) by svp RNAi (Figure 3D,E), is required for melanin production (Biessmann 1985; Whittkopp et al. 2002). Of the clotting genes regulated by Svp (Figure 3D,E), Fbp1 (FBgn0000639) encodes a known clotting factor originally isolated from larval hemolymph clots (Scherfer et al. 2004), and obst-j (FBgn0036940) encodes a chitin-binding protein that is upregulated in response to bacterial infection (Rynes et al. 2012). Lsp1α (FBgn0002562) and Lsp1β (FBgn0002563) encode predicted hemocyanins [proteins with known roles in regulating immunity (Coates and Nairn 2014)] and were also isolated from larval hemolymph clots (Scherfer et al. 2004). CG11313 (FBgn0039798), which is downregulated with a log2 fold change of -3.9 upon svp RNAi (Figure 3D,E), encodes a predicted serine protease that has potential roles in immunity (Karlsson et al. 2004).
Nuclear receptors have reported roles in regulating immunity (Glass and Ogawa 2006; Glass and Saijo 2010). For example, mammalian PPARγ is required for wound healing and promotes the transcription of anti-inflammatory genes in macrophages, whereas LXR is important for macrophage inflammatory responses [reviewed in (Leopold Wager et al. 2019)]. Immune response genes are differentially regulated in Drosophila mutants for Hr3 (FBgn0000448) (Ruaud et al. 2010), Hnf4 (Barry and Thummel 2016), and Hr4 (FBgn0264562) (King-Jones et al. 2005), suggesting that nuclear receptors also have immune response roles in invertebrates. However, only the developmental and metabolic roles of these nuclear receptors have been extensively studied [reviewed in (King-Jones and Thummel 2005)]. Our RNA sequencing analysis suggests that Svp normally functions in the adult female fat body as the first line of defense against infections (e.g., microbial and viral recognition, peptidoglycan scavenging, and clotting/melanization upon wounding), potentially preventing activation of the IMD pathway (Lemaitre and Hoffmann 2007). Although we did not observe an increase in anti-microbial peptide synthesis associated with IMD activation (Lee and Lee 2018), it is possible that our time window of svp knockdown is insufficiently long. In fact, a previous study in the larval fat body also showed that Svp regulates immune response genes including PGRP-SC2 and additional anti-microbial peptides (Musselman et al. 2018); however, Svp negatively regulates these genes in this context (Musselman et al. 2018). The seemingly opposite regulatory roles of Svp in larvae compared to adults could potentially be due to the organism’s distinct physiology at different developmental stages. For example, in larvae, nutrient availability plays a crucial role in growth and timing of metamorphosis (Colombani et al. 2003); by contrast, adult females have completed development but require large amounts of nutrients and energy to support egg production (Laws and Drummond-Barbosa 2017). The larval fat body is also remodeled during metamorphosis (Nelliot et al. 2006), which might alter the mechanisms linking immunity to physiology. Indeed, the nuclear receptor HNF4 was recently shown to be required in oenocytes for the conversion of larval lipid reservoirs into hydrocarbons for cuticular waterproofing in adults (Storelli et al. 2019), suggesting that the fat body is subject to different physiological demands depending on the developmental stage. Collectively, our results suggest that Svp may function in the adult female fat body to regulate genes that scavenge and eliminate foreign pathogens as a mechanism to prevent activation of the IMD pathway [which is an energy-intensive process (Buttgereit et al. 2000; Davoodi et al. 2019)], thereby freeing up resources to support reproduction in adult females.
Svp modulates the transcription of genes involved in xenobiotic responses
As a second strategy to identify a select group of candidate Svp targets, we also performed GO analysis [clusterProfiler R; (Yu et al. 2012)] of transcripts that were significantly altered when svp was knocked down in the adult female fat body. In accordance with the analysis described above (of genes with a log2 fold change of at least ±1.5), the GO analysis of our RNA sequencing data did not identify gene enrichment for any specific nutrient or metabolic pathway. Instead, GO analysis showed that a subset of genes involved in the response to xenobiotics (pollutants, insecticides, and drugs) are significantly regulated in response to svp knockdown (Figure 4A).
Differentially expressed transcripts are significantly enriched for genes involved in xenobiotic responses. (A) Gene ontology (GO) analysis of significantly upregulated (purple) and downregulated (green) genes in svp RNAi females compared to Luc control. (B) Some of the genes significantly enriched based on GO analysis that are up- or downregulated in the absence of svp. (C) Quantification of normalized transcript expression (FPKM) for significantly enriched genes in control and svp RNAi fat bodies. Data shown as mean±SEM. *P <0.05; **P <0.01; ***P <0.001, two-tailed Student’s t-test. (D) Simplified cartoon of detoxification of xenobiotics. Cyp4e3 and Cyp4p2 are predicted to localize to the endoplasmic reticulum (UniProt.org), whereas Cyp12d1-d is predicted to localize to the mitochondrial inner membrane (UniProt.org).
The breakdown of toxic molecules involves an elaborate three-phase system to metabolize xenobiotics into innocuous molecules and facilitate their excretion (Xu et al. 2005; Yu 2008). During phase I, xenobiotics are oxidized by cytochrome P450 (Cyp450) monooxygenases to introduce reactive and polar groups to substrates (Yu 2008). These proteins are membrane-bound monooxygenases that are involved in a wide array of physiological processes including detoxification, steroid metabolism, and fatty acid metabolism (Feyereisen 1999; Zangar et al. 2004; Xu et al. 2005). Xenobiotic metabolites generated during phase I are further converted by phase II enzymes [glutathione-S-transferases (GSTs), UDP-glucuronosyltransferases (UGTs), and carboxylases] through conjugation reactions that add functional side groups (such as hydroxyl, carboxyl, and epoxide) to increase hydrophobicity (Yu 2008). Phase III transporters [ATP-binding cassette (ABC) transporters and Organic anion transport proteins (Oatps)] act in the final phase of detoxification to export converted organic products out of the cell (Groen et al. 2017).
Genes involved in each step of the xenobiotic response were altered when svp was knocked down in the adult female fat body. Genes encoding members of the Cyp450 family (Cyp12d1-d, Cyp4e3, and Cyp4p2), which initiate phase I of the detoxification process (Xu et al. 2005), were significantly downregulated in svp knockdown fat bodies (Figure 4B-D). One exception was the gene phm (FBgn0004959), encoding a Cyp450 family member involved in ecdysteroid biosynthesis (Chávez et al. 2000), which was upregulated in the absence of svp (Table S5). Transcription of the phase II enzyme gene, GstD5 (FBgn0010041), was also significantly decreased in svp RNAi fat bodies (Figure 4B,C), suggesting that Svp normally induces expression of these genes. By contrast, genes encoding metallothionein (Mtn) proteins were significantly upregulated in svp knockdown fat bodies (Figure 4B-D), suggesting that Svp activity in the adult female fat body represses these genes. Mtn proteins are xenobiotic-induced enzymes involved in heavy metal detoxification and protection against free radicals, and have been implicated in the response to xenobiotic and immune stress (Figure 4D) (Bonneton et al. 1996). Additionally, genes encoding detoxification phase III transporters (Oatps) were also upregulated in the fat body in the absence of svp (Figure 4B-D; Table S5). Interestingly, svp knockdown did not affect the transcript levels of cap-n-collar [cnc (FBgn0262975); encodes Nrf2 homolog] or forkhead box, sub-group O [foxo (FBgn0038197)](Table S2), which encode known regulators of detoxifying and antioxidant genes (Ma 2013; Klotz et al. 2015). Comparison of known Cnc targets identified by microarray (Misra et al. 2011) with those determine by our RNA sequencing analysis identified 22 common transcripts (Table S6), consisting mostly of the detoxifying proteins described above. In addition, comparison of Svp-regulated genes with FOXO targets identified by ChIP-seq (Birnbaum et al. 2019) revealed at least 10 common transcriptional targets, including Cyp4e3 (FBgn0015035) (Table S7). Thus, Svp appears to share some common targets involved in xenobiotic responses with Cnc and Foxo.
It is conceivable that the upregulation of Mtn and Oatp transcripts is a secondary consequence of the downregulation of Cyp4e3 when svp is knocked down in the adult female fat body. It was previously shown that loss of Cyp4e3 in Malpighian tubules increases the levels of hydrogen peroxide and induces JAK/STAT and NF-κB-mediated stress responses (Terhzaz et al. 2015). Mtn transcription is induced in mammalian hepatic cell lines in response to hydrogen peroxide (Dalton et al. 1994). Mtns are able to scavenge a variety of reactive oxygen species (ROS), including hydrogen peroxide (Inoue et al. 2009), suggesting that Mtns protect against oxidative stress in addition to regulating metal abundance. Therefore, we speculate that svp knockdown in the fat body may cause the accumulation of toxic molecules (as a result of Cyp4e3 downregulation), leading to increased expression of Mtns and Oatps to eliminate these compounds from the cell.
Exposure to xenobiotics has long been known to result in perturbations of the immune system (Luster et al. 1989). In addition to their role in metal metabolism, Mtns have also been implicated in immune functions. Specifically, transcription of mammalian hepatocyte MT-1 is activated by STAT binding in response to lipopolysaccharide exposure (Arizono et al. 1995; Coyle et al. 2002), suggesting that under increased immune challenge Mtns possibly act as antioxidants in response to inflammation (Arizono et al. 1995). It is unclear whether Drosophila Mtn proteins have roles in regulating the immune response or whether they are induced downstream of the JAK/STAT pathway [potentially also downstream of reduction of Cyp4e3 (Terhzaz et al. 2015)]. In addition, it is unclear whether knockdown of svp in adult female fat bodies activates the JAK/STAT pathway. Nevertheless, it would be interesting to investigate whether the increased transcription of Mtn and Oatp genes observed when svp is knocked down might reflect the regulatory cross-talk between Mtns and immune response pathways. Collectively, our RNA sequencing results suggest that loss of svp and the reduced expression Cyp450 xenobiotic response genes in the adult female fat body could potentially result in accumulation of toxic compounds, causing activation of secondary downstream stress mechanisms.
Oogenesis is an energy-intensive process that is tightly regulated to ensure reproductive success (Schwenke et al. 2016; Laws and Drummond-Barbosa 2017). Mounting a full immune response and chemically inactivating harmful toxins from the body also require a significant amount of energy (Buttgereit et al. 2000). Our RNA sequencing analysis of potential Svp targets in the adult female fat body suggests that Svp normally functions to: 1) enhance the initial organismal defense against microorganisms and thus prevent a full immune response (e.g., involving the IMD pathway); and 2) optimize the levels of Cyp450s for detoxification of xenobiotics to prevent activation of stress response pathways. Through these actions, Svp might help allocate resources to promote distinct aspects of oogenesis (Figure 5). This model will be tested in future studies, and many additional questions remain regarding this potential model and beyond. For example, it is unknown whether, upon svp knockdown, reduced expression of bacterial recognition proteins (and thereby hampered initial bacterial defense) might lead to the activation of the humoral/IMD immune system at a later time point in the fat body. It is also possible that reduced levels of detoxification enzymes in the absence of svp might result in the accumulation of xenobiotics that activate an immune response, reminiscent of mammalian innate T-cells that recognize and process allergens and other environmental chemicals prior to their presentation to lymphocytes (Minnicozzi et al. 2011; Germolec et al. 2017; Maeda et al. 2019). It is also not known at this point whether these changes in immune function and xenobiotic response in the absence of svp occur in adipocytes, oenocytes, or both cell types, or what their functional consequence is to the steps of oogenesis affected by adipocyte/oenocyte Svp activity (Weaver and Drummond-Barbosa 2019). Finally, it remains to be tested whether the differentially expressed genes identified in our dataset contribute to the regulation of the processes of oogenesis that are affected by svp knockdown in adipocytes and oenocytes (Weaver and Drummond-Barbosa 2019). Deciphering the complex mechanisms of Svp activity in the adult female fat body and how they regulate distinct aspects of oogenesis will be a fertile area for future research.
Model for proposed role of Svp in the adult female fat body to regulate oogenesis. Our previous work showed that Svp is required in adult female adipocytes to control GSC number and early germline cyst survival and in oenocytes for vitellogenic follicle survival. This study identifies potential downstream targets for Svp and suggests the model that Svp activity in the fat body might promote oogenesis through a mechanism involving an enhancement of the initial defense against bacteria (to prevent full immune activation) and of Cyp450 detoxification mechanisms (to inhibit secondary stress response pathways).
Acknowledgments
Stocks obtained from the Bloomington Drosophila Stock Center (NIH P40 OD018537) and the Vienna Drosophila Resource Center (VDRC, www.vdrc.at) (Dietzl et al. 2007) were used in this study. We are thankful to Flybase (www.flybase.org), an essential Drosophila research resource. We are grateful to members of the Drummond-Barbosa Lab for critical reading of the manuscript. This work was supported by National Institutes of Health R01 GM069875 (D.D.-B.) and Pathway to Independence Award K99 GM127605 and R00 GM127605 (L.N.W.) grants.
Footnotes
Supplemental material available at figshare: https://doi.org/10.25387/g3.13122728.
Communicating editor: H. Salz
- Received August 16, 2020.
- Accepted October 17, 2020.
- Copyright © 2020 Weaver, Drummond-Barbosa
This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.