The Nuclear Receptor Seven Up Regulates Genes Involved in Immunity and Xenobiotic Response in the Adult Drosophila Female Fat Body

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.

Keywords: Drosophila; Seven up; fat body; immunity; oogenesis; xenobiotics.

Figure 1

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.

Figure 2

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.

Figure 3

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.

Figure 4

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).

Figure 5

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).