ABSTRACT
In cells, organs and whole organisms, nutrient sensing is key to maintaining homeostasis and adapting to a fluctuating environment1. In many animals, nutrient sensors are found within the enteroendocrine cells of the digestive system; however, less is known about nutrient sensing in their cellular siblings, the absorptive enterocytes1. Here we use a genetic screen in Drosophila melanogaster to identify Hodor, an ionotropic receptor in enterocytes that sustains larval development, particularly in nutrient-scarce conditions. Experiments in Xenopus oocytes and flies indicate that Hodor is a pH-sensitive, zinc-gated chloride channel that mediates a previously unrecognized dietary preference for zinc. Hodor controls systemic growth from a subset of enterocytes-interstitial cells-by promoting food intake and insulin/IGF signalling. Although Hodor sustains gut luminal acidity and restrains microbial loads, its effect on systemic growth results from the modulation of Tor signalling and lysosomal homeostasis within interstitial cells. Hodor-like genes are insect-specific, and may represent targets for the control of disease vectors. Indeed, CRISPR-Cas9 genome editing revealed that the single hodor orthologue in Anopheles gambiae is an essential gene. Our findings highlight the need to consider the instructive contributions of metals-and, more generally, micronutrients-to energy homeostasis.
Subject(s)
Chloride Channels/metabolism , Drosophila Proteins/metabolism , Drosophila melanogaster/growth & development , Drosophila melanogaster/metabolism , Eating/physiology , Intestines/physiology , Zinc/metabolism , Animals , Drosophila melanogaster/genetics , Enterocytes/metabolism , Female , Food Preferences , Homeostasis , Insect Vectors , Insulin/metabolism , Ion Channel Gating , Larva/genetics , Larva/growth & development , Larva/metabolism , Lysosomes/metabolism , Male , Oocytes/metabolism , Receptor Protein-Tyrosine Kinases/metabolism , Signal Transduction , XenopusABSTRACT
The gut sets the immune and metabolic parameters for the survival of commensal bacteria. We report that in Drosophila, deficiency in bacterial recognition upstream of Toll/NF-κB signalling resulted in reduced density and diversity of gut bacteria. Translational regulation factor 4E-BP, a transcriptional target of Toll/NF-κB, mediated this host-bacteriome interaction. In healthy flies, Toll activated 4E-BP, which enabled fat catabolism, which resulted in sustaining of the bacteriome. The presence of gut bacteria kept Toll signalling activity thus ensuring the feedback loop of their own preservation. When Toll activity was absent, TOR-mediated suppression of 4E-BP made fat resources inaccessible and this correlated with loss of intestinal bacterial density. This could be overcome by genetic or pharmacological inhibition of TOR, which restored bacterial density. Our results give insights into how an animal integrates immune sensing and metabolism to maintain indigenous bacteria in a healthy gut.
Subject(s)
Bacteria/growth & development , Carrier Proteins/metabolism , DNA-Binding Proteins/metabolism , Drosophila Proteins/metabolism , Drosophila melanogaster/microbiology , Toll-Like Receptors/metabolism , Transcription Factors/metabolism , Animals , Bacteria/immunology , Carrier Proteins/genetics , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Feedback, Physiological , Gastrointestinal Microbiome , NF-kappa B/metabolism , Signal Transduction , SymbiosisABSTRACT
[This corrects the article DOI: 10.1371/journal.pgen.1009992.].
ABSTRACT
Male reproductive glands like the mammalian prostate and the paired Drosophila melanogaster accessory glands secrete seminal fluid components that enhance fecundity. In humans, the prostate, stimulated by environmentally regulated endocrine and local androgens, grows throughout adult life. We previously showed that in fly accessory glands, secondary cells (SCs) and their nuclei also grow in adults, a process enhanced by mating and controlled by bone morphogenetic protein (BMP) signalling. Here, we demonstrate that BMP-mediated SC growth is dependent on the receptor for the developmental steroid ecdysone, whose concentration is reported to reflect sociosexual experience in adults. BMP signalling appears to regulate ecdysone receptor (EcR) levels via one or more mechanisms involving the EcR's N terminus or the RNA sequence that encodes it. Nuclear growth in virgin males is dependent on ecdysone, some of which is synthesised in SCs. However, mating induces additional BMP-mediated nuclear growth via a cell type-specific form of hormone-independent EcR signalling, which drives genome endoreplication in a subset of adult SCs. Switching to hormone-independent endoreplication after mating allows growth and secretion to be hyperactivated independently of ecdysone levels in SCs, permitting more rapid replenishment of the accessory gland luminal contents. Our data suggest mechanistic parallels between this physiological, behaviour-induced signalling switch and altered pathological signalling associated with prostate cancer progression.
Subject(s)
Bone Morphogenetic Proteins/genetics , Drosophila Proteins/genetics , Drosophila melanogaster/genetics , Ecdysone/metabolism , Genome, Insect , Insect Proteins/genetics , Receptors, Steroid/genetics , Animals , Bone Morphogenetic Proteins/metabolism , Cell Nucleus/metabolism , Cell Nucleus/ultrastructure , Copulation/physiology , Drosophila Proteins/metabolism , Drosophila melanogaster/cytology , Drosophila melanogaster/growth & development , Drosophila melanogaster/metabolism , Female , Gene Expression Regulation, Developmental , Insect Proteins/antagonists & inhibitors , Insect Proteins/metabolism , Male , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Receptors, Steroid/antagonists & inhibitors , Receptors, Steroid/metabolism , Signal TransductionABSTRACT
Regulated secretion by glands and neurons involves release of signalling molecules and enzymes selectively concentrated in dense-core granules (DCGs). Although we understand how many secretagogues stimulate DCG release, how DCG biogenesis is then accelerated to replenish the DCG pool remains poorly characterised. Here we demonstrate that each prostate-like secondary cell (SC) in the paired adult Drosophila melanogaster male accessory glands contains approximately ten large DCGs, which are loaded with the Bone Morphogenetic Protein (BMP) ligand Decapentaplegic (Dpp). These DCGs can be marked in living tissue by a glycophosphatidylinositol (GPI) lipid-anchored form of GFP. In virgin males, BMP signalling is sporadically activated by constitutive DCG secretion. Upon mating, approximately four DCGs are typically released immediately, increasing BMP signalling, primarily via an autocrine mechanism. Using inducible knockdown specifically in adult SCs, we show that secretion requires the Soluble NSF Attachment Protein, SNAP24. Furthermore, mating-dependent BMP signalling not only promotes cell growth, but is also necessary to accelerate biogenesis of new DCGs, restoring DCG number within 24 h. Our analysis therefore reveals an autocrine BMP-mediated feedback mechanism for matching DCG release to replenishment as secretion rates fluctuate, and might explain why in other disease-relevant systems, like pancreatic ß-cells, BMP signalling is also implicated in the control of secretion.
Subject(s)
Bone Morphogenetic Proteins/genetics , Drosophila Proteins/genetics , Drosophila melanogaster/genetics , Secretory Vesicles/genetics , Animals , Autocrine Communication/genetics , Drosophila Proteins/biosynthesis , Drosophila melanogaster/growth & development , Gene Expression Regulation, Developmental/genetics , Male , Neurons/metabolism , Prostate/growth & development , Prostate/metabolism , Secretory Vesicles/metabolism , Sexual Behavior, Animal/physiology , Signal Transduction/geneticsABSTRACT
The intestine maintains homeostasis by coordinating internal biological processes to adjust to fluctuating external conditions. The intestinal epithelium is continuously renewed and comprises multiple cell types, including absorptive cells, secretory cells, and resident stem cells. An important feature of this organ is its ability to coordinate many processes including cell proliferation, differentiation, regeneration, damage/stress response, immune activity, feeding behavior, and age-related changes by using conserved signaling pathways. However, the subcellular spatial organization of these signaling events and the organelles involved has only recently been studied in detail. Here we discuss how organelles of intestinal cells serve to initiate, mediate, and terminate signals, that are vital for homeostasis.
Subject(s)
Intestinal Mucosa , Stem Cells , Cell Differentiation , Cell Proliferation , OrganellesABSTRACT
CRISPR-Cas has revolutionized genetics and extensive efforts have been made to enhance its editing efficiency by developing increasingly more elaborate tools. Here, we evaluate the CRISPR-Cas9 system in Drosophila melanogaster to assess its ability to induce stem cell-derived tumors in the intestine. We generated conditional tissue-specific CRISPR knockouts using different Cas9 expression vectors with guide RNAs targeting the BMP, Notch, and JNK pathways in intestinal progenitors such as stem cells (ISCs) and enteroblasts (EBs). Perturbing Notch and BMP signaling increased the proliferation of ISCs/EBs and resulted in the formation of intestinal tumors, albeit with different efficiencies. By assessing both the anterior and posterior regions of the midgut, we observed regional differences in ISC/EB proliferation and tumor formation upon mutagenesis. Surprisingly, high continuous expression of Cas9 in ISCs/EBs blocked age-dependent increase in ISCs/EBs proliferation and when combined with gRNAs targeting tumor suppressors, it prevented tumorigenesis. However, no such effects were seen when temporal parameters of Cas9 were adjusted to regulate its expression levels or with a genetically modified version, which expresses Cas9 at lower levels, suggesting that fine-tuning Cas9 expression is essential to avoid deleterious effects. Our findings suggest that modifications to Cas9 expression results in differences in editing efficiency and careful considerations are required when choosing reagents for CRISPR-Cas9 mutagenesis studies. In summary, Drosophila can serve as a powerful model for context-dependent CRISPR-Cas based perturbations and to test genome-editing systems in vivo.
Subject(s)
CRISPR-Cas Systems/genetics , Drosophila melanogaster/genetics , Gene Editing , Intestinal Neoplasms/genetics , Animals , CRISPR-Associated Protein 9/metabolism , Digestive System/metabolism , Digestive System/pathology , Genes, Tumor Suppressor , Intestinal Neoplasms/pathology , Mitosis , Mutation/genetics , Stem Cells/metabolism , Time FactorsABSTRACT
Male reproductive glands secrete signals into seminal fluid to facilitate reproductive success. In Drosophila melanogaster, these signals are generated by a variety of seminal peptides, many produced by the accessory glands (AGs). One epithelial cell type in the adult male AGs, the secondary cell (SC), grows selectively in response to bone morphogenetic protein (BMP) signaling. This signaling is involved in blocking the rapid remating of mated females, which contributes to the reproductive advantage of the first male to mate. In this paper, we show that SCs secrete exosomes, membrane-bound vesicles generated inside late endosomal multivesicular bodies (MVBs). After mating, exosomes fuse with sperm (as also seen in vitro for human prostate-derived exosomes and sperm) and interact with female reproductive tract epithelia. Exosome release was required to inhibit female remating behavior, suggesting that exosomes are downstream effectors of BMP signaling. Indeed, when BMP signaling was reduced in SCs, vesicles were still formed in MVBs but not secreted as exosomes. These results demonstrate a new function for the MVB-exosome pathway in the reproductive tract that appears to be conserved across evolution.