Cell-Specific Imd-NF-κB Responses Enable Simultaneous Antibacterial Immunity and Intestinal Epithelial Cell Shedding upon Bacterial Infection.

Intestinal infection triggers potent immune responses to combat pathogens and concomitantly drives epithelial renewal to maintain barrier integrity. Current models propose that epithelial renewal is primarily driven by damage caused by reactive oxygen species (ROS). Here we found that in Drosophila, the Imd-NF-κB pathway controlled enterocyte (EC) shedding upon infection, via a mechanism independent of ROS-associated apoptosis. Mechanistically, the Imd pathway synergized with JNK signaling to induce epithelial cell shedding specifically in the context of bacterial infection, requiring also the reduced expression of the transcription factor GATAe. Furthermore, cell-specific NF-κB responses enabled simultaneous production of antimicrobial peptides (AMPs) and epithelial shedding in different EC populations. Thus, the Imd-NF-κB pathway is central to the intestinal antibacterial response by mediating both AMP production and the maintenance of barrier integrity. Considering the similarities between Drosophila Imd signaling and mammalian TNFR pathway, our findings suggest the existence of an evolutionarily conserved genetic program in immunity-induced epithelial shedding.

The Drosophila NPY-like system protects against chronic stress-induced learning deficit by preventing the disruption of autophagic flux.

Chronic stress may induce learning and memory deficits that are associated with a depression-like state in Drosophila melanogaster. The molecular and neural mechanisms underlying the etiology of chronic stress-induced learning deficit (CSLD) remain elusive. Here, we show that the autophagy-lysosomal pathway, a conserved cellular signaling mechanism, is associated with chronic stress in Drosophila, as indicated by time-series transcriptome profiling. Our findings demonstrate that chronic stress induces the disruption of autophagic flux, and chronic disruption of autophagic flux could lead to a learning deficit. Remarkably, preventing the disruption of autophagic flux by up-regulating the basal autophagy level is sufficient to protect against CSLD. Consistent with the essential role of the dopaminergic system in modulating susceptibility to CSLD, dopamine neuronal activity is also indispensable for chronic stress to induce the disruption of autophagic flux. By screening knockout mutants, we found that neuropeptide F, the Drosophila homolog of neuropeptide Y, is necessary for normal autophagic flux and promotes resilience to CSLD. Moreover, neuropeptide F signaling during chronic stress treatment promotes resilience to CSLD by preventing the disruption of autophagic flux. Importantly, neuropeptide F receptor activity in dopamine neurons also promotes resilience to CSLD. Together, our data elucidate a mechanism by which stress-induced excessive dopaminergic activity precipitates the disruption of autophagic flux, and chronic disruption of autophagic flux leads to CSLD, while inhibitory neuropeptide F signaling to dopamine neurons promotes resilience to CSLD by preventing the disruption of autophagic flux.

Opsin1 regulates light-evoked avoidance behavior in Aedes albopictus.

BACKGROUND: Mosquitoes locate a human host by integrating various sensory cues including odor, thermo, and vision. However, their innate light preference and its genetic basis that may predict the spatial distribution of mosquitoes, a prerequisite to encounter a potential host and initiate host-seeking behaviors, remains elusive. RESULTS: Here, we first studied mosquito visual features and surprisingly uncovered that both diurnal (Aedes aegypti and Aedes albopictus) and nocturnal (Culex quinquefasciatus) mosquitoes significantly avoided stronger light when given choices. With consistent results from multiple assays, we found that such negative phototaxis maintained throughout development to adult stages. Notably, female mosquitoes significantly preferred to bite hosts in a shaded versus illuminated area. Furthermore, silencing Opsin1, a G protein-coupled receptor that is most enriched in compound eyes, abolished light-evoked avoidance behavior of Aedes albopictus and attenuated photonegative behavior in Aedes aegypti. Finally, we found that field-collected Aedes albopictus also prefers darker area in an Opsin1-dependent manner. CONCLUSIONS: This study reveals that mosquitoes consistently prefer darker environment and identifies the first example of a visual molecule that modulates mosquito photobehavior.

A genetic framework controlling the differentiation of intestinal stem cells during regeneration in Drosophila.

The speed of stem cell differentiation has to be properly coupled with self-renewal, both under basal conditions for tissue maintenance and during regeneration for tissue repair. Using the Drosophila midgut model, we analyze at the cellular and molecular levels the differentiation program required for robust regeneration. We observe that the intestinal stem cell (ISC) and its differentiating daughter, the enteroblast (EB), form extended cell-cell contacts in regenerating intestines. The contact between progenitors is stabilized by cell adhesion molecules, and can be dynamically remodeled to elicit optimal juxtacrine Notch signaling to determine the speed of progenitor differentiation. Notably, increasing the adhesion property of progenitors by expressing Connectin is sufficient to induce rapid progenitor differentiation. We further demonstrate that JAK/STAT signaling, Sox21a and GATAe form a functional relay to orchestrate EB differentiation. Thus, our study provides new insights into the complex and sequential events that are required for rapid differentiation following stem cell division during tissue replenishment.

Antagonistic regulation of apoptosis and differentiation by the Cut transcription factor represents a tumor-suppressing mechanism in Drosophila.

Apoptosis is essential to prevent oncogenic transformation by triggering self-destruction of harmful cells, including those unable to differentiate. However, the mechanisms linking impaired cell differentiation and apoptosis during development and disease are not well understood. Here we report that the Drosophila transcription factor Cut coordinately controls differentiation and repression of apoptosis via direct regulation of the pro-apoptotic gene reaper. We also demonstrate that this regulatory circuit acts in diverse cell lineages to remove uncommitted precursor cells in status nascendi and thereby interferes with their potential to develop into cancer cells. Consistent with the role of Cut homologues in controlling cell death in vertebrates, we find repression of apoptosis regulators by Cux1 in human cancer cells. Finally, we present evidence that suggests that other lineage-restricted specification factors employ a similar mechanism to put the brakes on the oncogenic process.

High-fat diets induce inflammatory IMD/NFκB signaling via gut microbiota remodeling in Drosophila.

High-fat diets (HFDs), a prevailing daily dietary style worldwide, induce chronic low-grade inflammation in the central nervous system and peripheral tissues, promoting a variety of diseases including pathologies associated with neuroinflammation. However, the mechanisms linking HFDs to inflammation are not entirely clear. Here, using a Drosophila HFD model, we explored the mechanism of HFD-induced inflammation in remote tissues. We found that HFDs activated the IMD/NFκB immune pathway in the head through remodeling of the commensal gut bacteria. Removal of gut microbiota abolished such HFD-induced remote inflammatory response. Further experiments revealed that HFDs significantly increased the abundance of Acetobacter malorum in the gut, and the re-association of this bacterium was sufficient to elicit inflammatory response in remote tissues. Mechanistically, Acetobacter malorum produced a greater amount of peptidoglycan (PGN), a well-defined microbial molecular pattern that enters the circulation and remotely activates an inflammatory response. Our results thus show that HFDs trigger inflammation mediated by a bacterial molecular pattern that elicits host immune response.

Dietary L-Glu sensing by enteroendocrine cells adjusts food intake via modulating gut PYY/NPF secretion.

Amino acid availability is monitored by animals to adapt to their nutritional environment. Beyond gustatory receptors and systemic amino acid sensors, enteroendocrine cells (EECs) are believed to directly percept dietary amino acids and secrete regulatory peptides. However, the cellular machinery underlying amino acid-sensing by EECs and how EEC-derived hormones modulate feeding behavior remain elusive. Here, by developing tools to specifically manipulate EECs, we find that Drosophila neuropeptide F (NPF) from mated female EECs inhibits feeding, similar to human PYY. Mechanistically, dietary L-Glutamate acts through the metabotropic glutamate receptor mGluR to decelerate calcium oscillations in EECs, thereby causing reduced NPF secretion via dense-core vesicles. Furthermore, two dopaminergic enteric neurons expressing NPFR perceive EEC-derived NPF and relay an anorexigenic signal to the brain. Thus, our findings provide mechanistic insights into how EECs assess food quality and identify a conserved mode of action that explains how gut NPF/PYY modulates food intake.

Regulation of intestinal stem cell activity by a mitotic cell cycle regulator Polo in Drosophila.

Maintaining a definite and stable pool of dividing stem cells plays an important role in organ development. This process requires an appropriate progression of mitosis for proper spindle orientation and polarity to ensure the ability of stem cells to proliferate and differentiate correctly. Polo-like kinases (Plks)/Polo are the highly conserved serine/threonine kinases involved in the initiation of mitosis as well as in the progression of the cell cycle. Although numerous studies have investigated the mitotic defects upon loss of Plks/Polo in cells, little is known about the in vivo consequences of stem cells with abnormal Polo activity in the context of tissue and organism development. The current study aimed to investigate this question using the Drosophila intestine, an organ dynamically maintained by the intestinal stem cells (ISCs). The results indicated that the polo depletion caused a reduction in the gut size due to a gradual decrease in the number of functional ISCs. Interestingly, the polo-deficient ISCs showed an extended G2/M phase and aneuploidy and were subsequently eliminated by premature differentiation into enterocytes (ECs). In contrast, the constitutively active Polo (poloT182D) suppressed ISC proliferation, induced abnormal accumulation of ß-tubulin in cells, and drove ISC loss via apoptosis. Therefore, Polo activity should be properly maintained for optimal stem cell function. Further analysis suggested that polo was a direct target gene of Sox21a, a Sox transcription factor that critically regulates stem cell activity. Together, this study provided a novel perspective on the correlation between the progression of mitosis and the ISC function in Drosophila.

Transcriptome analysis of sex-biased gene expression in the spotted-wing Drosophila, Drosophila suzukii (Matsumura).

Sexual dimorphism occurs widely throughout insects and has profound influences on evolutionary path. Sex-biased genes are considered to account for most of phenotypic differences between sexes. In order to explore the sex-biased genes potentially associated with sexual dimorphism and sexual development in Drosophila suzukii, a major devastating and invasive crop pest, we conducted whole-organism transcriptome profiling and sex-biased gene expression analysis on adults of both sexes. We identified transcripts of genes involved in several sex-specific physiological and functional processes, including transcripts involved in sex determination, reproduction, olfaction, and innate immune signals. A total of 11,360 differentially expressed genes were identified in the comparison, and 1,957 differentially expressed genes were female-biased and 4,231 differentially expressed genes were male-biased. The pathway predominantly enriched for differentially expressed genes was related to spliceosome, which might reflect the differences in the alternative splicing mechanism between males and females. Twenty-two sex determination and 16 sex-related reproduction genes were identified, and expression pattern analysis revealed that the majority of genes were differentially expressed between sexes. Additionally, the differences in sex-specific olfactory and immune processes were analyzed and the sex-biased expression of these genes may play important roles in pheromone and odor detection, and immune response. As a valuable dataset, our sex-specific transcriptomic data can significantly contribute to the fundamental elucidation of the molecular mechanisms of sexual dimorphism in fruit flies, and may provide candidate genes potentially useful for the development of genetic sexing strains, an important tool for sterile insect technique applications against this economically important species.

Antioxidant potential of Pediococcus pentosaceus strains from the sow milk bacterial collection in weaned piglets.

BACKGROUND: In modern animal husbandry, breeders pay increasing attention to improving sow nutrition during pregnancy and lactation to favor the health of neonates. Sow milk is a main food source for piglets during their first three weeks of life, which is not only a rich repository of essential nutrients and a broad range of bioactive compounds, but also an indispensable source of commensal bacteria. Maternal milk microorganisms are important sources of commensal bacteria for the neonatal gut. Bacteria from maternal milk may confer a health benefit on the host. METHODS: Sow milk bacteria were isolated using culturomics followed by identification using 16S rRNA gene sequencing. To screen isolates for potential probiotic activity, the functional evaluation was conducted to assess their antagonistic activity against pathogens in vitro and evaluate their resistance against oxidative stress in damaged Drosophila induced by paraquat. In a piglet feeding trial, a total of 54 newborn suckling piglets were chosen from nine sows and randomly assigned to three treatments with different concentrations of a candidate strain. Multiple approaches were carried out to verify its antioxidant function including western blotting, enzyme activity analysis, metabolomics and 16S rRNA gene amplicon sequencing. RESULTS: The 1240 isolates were screened out from the sow milk microbiota and grouped into 271 bacterial taxa based on a nonredundant set of 16S rRNA gene sequencing. Among 80 Pediococcus isolates, a new Pediococcus pentosaceus strain (SMM914) showed the best performance in inhibition ability against swine pathogens and in a Drosophila model challenged by paraquat. Pretreatment of piglets with SMM914 induced the Nrf2-Keap1 antioxidant signaling pathway and greatly affected the pathways of amino acid metabolism and lipid metabolism in plasma. In the colon, the relative abundance of Lactobacillus was significantly increased in the high dose SMM914 group compared with the control group. CONCLUSION: P. pentosaceus SMM914 is a promising probiotic conferring antioxidant capacity by activating the Nrf2-Keap1 antioxidant signaling pathway in piglets. Our study provided useful resources for better understanding the relationships between the maternal microbiota and offspring. Video Abstract.

Functional dissection of the Hox protein Abdominal-B in Drosophila cell culture.

Hox transcription factors regulate the morphogenesis along the anterior-posterior (A/P) body axis through the interaction with small cis-regulatory modules (CRMs) of their target gene, however so far very few Hox CRMs are known and have been analyzed in detail. In this study we have identified a new Hox CRM, ct340, which guides the expression of the cell type specification gene cut (ct) in the posterior spiracle under the direct control of the Hox protein Abdominal-B (Abd-B). Using the ct340 enhancer activity as readout, an efficient cloning system to generate VP16 activation domain fusion protein was developed to unambiguously test protein-DNA interaction in Drosophila cell culture. By functionally dissecting the Abd-B protein, new features of Abd-B dependent target gene regulation were detected. Due to its easy adaptability, this system can be generally used to map functional domains within sequence-specific transcriptional factors in Drosophila cell culture, and thus provide preliminary knowledge of the protein functional domain structure for further in vivo analysis.

Vectors for efficient and high-throughput construction of fluorescent drosophila reporters using the PhiC31 site-specific integration system.

The fruit fly Drosophila is a leading model system for the study of transcriptional control by cis-regulatory elements or enhancers. Here, we present a rapid and highly efficient system for the large-scale analysis of enhancer elements, site-specifically integrated into the Drosophila genome. This system, which is scalable for either small projects or high-throughput approaches, makes use of the Gateway cloning technology and the PhiC31 site-specific integration system, which allows the insertion of constructs at predetermined genomic locations. Thus, this system allows not only a fast and easy analysis of reporter gene expression in live animals, but also the simultaneous analysis of different regulatory outputs on a cellular resolution by recombining in the same animal distinct enhancer elements fused to different fluorescent proteins.

Identification of mucins and their expression in the vector mosquito Aedes albopictus.

Mucins, the main structural components of vertebrate respiratory, digestive and reproductive tract mucus, as well as insect peritrophic matrix, play important roles in protecting host cells from invading microbes and difficult external environments. Mucins are characterized by highly glycosylated proteins constituting the mucin domain that is rich in repetitive sequences of threonine, serine, and proline (PTS). Despite potential important roles, mosquito mucins remain largely uncharacterized. Here, we performed bioinformatics analyses to identify proteins with PTS repeat domain and predicted 43 mucins or mucin-related proteins in Aedes albopictus. Gene expression analysis revealed that these mucins are dynamically expressed across different development stages and in different organs of Aedes albopictus. Of note, blood feeding upregulated AALF016448 and AALF013291 expression in the midgut, fat body, and ovary, raising the possibility that these mucins play potential roles in reproduction, digestion, and intestinal defense against invading pathogens upon blood feeding. Our in silico identification, followed by expressional validation, thus established a valuable resource for further dissecting the functions of mucins for vector control.

Expression of the apoptosis gene reaper in homeotic, segmentation and other mutants in Drosophila.

Apoptosis is an essential process required for development and morphogenesis in metazoan organisms. The apoptosis pathway and cell death machinery have been extensively studied, but little is known how apoptosis genes are regulated in the course of development . In this study, we analyzed the transcriptional regulation of the pro-apoptotic gene reaper (rpr) by performing whole-mount in situ hybridization in embryos mutant for a number of transcription factor genes in Drosophila melanogaster. In sum, our data show that all factors studied have very specific temporal and spatial effects on rpr transcription . Thus, our results reinforce the concept that apoptosis is an essential process for morphogenesis and that apoptosis related genes very tight developmental factors identified in sculpting the morphology of various embryonic structures by modulating the apoptosis pathway.

Beyond immunity: The Imd pathway as a coordinator of host defense, organismal physiology and behavior.

The humoral arm of host defense in Drosophila relies on two evolutionarily conserved NFκB signaling cascades, the Toll and the immune deficiency (Imd) pathways. The Imd signaling pathway senses and neutralizes Gram-negative bacteria. Its activity is tightly adjusted, allowing the host to simultaneously prevent infection by pathogenic bacteria and tolerate beneficial gut microbiota. Over-activation of Imd signaling is detrimental at least in part by causing gut dysbiosis that further exacerbates intestinal pathologies. Furthermore, it is increasingly recognized that the Imd pathway or its components also play non-immune roles. In this review, we summarize recent advances in Imd signal transduction, discuss the gut-microbiota interactions mediated by Imd signaling, and finally elaborate on its diverse physiological functions beyond immunity. Understanding the multifaceted physiological outputs of Imd activation will help integrate its immune role into the regulation of whole organismal physiology.

Accumulation of differentiating intestinal stem cell progenies drives tumorigenesis.

Stem cell self-renewal and differentiation are coordinated to maintain tissue homeostasis and prevent cancer. Mutations causing stem cell proliferation are traditionally the focus of cancer studies. However, the contribution of the differentiating stem cell progenies in tumorigenesis is poorly characterized. Here we report that loss of the SOX transcription factor, Sox21a, blocks the differentiation programme of enteroblast (EB), the intestinal stem cell progeny in the adult Drosophila midgut. This results in EB accumulation and formation of tumours. Sox21a tumour initiation and growth involve stem cell proliferation induced by the unpaired 2 mitogen released from accumulating EBs generating a feed-forward loop. EBs found in the tumours are heterogeneous and grow towards the intestinal lumen. Sox21a tumours modulate their environment by secreting matrix metalloproteinase and reactive oxygen species. Enterocytes surrounding the tumours are eliminated through delamination allowing tumour progression, a process requiring JNK activation. Our data highlight the tumorigenic properties of transit differentiating cells.

Cellular analysis of newly identified Hox downstream genes in Drosophila.

Hox genes code for conserved homeodomain transcription factors, which act as regional regulators for the specification of segmental identities along the anterior-posterior axis in all animals studied. They execute their function mainly through the activation or repression of their downstream genes. We have recently identified a large number of genes to be directly or indirectly targeted by Hox proteins through gene expression profiling in the model organism Drosophila. However, the cell-specific regulation of these downstream genes and the functional significance of the regulation are largely unknown. We have validated and functionally studied many of the newly identified downstream genes of the Hox proteins Deformed (Dfd) and Abdominal-B (Abd-B), and provide evidence that Hox proteins regulate a diverse group of downstream genes, from transcription factors to realisators with major and minor roles during morphogenesis.