ABSTRACT
Bacillus thuringiensis subsp. kurstaki (Btk) is a strong pathogen toward lepidopteran larvae thanks to specific Cry toxins causing leaky gut phenotypes. Hence, Btk and its toxins are used worldwide as microbial insecticide and in genetically modified crops, respectively, to fight crop pests. However, Btk belongs to the B. cereus group, some strains of which are well known human opportunistic pathogens. Therefore, ingestion of Btk along with food may threaten organisms not susceptible to Btk infection. Here we show that Cry1A toxins induce enterocyte death and intestinal stem cell (ISC) proliferation in the midgut of Drosophila melanogaster, an organism non-susceptible to Btk. Surprisingly, a high proportion of the ISC daughter cells differentiate into enteroendocrine cells instead of their initial enterocyte destiny. We show that Cry1A toxins weaken the E-Cadherin-dependent adherens junction between the ISC and its immediate daughter progenitor, leading the latter to adopt an enteroendocrine fate. Hence, although not lethal to non-susceptible organisms, Cry toxins can interfere with conserved cell adhesion mechanisms, thereby disrupting intestinal homeostasis and endocrine functions.
Subject(s)
Bacillus thuringiensis Toxins , Drosophila melanogaster , Stem Cells , Animals , Bacillus thuringiensis , Bacillus thuringiensis Toxins/adverse effects , Cell Adhesion , Crops, Agricultural , Plants, Genetically Modified , Stem Cells/drug effectsABSTRACT
Tissues must adapt to the different external stimuli so that organisms can survive in their environments. The intestine is a vital organ involved in food processing and absorption, as well as in innate immune response. Its adaptation to environmental cues such as diet and biotic/abiotic stress involves regulation of the proliferative rate and a switch of division mode (asymmetric versus symmetric) of intestinal stem cells (ISC). In this review, we outline the current comprehension of the physiological and molecular mechanisms implicated in stem cell division modes in the adult Drosophila midgut. We present the signaling pathways and polarity cues that control the mitotic spindle orientation, which is the terminal determinant ensuring execution of the division mode. We review these events during gut homeostasis, as well as during its response to nutrient availability, bacterial infection, chemical damage, and aging. JNK signaling acts as a central player, being involved in each of these conditions as a direct regulator of spindle orientation. The studies of the mechanisms regulating ISC divisions allow a better understanding of how adult stem cells integrate different signals to control tissue plasticity, and of how various diseases, notably cancers, arise from their alterations.
Subject(s)
Adaptation, Physiological , Cell Division , Cues , Homeostasis , Intestinal Mucosa/cytology , Stem Cells/cytology , Stress, Physiological , Animals , Asymmetric Cell Division , Diet , Humans , Intestinal Mucosa/metabolism , Signal Transduction , Stem Cells/metabolismABSTRACT
In segmented tissues, anterior and posterior compartments represent independent morphogenetic domains, which are made of distinct lineages separated by boundaries. During dorsal closure of the Drosophila embryo, specific "mixer cells" (MCs) are reprogrammed in a JNK-dependent manner to express the posterior determinant engrailed (en) and cross the segment boundary. Here, we show that JNK signaling induces de novo expression of en in the MCs through repression of Polycomb (Pc) and release of the en locus from the silencing PcG bodies. Whereas reprogramming occurs in MCs from all thoracic and abdominal segments, cell mixing is restricted to the central abdominal region. We demonstrate that this spatial control of MC remodeling depends on the antagonist activity of the Hox genes abdominal-A and Abdominal-B. Together, these results reveal an essential JNK/en/Pc/Hox gene regulatory network important in controlling both the plasticity of segment boundaries and developmental reprogramming.
Subject(s)
Cellular Reprogramming , Drosophila melanogaster/embryology , Genes, Homeobox/physiology , MAP Kinase Signaling System , Morphogenesis , Polycomb-Group Proteins/metabolism , Animals , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Gene Expression Regulation, Developmental , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Nuclear Proteins/metabolism , Phosphoproteins/metabolism , Polycomb-Group Proteins/genetics , Transcription Factors/metabolismABSTRACT
Tissue morphogenesis relies on proper differentiation of morphogenetic domains, adopting specific cell behaviours. Yet, how signalling pathways interact to determine and coordinate these domains remains poorly understood. Dorsal closure (DC) of the Drosophila embryo represents a powerful model to study epithelial cell sheet sealing. In this process, JNK (JUN N-terminal Kinase) signalling controls leading edge (LE) differentiation generating local forces and cell shape changes essential for DC. The LE represents a key morphogenetic domain in which, in addition to JNK, a number of signalling pathways converges and interacts (anterior/posterior -AP- determination; segmentation genes, such as Wnt/Wingless; TGFß/Decapentaplegic). To better characterize properties of the LE morphogenetic domain, we sought out new JNK target genes through a genomic approach: 25 were identified of which 8 are specifically expressed in the LE, similarly to decapentaplegic or puckered. Quantitative in situ gene profiling of this new set of LE genes reveals complex patterning of the LE along the AP axis, involving a three-way interplay between the JNK pathway, segmentation and HOX genes. Patterning of the LE into discrete domains appears essential for coordination of tissue sealing dynamics. Loss of anterior or posterior HOX gene function leads to strongly delayed and asymmetric DC, due to incorrect zipping in their respective functional domain. Therefore, in addition to significantly increasing the number of JNK target genes identified so far, our results reveal that the LE is a highly heterogeneous morphogenetic organizer, sculpted through crosstalk between JNK, segmental and AP signalling. This fine-tuning regulatory mechanism is essential to coordinate morphogenesis and dynamics of tissue sealing.
Subject(s)
Cell Differentiation/genetics , Embryonic Development/genetics , JNK Mitogen-Activated Protein Kinases/genetics , Morphogenesis/genetics , Animals , Body Patterning/genetics , Drosophila melanogaster/genetics , Drosophila melanogaster/growth & development , Ectoderm/growth & development , Ectoderm/metabolism , Embryo, Nonmammalian , Epithelial Cells/cytology , Epithelial Cells/metabolism , Gene Expression Regulation, Developmental , JNK Mitogen-Activated Protein Kinases/biosynthesis , MAP Kinase Signaling System/genetics , PhenotypeABSTRACT
Segments are fundamental units in animal development which are made of distinct cell lineages separated by boundaries. Although boundaries show limited plasticity during their formation for sharpening, cell lineages make compartments that become tightly restricted as development goes on. Here, we characterize a unique case of breaking of the segment boundary in late drosophila embryos. During dorsal closure, specific cells from anterior compartments cross the segment boundary and enter the adjacent posterior compartments. This cell mixing behaviour is driven by an anterior-to-posterior reprogramming mechanism involving de novo expression of the homeodomain protein Engrailed. Mixing is accompanied by stereotyped local cell intercalation, converting the segment boundary into a relaxation compartment important for tension-release during morphogenesis. This process of lineage switching and cell remodelling is controlled by JNK signalling. Our results reveal plasticity of segment boundaries during late morphogenesis and a role for JNK-dependent developmental reprogramming in this process.
Subject(s)
Drosophila/growth & development , MAP Kinase Kinase 4/metabolism , Morphogenesis , Signal Transduction , Animals , Drosophila/cytologyABSTRACT
In Drosophila melanogaster, dorsal closure is a model of tissue morphogenesis leading to the dorsal migration and sealing of the embryonic ectoderm. The activation of the JNK signal transduction pathway, specifically in the leading edge cells, is essential to this process. In a genome-wide microarray screen, we identified new JNK target genes during dorsal closure. One of them is the gene scarface (scaf), which belongs to the large family of trypsin-like serine proteases. Some proteins of this family, like Scaf, bear an inactive catalytic site, representing a subgroup of serine protease homologues (SPH) whose functions are poorly understood. Here, we show that scaf is a general transcriptional target of the JNK pathway coding for a secreted SPH. scaf loss-of-function induces defects in JNK-controlled morphogenetic events such as embryonic dorsal closure and adult male terminalia rotation. Live imaging of the latter process reveals that, like for dorsal closure, JNK directs the dorsal fusion of two epithelial layers in the pupal genital disc. Genetic data show that scaf loss-of-function mimics JNK over-activity. Moreover, scaf ectopic expression aggravates the effect of the JNK negative regulator puc on male genitalia rotation. We finally demonstrate that scaf acts as an antagonist by negatively regulating JNK activity. Overall, our results identify the SPH-encoding gene scaf as a new transcriptional target of JNK signalling and reveal the first secreted regulator of the JNK pathway acting in a negative-feedback loop during epithelial morphogenesis.
Subject(s)
Drosophila melanogaster/embryology , Drosophila melanogaster/metabolism , Epithelial Cells/metabolism , JNK Mitogen-Activated Protein Kinases/metabolism , Signal Transduction , Animals , Embryo, Nonmammalian/metabolism , Feedback , Genitalia, Male/embryology , Male , MorphogenesisABSTRACT
JNK-mediated closure of the Drosophila dorsal epidermis during embryogenesis is a well-characterised model for morphogenesis. However, little is known about how JNK signalling modifies particular cellular behaviours such as intracellular transport. Here we demonstrate that the gene encoding the small GTPase Rab30 is a new JNK transcriptional target whose function is required during embryonic and adult morphogenesis including JNK-dependent dorsal closure, embryonic head involution and thorax closure. Using immuno-fluorescence and live imaging, we show that EGFP-Rab30 localises to trans-Golgi in addition to small unidentified vesicles, and moves in a microtubule-dependent, polarised dorso-ventral manner in the leading edge during dorsal closure. We propose that JNK activity upregulates genes involved in intracellular transport in order to provide an increased level of trafficking activity in cells undergoing complex morphogenetic arrangements such as dorsal closure.
Subject(s)
Drosophila Proteins/physiology , Drosophila/embryology , Head/physiology , JNK Mitogen-Activated Protein Kinases/physiology , Monomeric GTP-Binding Proteins/physiology , rab GTP-Binding Proteins/physiology , Animals , Biological Transport , Body Patterning , Drosophila/metabolism , Golgi Apparatus/metabolism , Head/embryology , Morphogenesis , Signal Transduction , Up-Regulation/physiologyABSTRACT
Precise control of Wnt/beta-catenin signaling is critical for animal development, stem cell renewal, and prevention of disease. In the fruit fly Drosophila melanogaster, the naked cuticle (nkd) gene limits signaling by the Wnt ligand Wingless (Wg) during embryo segmentation. Nkd is an intracellular protein that is composed of separable membrane- and nuclear-localization sequences (NLS) as well as a conserved EF-hand motif that binds the Wnt receptor-associated scaffold protein Dishevelled (Dsh), but the mechanism by which Nkd inhibits Wnt signaling remains a mystery. Here we identify a second NLS in Nkd that is required for full activity and that binds to the canonical nuclear import adaptor Importin-alpha3. The Nkd NLS is similar to the Importin-alpha3-binding NLS in the Drosophila heat-shock transcription factor (dHSF), and each Importin-alpha3-binding NLS required intact basic residues in similar positions for nuclear import and protein function. Our results provide further support for the hypothesis that Nkd inhibits nuclear step(s) in Wnt/beta-catenin signaling and broaden our understanding of signaling pathways that engage the nuclear import machinery.
Subject(s)
Drosophila Proteins/metabolism , Drosophila melanogaster , Signal Transduction/physiology , Wnt Proteins/metabolism , alpha Karyopherins/metabolism , beta Catenin/metabolism , Active Transport, Cell Nucleus/physiology , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Amino Acid Sequence , Animals , Dishevelled Proteins , Drosophila Proteins/genetics , Drosophila melanogaster/anatomy & histology , Drosophila melanogaster/embryology , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Female , Molecular Sequence Data , Nuclear Localization Signals , Phosphoproteins/genetics , Phosphoproteins/metabolism , RNA Interference , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Salivary Glands/cytology , Salivary Glands/embryology , Salivary Glands/metabolism , Sequence Alignment , Two-Hybrid System Techniques , Wnt Proteins/genetics , alpha Karyopherins/genetics , beta Catenin/geneticsABSTRACT
Wnt/beta-catenin signals orchestrate cell fate and behavior throughout the animal kingdom. Aberrant Wnt signaling impacts nearly the entire spectrum of human disease, including birth defects, cancer, and osteoporosis. If Wnt signaling is to be effectively manipulated for therapeutic advantage, we first must understand how Wnt signals are normally controlled. Naked cuticle (Nkd) is a novel and evolutionarily conserved inducible antagonist of Wnt/beta-catenin signaling that is crucial for segmentation in the model genetic organism, the fruit fly Drosophila melanogaster. Nkd can bind and inhibit the Wnt signal transducer Dishevelled (Dsh), but the mechanism by which Nkd limits Wnt signaling in the fly embryo is not understood. Here we show that nkd mutants exhibit elevated levels of the beta-catenin homolog Armadillo but no alteration in Dsh abundance or distribution. In the fly embryo, Nkd and Dsh are predominantly cytoplasmic, although a recent report suggests that vertebrate Dsh requires nuclear localization for activity in gain-of-function assays. While Dsh-binding regions of Nkd contribute to its activity, we identify a conserved 30-amino-acid motif, separable from Dsh-binding regions, that is essential for Nkd function and nuclear localization. Replacement of the 30-aa motif with a conventional nuclear localization sequence rescued a small fraction of nkd mutant animals to adulthood. Our studies suggest that Nkd targets Dsh-dependent signal transduction steps in both cytoplasmic and nuclear compartments of cells receiving the Wnt signal.
Subject(s)
Drosophila Proteins/antagonists & inhibitors , Drosophila Proteins/chemistry , Drosophila/metabolism , Nuclear Localization Signals/physiology , Proto-Oncogene Proteins/antagonists & inhibitors , Wnt Proteins/antagonists & inhibitors , Active Transport, Cell Nucleus/physiology , Adaptor Proteins, Signal Transducing/chemistry , Adaptor Proteins, Signal Transducing/metabolism , Amino Acid Motifs/physiology , Amino Acid Sequence , Animals , Anopheles/genetics , Armadillo Domain Proteins/metabolism , Cytoplasm/metabolism , Dishevelled Proteins , Drosophila/embryology , Drosophila/genetics , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Drosophila Proteins/physiology , Embryo, Nonmammalian , Mice , Models, Biological , Molecular Sequence Data , Mutant Proteins/physiology , Mutation , Phosphoproteins/chemistry , Phosphoproteins/metabolism , Protein Binding , Proto-Oncogene Proteins/metabolism , Sequence Homology, Amino Acid , Signal Transduction , Tissue Distribution , Transcription Factors/metabolism , Wnt Proteins/metabolism , Wnt1 Protein , beta Catenin/metabolismSubject(s)
Drosophila/embryology , Morphogenesis , Animals , Epithelium/embryology , Humans , Wound HealingABSTRACT
Recent quantitative modeling of dorsal closure in the fruitfly Drosophila has revealed how multiple forces drive sealing of the two symmetrical epithelial sheets. A predictive model based on the new data allows gene function to be linked to the forces that drive tissue movement.
Subject(s)
Body Patterning/physiology , Cell Movement/physiology , Drosophila/embryology , Models, Biological , Animals , Biomechanical Phenomena , MorphogenesisABSTRACT
During Drosophila development, the naked cuticle (nkd) gene attenuates wingless/Wnt signaling through a negative feedback loop mechanism. Fly and vertebrate Nkd proteins contain a putative calcium-binding EF-hand motif, the EFX domain, that interacts with the basic/PDZ region of the Wnt signal transducer, dishevelled (Dsh). Here we show that Dsh binding by Drosophila Nkd in vitro is mediated by the EFX domain as well as an adjacent C-terminal sequence. In vivo data suggest that both of these regions contribute to the ability of Nkd to antagonize Wnt signaling. Mutations in the Nkd EF-hand designed to eliminate potential ion binding affected Nkd-Dsh interactions in the yeast two-hybrid assay but not in the glutathione S-transferase pull-down assay. Addition of the chelating agent EDTA abolished the in vitro Nkd-Dsh interaction. Surprisingly zinc, but not calcium, was able to restore Nkd-Dsh binding, suggesting a zinc-mediated interaction. Calcium 45- and zinc 65-blotting experiments show that Nkd is a zinc-binding metalloprotein. The results further clarify how Nkd may antagonize Wnt signaling via interaction with Dsh, and identify a novel zinc-binding domain in Drosophila Nkd that collaborates with the conserved EFX domain to bind Dsh.
Subject(s)
Drosophila Proteins , Insect Proteins/metabolism , Phosphoproteins/metabolism , Proto-Oncogene Proteins/antagonists & inhibitors , Zebrafish Proteins , Zinc/physiology , Adaptor Proteins, Signal Transducing , Animals , Calcium/physiology , Cations, Divalent , Dishevelled Proteins , Drosophila , Magnesium/physiology , Protein Binding , Two-Hybrid System Techniques , Wnt ProteinsABSTRACT
Planar cell polarity signaling in Drosophila requires the receptor Frizzled and the cytoplasmic proteins Dishevelled and Prickle. From initial, symmetric subcellular distributions in pupal wing cells, Frizzled and Dishevelled become highly enriched at the distal portion of the cell cortex. We describe a Prickle-dependent intercellular feedback loop that generates asymmetric Frizzled and Dishevelled localization. In the absence of Prickle, Frizzled and Dishevelled remain symmetrically distributed. Prickle localizes to the proximal side of pupal wing cells and binds the Dishevelled DEP domain, inhibiting Dishevelled membrane localization and antagonizing Frizzled accumulation. This activity is linked to Frizzled activity on the adjacent cell surface. Prickle therefore functions in a feedback loop that amplifies differences between Frizzled levels on adjacent cell surfaces.