Intestinal stem cells in the adult Drosophila midgut

Drosophila has long been an excellent model organism for studying stem cell biology. Notably, studies of Drosophila's germline stem cells have been instrumental in developing the stem cell niche concept. The recent discovery of somatic stem cells in adult Drosophila, particularly the intestinal stem cells (ISCs) of the midgut, has established Drosophila as an exciting model to study stem cell-mediated adult tissue homeostasis and regeneration. Here, we review the major signaling pathways that regulate the self-renewal, proliferation and differentiation of Drosophila ISCs, discussing how this regulation maintains midgut homeostasis and mediates regeneration of the intestinal epithelium after injury.     

Tiny Drosophila intestinal stem cells,big power

The signaling pathways are highly conserved between Drosophila and mammals concerning intestinal development, regeneration, and disease. The powerful genetic tools of Drosophila make it a valuable and convenient alternative to answer basic biological questions that can not be addressed using mammalian models. In this review, we discuss recent advances in how we use fly midgut to answer the following key questions: (1) How intestine stem cell niches are established; (2) which factors control asymmetric division of stem cells; (3) how intestinal cells interact with environmental factors, such as tissue damage, microbiota, and diet; (4) how to screen aging/cancer-related factors or drugs by fly intestine stem cells.     

Drosophila melanogaster as a model for human intestinal infection and pathology

Recent findings concerning Drosophila melanogaster intestinal pathology suggest that this model is well suited for the study of intestinal stem cell physiology during aging, stress and infection. Despite the physiological divergence between vertebrates and insects, the modeling of human intestinal diseases is possible in Drosophila because of the high degree of conservation between Drosophila and mammals with respect to the signaling pathways that control intestinal development, regeneration and disease. Furthermore, the genetic amenability of Drosophila makes it an advantageous model species. The well-studied intestinal stem cell lineage, as well as the tools available for its manipulation in vivo, provide a promising framework that can be used to elucidate many aspects of human intestinal pathology. In this Perspective, we discuss recent advances in the study of Drosophila intestinal infection and pathology, and briefly review the parallels and differences between human and Drosophila intestinal regeneration and disease.     

Eco-genetics of desiccation resistance in Drosophila

Climate change globally perturbs water circulation thereby influencing ecosystems including cultivated land. Both harmful and beneficial species of insects are likely to be vulnerable to such changes in climate. As small animals with a disadvantageous surface area to body mass ratio, they face a risk of desiccation. A number of behavioural, physiological and genetic strategies are deployed to solve these problems during adaptation in various Drosophila species. Over 100 desiccation-related genes have been identified in laboratory and wild populations of the cosmopolitan fruit fly Drosophila melanogaster and its sister species in large-scale and single-gene approaches. These genes are involved in water sensing and homeostasis, and barrier formation and function via the production and composition of surface lipids and via pigmentation. Interestingly, the genetic strategy implemented in a given population appears to be unpredictable. In part, this may be due to different experimental approaches in different studies. The observed variability may also reflect a rich standing genetic variation in Drosophila allowing a quasi-random choice of response strategies through soft-sweep events, although further studies are needed to unravel any underlying principles. These findings underline that D. melanogaster is a robust species well adapted to resist climate change-related desiccation. The rich data obtained in Drosophila research provide a framework to address and understand desiccation resistance in other insects. Through the application of powerful genetic tools in the model organism D. melanogaster, the functions of desiccation-related genes revealed by correlative studies can be tested and the underlying molecular mechanisms of desiccation tolerance understood. The combination of the wealth of available data and its genetic accessibility makes Drosophila an ideal bioindicator. Accumulation of data on desiccation resistance in Drosophila may allow us to create a world map of genetic evolution in response to climate change in an insect genome. Ultimately these efforts may provide guidelines for dealing with the effects of climate-related perturbations on insect population dynamics in the future.     

The benefits of oxidative stress for tissue repair and regeneration

Recent work has strengthened Drosophila imaginal discs as a model system for regeneration studies. Evidence is accumulating that oxidative stress drives the cellular responses for repair and regeneration. Drosophila imaginal discs generate a burst of reactive oxygen species (ROS) upon damage that is necessary for the activation of the Jun N-terminal kinase (JNK) and p38 MAP kinase signaling pathways. Moreover, these pathways are pivotal in the activation of regenerative growth. A hypothetical mechanism of how the ROS are initiated, and how repair and regeneration is activated is discussed here.     

A genomic investigation of ecological differentiation between free‐living and Drosophila‐associated bacteria

Various bacterial taxa have been identified both in association with animals and in the external environment, but the extent to which related bacteria from the two habitat types are ecologically and evolutionarily distinct is largely unknown. This study investigated the scale and pattern of genetic differentiation between bacteria of the family Acetobacteraceae isolated from the guts of Drosophila fruit flies, plant material and industrial fermentations. Genome‐scale analysis of the phylogenetic relationships and predicted functions was conducted on 44 Acetobacteraceae isolates, including newly sequenced genomes from 18 isolates from wild and laboratory Drosophila. Isolates from the external environment and Drosophila could not be assigned to distinct phylogenetic groups, nor are their genomes enriched for any different sets of genes or category of predicted gene functions. In contrast, analysis of bacteria from laboratory Drosophila showed they were genetically distinct in their universal capacity to degrade uric acid (a major nitrogenous waste product of Drosophila) and absence of flagellar motility, while these traits vary among wild Drosophila isolates. Analysis of the competitive fitness of Acetobacter discordant for these traits revealed a significant fitness deficit for bacteria that cannot degrade uric acid in culture with Drosophila. We propose that, for wild populations, frequent cycling of Acetobacter between Drosophila and the external environment prevents genetic differentiation by maintaining selection for traits adaptive in both the gut and external habitats. However, laboratory isolates bear the signs of adaptation to persistent association with the Drosophila host under tightly defined environmental conditions.     

Drosophila growth cones: A genetically tractable platform for the analysis of axonal growth dynamics

The formation of neuronal networks, during development and regeneration, requires outgrowth of axons along reproducible paths toward their appropriate postsynaptic target cells. Axonal extension occurs at growth cones (GCs) at the tips of axons. GC advance and navigation requires the activity of their cytoskeletal networks, comprising filamentous actin (F‐actin) in lamellipodia and filopodia as well as dynamic microtubules (MTs) emanating from bundles of the axonal core. The molecular mechanisms governing these two cytoskeletal networks, their cross‐talk, and their response to extracellular signaling cues are only partially understood, hindering our conceptual understanding of how regulated changes in GC behavior are controlled. Here, we introduce Drosophila GCs as a suitable model to address these mechanisms. Morphological and cytoskeletal readouts of Drosophila GCs are similar to those of other models, including mammals, as demonstrated here for MT and F‐actin dynamics, axonal growth rates, filopodial structure and motility, organizational principles of MT networks, and subcellular marker localization. Therefore, we expect fundamental insights gained in Drosophila to be translatable into vertebrate biology. The advantage of the Drosophila model over others is its enormous amenability to combinatorial genetics as a powerful strategy to address the complexity of regulatory networks governing axonal growth. Thus, using pharmacological and genetic manipulations, we demonstrate a role of the actin cytoskeleton in a specific form of MT organization (loop formation), known to regulate GC pausing behavior. We demonstrate these events to be mediated by the actin‐MT linking factor Short stop, thus identifying an essential molecular player in this context. © 2009 Wiley Periodicals, Inc. Develop Neurobiol 2010     

Progress inDrosophila genome manipulation

The introduction of cloned and manipulated genetic material into the germline of an experimental organism is one of the most powerful tools of modern biology. In the case of the fruit fly,Drosophila melanogaster, there is also an unparalleled range of sophisticated genetic tools to facilitate subsequent analysis. In consequence,Drosophila remains a most favourable model organism for the dissection of gene structure and functionin vivo. In this review we look at some of the achievements to date inDrosophila genome manipulation, and at what may be possible in the near future.     

Interplay of cell proliferation and cell death in Drosophila tissue regeneration

Regeneration is a fascinating process that allows some organisms to reconstruct damaged tissues. In addition to the classical regeneration model of the Drosophila larval imaginal discs, the genetically induced tissue ablation model has promoted the understanding of molecular mechanisms underlying cell death, proliferation, and remodeling for tissue regeneration. Recent studies have also revealed that tissue injury responses occur not only locally but also systemically, even in the uninjured region. Genetic studies in Drosophila have demonstrated the dynamic role of the cell death‐induced tissue response in the reconstruction of damaged tissues.     

Pharmacological analysis of heartbeat in Drosophila

Analysis of the mechanisms underlying cardiac excitability can be faciliated greatly by mutations that disrupt ion channels and receptors involved in this excitability. With an extensive repertoire of such mutations, Drosophila provides the best available genetic model for these studies. However, the use of Drosophila for this purpose has been severely handicapped by lack of a suitable preparation of heart and a complete lack of knowledge about the ionic currents that underlie its excitability. We describe a simple preparation to measure heartbeat in Drosophila. This preparation was used to ask if heartbeat in Drosophila is myogenic in origin, and to determine the types of ion channels involved in influencing the heart rate. Tetrodotoxin, even at a high concentration of 40 μM, did not affect heart rate, indicating that heartbeat may be myogenic in origin and that it may not be determined by Na⁺ channels. Heart rate was affected by PN200–110, verapamil, and diltiazem, which block vertebrate L-type Ca²⁺ channels. Thus, L-type channels, which contribute to the prolonged plateau of action potentials in vertebrate heart, may play a role in Drosophila cardiac excitability. It also suggests that Drosophila heart is subject to a similar intervention by organic Ca²⁺ channel blockers as the vertebrate heart. A role for K⁺ currents in the function of Drosophila heart was suggested by an effect of tetraethylammonium, which blocks all the four identified K⁺ currents in the larval body wall muscles, and quinidine, which blocks the delayed rectifier K⁺ current in these muscles. The preparation described here also provides an extremely simple method for identifying mutations that affect heart rate. Such mutations and pharmacological agents will be very useful for analyzing molecular components of cardiac excitability in Drosophila. © 1995 John Wiley & Sons, Inc.     

Cholera toxin and pertussis toxin substrates and endogenous ADP-ribosyltransferase activity in Drosophila melanogaster

Cholera toxin- and pertussis toxin-catalyzed ADP-ribosylation were used to identify and localize G protein substrates in Drosophila melanogaster and in Manduca sexta. Cholera toxin catalyzes ADP-ribosylation of 37 kDa and 50 kDa polypeptides, but these polypeptides are also substrates for an ADP-ribosyltransferase (EC 2.4.2.30) activity endogenous to the Drosophila extracts. Pertussis toxin modifies 37 kDa and 39 kDa polypeptides in Drosophila homogenates. The pattern of proteolysis of the 39 kDa pertussis toxin substrate is similar to that of mammalian G_o and is influenced by guanyl nucleotide binding. The 39 kDa G_o-like Drosophila and Manduca pertussis toxin substrates are found primarily in neural tissues. These studies provide further evidence that G proteins are present in Drosophila and that this organism can therefore be used to investigate the physiological roles of these enzymes using advanced genetic manipulations.     

Sialylation in protostomes: a perspective from <Emphasis Type="Italic">Drosophila</Emphasis> genetics and biochemistry

Numerous studies have revealed important functions for sialylation in both prokaryotes and higher animals. However, the genetic and biochemical potential for sialylation in Drosophila has only been confirmed recently. Recent studies suggest significant similarities between the sialylation pathways of vertebrates and insects and provide evidence for their common evolutionary origin. These new data support the hypothesis that sialylation in insects is a specialized and developmentally regulated process which likely plays a prominent role in the nervous system. Yet several key issues remain to be addressed in Drosophila, including the initiation of sialic acid de novo biosynthesis and understanding the structure and function of sialylated glycoconjugates. This review discusses our current knowledge of the Drosophila sialylation pathway, as compared to the pathway in bacteria and vertebrates. We arrive at the conclusion that Drosophila is emerging as a useful model organism that is poised to shed new light on the function of sialylation not only in protostomes, but also in a larger evolutionary context. K. Koles and E. Repnikova contributed equally to this work.     

Analysis of the 3-hydroxyl group in Drosophila siRNA function

Members of the RNA-dependent RNA polymerase (RdRP) gene family have been shown to be essential for dsRNA-mediated gene silencing based on genetic screens in a variety of organisms, including Caenorhabditis elegans, Arabidopsis, Neurospora, and Dictyostelium. A hallmark of this process is the formation of small 21- to 25-bp dsRNAs, termed siRNAs for small interfering RNAs, which are derived from the dsRNA that initiates gene silencing. We have developed methods to demonstrate that these siRNAs produced in Drosophila embryo extract can be uniformly incorporated into dsRNA in a template-specific manner that is subsequently degraded by RNase III-related enzyme activity to create a second generation of siRNAs. SiRNA function in dsRNA synthesis and mRNA degradation depends upon the integrity of the 3^′-hydroxyl of the siRNA, consistent with the interpretation that siRNAs serve as primers for RdRP activity in the formation of dsRNA. This process of siRNA incorporation into dsRNA followed by degradation and the formation of new siRNAs has been termed “degradative PCR” and the proposed mechanism is consistent with the genetic and biochemical data derived from studies in C. elegans, Arabidopsis, Drosophila, and Dictyostelium. The methods used to study the function of both natural and synthetic siRNAs in RNA interference in Drosophila embryo extracts are detailed. The importance of the 3^′-hydroxyl group for siRNA function and its incorporation into dsRNA is emphasized and the results support a model that places RNA-dependent RNA polymerase as a key mediator in the RNA interference mechanism in Drosophila.     

c‐Src drives intestinal regeneration and transformation

The non‐receptor tyrosine kinase c‐Src, hereafter referred to as Src, is overexpressed or activated in multiple human malignancies. There has been much speculation about the functional role of Src in colorectal cancer (CRC), with Src amplification and potential activating mutations in up to 20% of the human tumours, although this has never been addressed due to multiple redundant family members. Here, we have used the adult Drosophila and mouse intestinal epithelium as paradigms to define a role for Src during tissue homeostasis, damage‐induced regeneration and hyperplasia. Through genetic gain and loss of function experiments, we demonstrate that Src is necessary and sufficient to drive intestinal stem cell (ISC) proliferation during tissue self‐renewal, regeneration and tumourigenesis. Surprisingly, Src plays a non‐redundant role in the mouse intestine, which cannot be substituted by the other family kinases Fyn and Yes. Mechanistically, we show that Src drives ISC proliferation through upregulation of EGFR and activation of Ras/MAPK and Stat3 signalling. Therefore, we demonstrate a novel essential role for Src in intestinal stem/progenitor cell proliferation and tumourigenesis initiation in vivo.     

<Emphasis Type="Italic">Drosophila</Emphasis> retinophilin contains MORN repeats and is conserved in humans

The function of conserved novel human genes can be efficiently addressed in genetic model organisms. From a collection of genes expressed in the Drosophila visual system, cDNAs expressed in vertebrates were identified and one similar to a novel human gene was chosen for further investigation. The results reported here characterize the Drosophila retinophilin gene and demonstrate that a similar gene is expressed in the human retina. The Drosophila and human retinophilin sequences are 50% identical, and they share an additional 16% conserved substitutions. Examination of the cDNA and genomic sequence indicates that it corresponds to the gene CG10233 of the annotated genome and predicts a 22.7 kDa protein. Polyclonal antibodies generated to a predicted retinophilin peptide recognize an antigen in Drosophila photoreceptor cells. The retinophilins encode 4 copies of a repeat associated with a Membrane Occupation and Recognition Nexus (MORN) function first discovered in junctophilins, which may interact with the plasma membrane. These results therefore show that Drosophila retinophilin is expressed in fly photoreceptor cells, demonstrate that a conserved human gene is expressed in human retina, and suggest that a mutational analysis of the Drosophila gene would be valuable.     

Evolution of the<Emphasis Type="Italic"> Drosophila broad</Emphasis> locus: the<Emphasis Type="Italic"> Manduca sexta broad</Emphasis> Z4 isoform has biological activity in<Emphasis Type="Italic"> Drosophila</Emphasis>

The Drosophila melanogaster broad locus is essential for normal metamorphic development. Broad encodes three genetically distinct functions (rbp, br, and 2Bc) and a family of four zinc-finger DNA-binding proteins (Z1-Z4). The Z1, Z2, and Z3 protein isoforms are primarily associated with the rbp, br, and 2Bc genetic functions respectively. The Z4 protein isoform also provides some rbp genetic function, however an essential function for the Z4 isoform in metamorphosis has not been identified. To determine the degree of conservation of Z4 function between the tobacco hornworm Manduca sexta and Drosophila we generated transgenic Drosophila expressing the Manduca broad Z4 isoform and used this transgene to rescue rbp mutant lethality during Drosophila metamorphosis. We find that the Manduca Z4 protein has significant biological activity in Drosophila with respect to rescue of rbp-associated lethality. There was also some overlap in effects on cuticle gene expression between the Manduca Z4 and Drosophila Z1 isoforms that was not shared with the Drosophila Z4 isoform. Our findings show that Z4 function has been conserved over the 260-million-year period since the divergence of Diptera and Lepidoptera, and are consistent with the hypothesis that the Drosophila Z4 and Manduca Z4 isoforms have essential roles in metamorphosis.Edited by M. Akam     

Conditional mutations in SERCA, the Sarco-endoplasmic reticulum Ca2+-ATPase, alter heart rate and rhythmicity in Drosophila

To analyze the role of cytosolic calcium in regulating heart beat frequency and rhythm, we studied conditional mutations in Drosophila Sarco-endoplasmic reticulum Ca²⁺-ATPase, believed to be predominantly responsible for sequestering free cytosolic calcium. Abnormalities in the amount or structure of the SERCA protein have been linked to cardiac malfunction in mammals. Drosophila SERCA protein (dSERCA) is highly enriched in Drosophila larval heart with a distinct membrane distribution of SERCA at cardiac Z-lines, suggesting evolutionarily conserved zones for calcium uptake into the sarcoplasmic reticulum. Heart beat frequency is strikingly reduced in mutant animals following dSERCA inactivation, (achieved by a brief exposure of these conditional mutants to non-permissive temperature). Cardiac contractions also show abnormal rhythmicity and electrophysiological recordings from the heart muscle reveal dramatic alterations in electrical activity. Overall, these studies underscore the utility of the Drosophila heart to model SERCA dysfunction dependent cardiac disorders and constitute an initial step towards developing Drosophila as a viable genetic model system to study conserved molecular determinants of cardiac physiology.     

Hsp22对SCA3/MJD转基因果蝇的神经保护作用研究

为了探讨Hsp22在SCA3/MJD发病机制中的作用．选用GMR-GAL4和elav-GAL4驱动子,利用经典的GAL4-UAS系统,将含有78个CAG重复扩增的ataxin-3蛋白片段(MJDtr-Q78)分别在果蝇眼睛和神经系统选择性表达,构建GMR-GAL4/UAS和elav-GAL4/UAS系统SCA3/MJD转基因果蝇模型, 然后利用遗传学方法和热休克反应使Hsp22在SCA3/ MJD转基因果蝇眼睛和神经系统以不同水平过表达．结果表明,Hsp22过表达显著抑制了MJDtr-Q78蛋白的神经毒性,果蝇眼睛视网膜光感受神经元变性明显缓解,果蝇存活能力也显著提高．Hsp22对SCA3/MJD具有保护作用,增强Hsp22表达对SCA3/MJD可能是一种潜在的治疗方法．