Drosophila Fragile X Protein controls cellular proliferation by regulating cbl levels in the ovary

FMRP is an RNA binding protein linked to the most common form of inherited mental retardation, Fragile X syndrome (FraX). In addition to severe cognitive deficits, FraX etiology includes postpubescent macroorchidism, which is thought to result from overproliferation. Using a Drosophila FraX model, we show that FMRP controls germline proliferation during oogenesis. dFmr1 null ovaries contain egg chambers with both fewer and supranumerary germ cells. The mutant germaria contain a significantly increased number of cyclin E and PhosphoHistone H3 positive cells, suggesting that loss of FMRP leads to defects in cell cycle progression. BrdU incorporation and flow cytometry data suggest that, in addition to proliferation, germline endoreplication and ploidy are also affected by the loss of FMRP during ovary development. Here we report that FMRP controls the levels of cbl mRNA in the ovary and that reducing cbl gene dosage by half rescues the dFmr1 oogenesis phenotypes. These data support a model whereby FMRP controls germline proliferation by regulating the expression of cbl in the developing ovary.     

The zinc finger homeodomain-2 gene of Drosophila controls Notch targets and regulates apoptosis in the tarsal segments

The development of the Drosophila leg is a good model to study processes of pattern formation, cell death and segmentation. Such processes require the coordinate activity of different genes and signaling pathways that progressively subdivide the leg territory into smaller domains. One of the main pathways needed for leg development is the Notch pathway, required for determining the proximo-distal axis of the leg and for the formation of the joints that separate different leg segments. The mechanisms required to coordinate such events are largely unknown. We describe here that the zinc finger homeodomain-2 (zfh-2) gene is highly expressed in cells that will form the leg joints and needed to establish a correct size and pattern in the distal leg. There is an early requirement of zfh-2 to establish the correct proximo-distal axis, but zfh-2 is also needed at late third instar to form the joint between the fourth and fifth tarsal segments. The expression of zfh-2 requires Notch activity but zfh-2 is necessary, in turn, to activate Notch targets such as Enhancer of split and big brain. zfh-2 is controlled by the Drosophila activator protein 2 gene and regulates the late expression of tarsal-less. In the absence of zfh-2 many cells ectopically express the pro-apoptotic gene head involution defective, activate caspase-3 and are positive for acridine orange, indicating they undergo apoptosis. Our results demonstrate the key role of zfh-2 in the control of cell death and Notch signaling during leg development.     

Characterization of a dominant-active STAT that promotes tumorigenesis in Drosophila

Little is known about the molecular mechanisms by which STAT proteins promote tumorigenesis. Drosophila is an ideal system for investigating this issue, as there is a single STAT (Stat92E), and its hyperactivation causes overgrowths resembling human tumors. Here we report the first identification of a dominant-active Stat92E protein, Stat92E^ΔNΔC, which lacks both N- and C-termini. Mis-expression of Stat92E^ΔNΔCin vivo causes melanotic tumors, while in vitro it transactivates a Stat92E-luciferase reporter in the absence of stimulation. These gain-of-function phenotypes require phosphorylation of Y⁷¹¹ and dimer formation with full-length Stat92E. Furthermore, a single point mutation, an R^442P substitution in the DNA-binding domain, abolishes Stat92E function. Recombinant Stat92E^R442P translocates to the nucleus following activation but fails to function in all assays tested. Interestingly, R⁴⁴² is conserved in most STATs in higher organisms, suggesting conservation of function. Modeling of Stat92E indicates that R⁴⁴² may contact the minor groove of DNA via invariant TC bases in the consensus binding element bound by all STAT proteins. We conclude that the N- and C- termini function unexpectedly in negatively regulating Stat92E activity, possibly by decreasing dimer dephosphorylation or increasing stability of DNA interaction, and that Stat92E^R442 has a nuclear function by altering dimer:DNA binding.     

The respiratory basis of locomotion in Drosophila

The respiratory system of insects has evolved to satisfy the oxygen supply during rest and energetically demanding processes such as locomotion. Flapping flight in particular is considered a key trait in insect evolution and requires an increase in metabolic activity of 10-15-fold the resting metabolism. Two major trade-offs are associated with the extensive development of the tracheal system and the function of spiracles in insects: the risk of desiccation because body water may leave the tracheal system when spiracles open for gas exchange and the risk of toxic tracheal oxygen levels at low metabolic activity. In resting animals there is an ongoing debate on the function and evolution of spiracle opening behavior, focusing mainly on discontinuous gas exchange patterns. During locomotion, large insects typically satisfy the increased respiratory requirements by various forms of ventilation, whereas in small insects such as Drosophila diffusive processes are thought to be sufficient. Recent data, however, have shown that during flight even small insects employ ventilatory mechanisms, potentially helping to balance respiratory currents inside the tracheal system. This review broadly summarizes our current knowledge on breathing strategies and spiracle function in the genus Drosophila, highlighting the gas exchange strategies in resting, running and flying animals.     

Thermal preference in Drosophila

Environmental temperature strongly affects physiology of ectotherms. Small ectotherms, like Drosophila, cannot endogenously regulate body temperature so must rely on behavior to maintain body temperature within a physiologically permissive range. Here we review what is known about Drosophila thermal preference. Work on thermal behavior in this group is particularly exciting because it provides the opportunity to connect genes to neuromolecular mechanisms to behavior to fitness in the wild.     

Myoblast fusion in Drosophila

The body wall musculature of a Drosophila larva is composed of an intricate pattern of 30 segmentally repeated muscle fibers in each abdominal hemisegment. Each muscle fiber has unique spatial and behavioral characteristics that include its location, orientation, epidermal attachment, size and pattern of innervation. Many, if not all, of these properties are dictated by founder cells, which determine the muscle pattern and seed the fusion process. Myofibers are then derived from fusion between a specific founder cell and several fusion competent myoblasts (FCMs) fusing with as few as 3-5 FCMs in the small muscles on the most ventral side of the embryo and as many as 30 FCMs in the larger muscles on the dorsal side of the embryo. The focus of the present review is the formation of the larval muscles in the developing embryo, summarizing the major issues and players in this process. We have attempted to emphasize experimentally-validated details of the mechanism of myoblast fusion and distinguish these from the theoretically possible details that have not yet been confirmed experimentally. We also direct the interested reader to other recent reviews that discuss myoblast fusion in Drosophila, each with their own perspective on the process [1], [2], [3] and [4]. With apologies, we use gene nomenclature as specified by Flybase (http://flybase.org) but provide Table 1 with alternative names and references.     

A genetic screen in Drosophila for regulators of human prostate cancer progression

To uncover the mechanism by which human prostate cancer progresses, we performed a genetic screen for regulators of human prostate cancer progression using the Drosophila accessory gland, a functional homolog of the mammalian prostate. Cell growth and migration of secondary cells in the adult male accessory gland were found to be regulated by paired, N-cadherin, and E-cadherin, which are Drosophila homologues of regulators of human prostate cancer progression. Using this screening system, we also identified three genes that promoted growth and migration of secondary cells in the accessory gland. The human homologues of these candidate genes – MRGBP, CNPY2, and MEP1A – were found to be expressed in human prostate cancer model cells and to promote replication and invasiveness in these cells. These findings suggest that the development of the Drosophila accessory gland and human prostate cancer cell growth and invasion are partly regulated through a common mechanism. The screening system using the Drosophila accessory gland can be a useful tool for uncovering the mechanisms of human prostate cancer progression.     

Defective gut function in drop-dead mutant Drosophila

Mutation of the gene drop-dead (drd) causes adult Drosophila to die within 2 weeks of eclosion and is associated with reduced rates of defecation and increased volumes of crop contents. In the current study, we demonstrate that flies carrying the strong allele drdlwf display a reduction in the transfer of ingested food from the crop to the midgut, as measured both as a change in the steady-state distribution of food within the gut and also in the rates of crop emptying and midgut filling following a single meal. Mutant flies have abnormal triglyceride (TG) and glycogen stores over the first 4 days post-eclosion, consistent with their inability to move food into the midgut for digestion and nutrient absorption. However, the lifespan of mutants was dependent upon food presence and quality, suggesting that at least some individual flies were able to digest some food. Finally, spontaneous motility of the crop was abnormal in drdlwf flies, with the crops of mutant flies contracting significantly more rapidly than those of heterozygous controls. We therefore hypothesize that mutation of drd causes a structural or regulatory defect that inhibits the entry of food into the midgut.     

Asymmetrical Reinforcement and Wolbachia Infection in Drosophila

Reinforcement refers to the evolution of increased mating discrimination against heterospecific individuals in zones of geographic overlap and can be considered a final stage in the speciation process. One the factors that may affect reinforcement is the degree to which hybrid matings result in the permanent loss of genes from a species' gene pool. Matings between females of Drosophila subquinaria and males of D. recens result in high levels of offspring mortality, due to interspecific cytoplasmic incompatibility caused by Wolbachia infection of D. recens. Such hybrid inviability is not manifested in matings between D. recens females and D. subquinaria males. Here we ask whether the asymmetrical hybrid inviability is associated with a corresponding asymmetry in the level of reinforcement. The geographic ranges of D. recens and D. subquinaria were found to overlap across a broad belt of boreal forest in central Canada. Females of D. subquinaria from the zone of sympatry exhibit much stronger levels of discrimination against males of D. recens than do females from allopatric populations. In contrast, such reproductive character displacement is not evident in D. recens, consistent with the expected effects of unidirectional cytoplasmic incompatibility. Furthermore, there is substantial behavioral isolation within D. subquinaria, because females from populations sympatric with D. recens discriminate against allopatric conspecific males, whereas females from populations allopatric with D. recens show no discrimination against any conspecific males. Patterns of general genetic differentiation among populations are not consistent with patterns of behavioral discrimination, which suggests that the behavioral isolation within D. subquinaria results from selection against mating with Wolbachia-infected D. recens. Interspecific cytoplasmic incompatibility may contribute not only to post-mating isolation, an effect already widely recognized, but also to reinforcement, particularly in the uninfected species. The resulting reproductive character displacement not only increases behavioral isolation from the Wolbachia-infected species, but may also lead to behavioral isolation between populations of the uninfected species. Given the widespread occurrence of Wolbachia among insects, it thus appears that there are multiple ways by which these endosymbionts may directly and indirectly contribute to reproductive isolation and speciation.     

Autophagy takes flight in Drosophila

Drosophila has been shown to be a powerful model to study autophagy, whose regulation involves a core machinery consisting of Atg proteins and upstream signaling regulators similar to those in yeast and mammals. The conserved role in degrading proteins and organelles gives autophagy an important function in coordinating several cellular processes as well as in a number of pathological conditions. This review summarizes key studies in Drosophila autophagy research and discusses potential questions that may lead to better understanding of the roles and regulation of autophagy in higher eukaryotes.     

Lipid signaling in Drosophila photoreceptors

Drosophila photoreceptors are sensory neurons whose primary function is the transduction of photons into an electrical signal for forward transmission to the brain. Photoreceptors are polarized cells whose apical domain is organized into finger like projections of plasma membrane, microvilli that contain the molecular machinery required for sensory transduction. The development of this apical domain requires intense polarized membrane transport during development and it is maintained by post developmental membrane turnover. Sensory transduction in these cells involves a high rate of G-protein coupled phosphatidylinositol 4,5 bisphosphate [PI(4,5)P₂] hydrolysis ending with the activation of ion channels that are members of the TRP superfamily. Defects in this lipid-signaling cascade often result in retinal degeneration, which is a consequence of the loss of apical membrane homeostasis. In this review we discuss the various membrane transport challenges of photoreceptors and their regulation by ongoing lipid signaling cascades in these cells. This article is part of a Special Issue entitled Lipids and Vesicular Transport.     

The function of courtship song rhythms in Drosophila

The courtship songs of Drosophila are produced by the male's wing vibration and consist of a series of pulses, with an inter-pulse interval (IPI) of 34 ms for D. melanogaster and 48 ms for D. simulans. The IPI's are not constant in length during courtship, but oscillate sinusoidally with 55-s cycles in the former species and 35-s cycles in the latter. We have stimulated D. melanogaster females with artificially generated courtship songs, and have observed that they mate fastest when the song incorporates a 55-s oscillation superimposed on a 34-ms IPI. D. simulans females, on the other hand, mate fastest with a 48-ms IPI and a 35-s oscillation period. Consequently these newly-discovered song cycles produce significant mating enhancement in these species, with the females showing a preference for songs which carry both the species-specific IPI and the species-specific IPI rhythm.     

Role of svp in Drosophila Pericardial Cell Growth

The Drosophila dorsal vessel is a segmentally repeated linear organ, in which seven-up (svp) is expressed in two pairs of cardioblasts and two pairs of pericardial cells in each segment. Under the control of hedgehog (hh) signaling from the dorsal ectoderm, svp participates in diversifying cardioblast identities within each segment. In this experiment, the homozygous embryos of svp mutants exhibited an increase in cell size of Eve positive pericardial cells (EPCs) and a disarranged expression pattern, while the cardioblasts pattern of svp-lacZ expression was normal. In the meantime, the DA1 muscle founders were absent in some segments in svp mutant embryos, and the dorsal somatic muscle patterning was also severely damaged in the late stage mutant embryos, suggesting that svp is required for the differentiation of Eve-positive pericardial cells and DA1 muscle founders and may have a role in EPC cell growth.     

The Golgi apparatus: Lessons from Drosophila

Historically, Drosophila has been a model organism for studying molecular and developmental biology leading to many important discoveries in this field. More recently, the fruit fly has started to be used to address cell biology issues including studies of the secretory pathway, and more specifically on the functional integrity of the Golgi apparatus. A number of advances have been made that are reviewed below. Furthermore, with the development of RNAi technology, Drosophila tissue culture cells have been used to perform genome-wide screens addressing similar issues. Last, the Golgi function has been involved in specific developmental processes, thus shedding new light on the functions of a number of Golgi proteins.     

New yeast/E. coli/Drosophila triple shuttle vectors for efficient generation of Drosophila P element transformation constructs

We have generated a set of novel triple shuttle vectors that facilitate the construction of Drosophila-P-element transformations vectors. These YED-vectors allow the insertion of any kind of sequence at any chosen position due to the presence of a yeast casette which ensures replication and allows for homologous recombination in Saccharomyces cerevisiae. As a proof of principle we generated several reporter constructs and tested them in transgenic flies for expression and correct subcellular localization. YED-vectors can be used for many purposes including promoter analysis or the expression of tagged or truncated proteins. Thus, time-consuming conventional restriction site based multi-step cloning procedures can be circumvented by using the new YED-vectors. The new set of triple shuttle vectors will be highly beneficial for the rapid construction of complex Drosophila transformation plasmids.