Genetic Model Organism Research Articles

Human Disease Models inDrosophila melanogasterand the Role of the Fly in Therapeutic Drug Discovery

The common fruit fly, Drosophila melanogaster, is a well studied and highly tractable genetic model organism for understanding molecular mechanisms of human diseases. Many basic biological, physiological, and neurological properties are conserved between mammals and D. melanogaster, and nearly 75% of human disease-causing genes are believed to have a functional homolog in the fly. In the discovery process for therapeutics, traditional approaches employ high-throughput screening for small molecules that is based primarily on in vitro cell culture, enzymatic assays, or receptor binding assays. The majority of positive hits identified through these types of in vitro screens, unfortunately, are found to be ineffective and/or toxic in subsequent validation experiments in whole-animal models. New tools and platforms are needed in the discovery arena to overcome these limitations. The incorporation of D. melanogaster into the therapeutic discovery process holds tremendous promise for an enhanced rate of discovery of higher quality leads. D. melanogaster models of human diseases provide several unique features such as powerful genetics, highly conserved disease pathways, and very low comparative costs. The fly can effectively be used for low- to high-throughput drug screens as well as in target discovery. Here, we review the basic biology of the fly and discuss models of human diseases and opportunities for therapeutic discovery for central nervous system disorders, inflammatory disorders, cardiovascular disease, cancer, and diabetes. We also provide information and resources for those interested in pursuing fly models of human disease, as well as those interested in using D. melanogaster in the drug discovery process.

Carbon dioxide concentrating mechanism in Chlamydomonas reinhardtii: inorganic carbon transport and CO2 recapture

Many microalgae are capable of acclimating to CO(2) limited environments by operating a CO(2) concentrating mechanism (CCM), which is driven by various energy-coupled inorganic carbon (Ci; CO(2) and HCO(3)(-)) uptake systems. Chlamydomonas reinhardtii (hereafter, Chlamydomonas), a versatile genetic model organism, has been used for several decades to exemplify the active Ci transport in eukaryotic algae, but only recently have many molecular details behind these Ci uptake systems emerged. Recent advances in genetic and molecular approaches, combined with the genome sequencing of Chlamydomonas and several other eukaryotic algae have unraveled some unique characteristics associated with the Ci uptake mechanism and the Ci-recapture system in eukaryotic microalgae. Several good candidate genes for Ci transporters in Chlamydomonas have been identified, and a few specific gene products have been linked with the Ci uptake systems associated with the different acclimation states. This review will focus on the latest studies on characterization of functional components involved in the Ci uptake and the Ci-recapture in Chlamydomonas.

Michael Dickinson

Michael Dickinson

Maintenance and regulation of extracellular volume and the ion environment in Drosophila larval nerves

In mammals and insects, paracellular blood barriers isolate the nervous system from the rest of the animal. Glia and accessory cells of the nervous system use pumps, channels, cotransporters, and exchangers collectively to maintain the extracellular ion environment and osmotic balance in the nervous system. At present, the molecular mechanisms that regulate this process remain unclear. In humans, loss of extracellular ion and volume regulation in the nervous system poses serious health threats. Drosophila is a model genetic organism with a proven track record for uncovering molecular mechanisms relevant to human health and disease. Here, we review what is known about extracellular ion and volume regulation in larval abdominal nerves, present some new data about the impact of neural activity on the extracellular environment, and relate the findings to mammalian systems. Homologies have been found at the level of morphology, physiology, molecular mechanisms, and mutant phenotypes. The Fray-Ncc69 module regulates extracellular volume in larval nerves. Genetic rescue experiments with the mammalian orthologs prove this module has a direct correlate in humans. This and other molecular homologies, together with the similar physiological needs, suggest that uncovering the molecular mechanisms of ion and volume regulation in larval nerves will likely provide significant insights into this process in mammalian systems.

Imaging in vivo Neuronal Transport in Genetic Model Organisms Using Microfluidic Devices

Microfluidic devices have been developed for imaging behavior and various cellular processes in Caenorhabditis elegans, but not subcellular processes requiring high spatial resolution. In neurons, essential processes such as axonal, dendritic, intraflagellar and other long-distance transport can be studied by acquiring fast time-lapse images of green fluorescent protein (GFP)-tagged moving cargo. We have achieved two important goals in such in vivo studies namely, imaging several transport processes in unanesthetized intact animals and imaging very early developmental stages. We describe a microfluidic device for immobilizing C. elegans and Drosophila larvae that allows imaging without anesthetics or dissection. We observed that for certain neuronal cargoes in C. elegans, anesthetics have significant and sometimes unexpected effects on the flux. Further, imaging the transport of certain cargo in early developmental stages was possible only in the microfluidic device. Using our device we observed an increase in anterograde synaptic vesicle transport during development corresponding with synaptic growth. We also imaged Q neuroblast divisions and mitochondrial transport during early developmental stages of C. elegans and Drosophila, respectively. Our simple microfluidic device offers a useful means to image high-resolution subcellular processes in C. elegans and Drosophila and can be readily adapted to other transparent or translucent organisms.

Calcium Imaging in Drosophila During Walking and Flight Behavior

Drosophila melanogaster is a genetic model organism with many experimental advantages, including the ability to genetically manipulate specific sub-populations of neurons. The combination of physiology and genetic tools is increasingly being applied to questions in systems neuroscience [1]. Our goal is to understand circuit computations underlying sensory-motor transformation in the fly brain. This requires recording not just neural activity but also the fly's behavior. Towards this end, we have developed a novel experimental setup [2] for two-photon calcium imaging while the fly is walking on an air-supported ‘Buchner’ ball [3, 4] in a virtual arena [5]. These recordings represent the first examples of functional imaging in behaving Drosophila and provide a platform for future explorations of decision-making and sensory-motor transformations in this powerful genetic model organism. We have also adapted the setup for calcium imaging in tethered flying Drosophila. In complementary work, a different group has succeeded in whole-cell patch-clamp recording from fruit flies during flight behavior [6]. Motion-sensitive interneurons in the visual system of walking and flying Drosophila show modulation of their responses depending on their behavioral state [6, 7]. Under natural conditions the visual input of the fly changes and is controlled by the movement of the fly. Closed-loop tethered behavior allows approaching this situation in a controlled experimental setting. We demonstrated the feasibility of closed loop flight behavior during calcium imaging and compare neuronal responses during open and closed-loop flight behavior. 1. Olsen and Wilson (Trends Neurosci., 2008) 2. Seelig, Chiappe, Lott, Dutta, Osborne, Reiser, Jayaraman (Nat Meth, 2010) 3. Gotz and Wenking (J Comp Physiol, 1973) 4. Bohm, Schildberger and Huber (JEB, 1991) 5. Reiser and Dickinson (J Neurosci Meth, 2008) 6. Maimon, Straw, Dickinson (Nat Neurosci, 2010) 7. Chiappe, Seelig, Reiser, Jayaraman, (Curr Biol, 2010)

Purification of Actin from Fission Yeast Schizosaccharomyces pombe and Characterization of Functional Differences from Muscle Actin

Fission yeast Schizosaccharomyces pombe is an important genetic model organism for studying the mechanisms of endocytosis and cytokinesis. However, most work on the biochemical properties of fission yeast actin-binding proteins has been done with skeletal muscle actin for matters of convenience. When simulations of mathematical models of the mechanism of endocytosis were compared with events in live cells, some of the reactions appeared to be much faster than observed in biochemical experiments with muscle actin. Here, we used gelsolin affinity chromatography to purify actin from fission yeast. S. pombe actin shares many properties with skeletal muscle actin but has higher intrinsic nucleotide exchange rate, faster trimer nucleus formation, faster phosphate dissociation rate from polymerized actin, and faster nucleation of actin filaments with Arp2/3 complex. These properties close the gap between the biochemistry and predictions made by mathematical models of endocytosis in S. pombe cells.

Reconstitution of human RNA interference in budding yeast

Although RNA-mediated interference (RNAi) is a widely conserved process among eukaryotes, including many fungi, it is absent from the budding yeast Saccharomyces cerevisiae. Three human proteins, Ago2, Dicer and TRBP, are sufficient for reconstituting the RISC complex in vitro. To examine whether the introduction of human RNAi genes can reconstitute RNAi in S. cerevisiae, genes encoding these three human proteins were introduced into S. cerevisiae. We observed both siRNA and siRNA- and RISC-dependent silencing of the target gene GFP. Thus, human Ago2, Dicer and TRBP can functionally reconstitute human RNAi in S. cerevisiae, in vivo, enabling the study and use of the human RNAi pathway in a facile genetic model organism.

A Grand Challenge for Genetics of Aging: Adding Healthy Years to Our Lives

A Grand Challenge for Genetics of Aging: Adding Healthy Years to Our Lives

The Functions of RNA-Dependent RNA Polymerases in Arabidopsis

One recently identified mechanism that regulates mRNA abundance is RNA silencing, and pioneering work in Arabidopsis thaliana and other genetic model organisms helped define this process. RNA silencing pathways are triggered by either self-complementary fold-back structures or the production of double-stranded RNA (dsRNA) that gives rise to small RNAs (smRNAs) known as microRNAs (miRNAs) or small-interfering RNAs (siRNAs). These smRNAs direct sequence-specific regulation of various gene transcripts, repetitive sequences, viruses, and mobile elements via RNA cleavage, translational inhibition, or transcriptional silencing through DNA methylation and heterochromatin formation. Early genetic screens in Arabidopsis were instrumental in uncovering numerous proteins required for these important regulatory pathways. Among the factors identified by these studies were RNA-dependent RNA polymerases (RDRs), which are proteins that synthesize siRNA-producing dsRNA molecules using a single-stranded RNA (ssRNA) molecule as a template. Recently, a growing body of evidence has implicated RDR-dependent RNA silencing in many different aspects of plant biology ranging from reproductive development to pathogen resistance. Here, we focus on the specific functions of the six Arabidopsis RDRs in RNA silencing, their ssRNA substrates and resulting RDR-dependent smRNAs, and the numerous biological functions of these proteins in plant development and stress responses.

A panorama of mammalian gene expression evolution

Thirty-six years ago, King and Wilson (1975) noticed in their comparison of humans and chimps that the two species, despite having numerous phenotypic differences, are extremely similar in protein sequences. They speculated that changes in gene expression levels and patterns must have played a more important role in phenotypic evolution than changes in protein sequences and functions. However, testing King and Wilson’s hypothesis has not been easy, in part because of the difficulty in comparing gene expressions between species, especially at the genomic scale. Since B10 years ago, microarray has been used to quantify and compare genome-wide gene expressions across species, but this technology has serious limitations. First, if different probes are used to measure the expressions of orthologous genes in two species, the estimated expression levels are usually not directly comparable because of differential affinities of different probes to their respective targets (Liao and Zhang, 2006a). Second, if the same probes are used for different species, the probe-targeting sequences must be identical between the species to avoid creating spurious expression differences. For these reasons, it has not been possible to examine genome-wide evolutionary patterns of gene expression except in a few closely related species (Khaitovich et al, 2005). RNA sequencing (RNA-seq) quantifies messenger RNA concentrations by directly sequencing their complementary DNAs, and therefore is immune to the hybridization-caused non-comparability problem of microarray. RNA-seq data have been generated from a number of genetic model organisms such as yeast, Arabidopsis, and mouse, and have been used to address a few evolutionary questions (Qian et al, 2010). What is lacking, however, are RNA-seq data from multiple conditions or tissues of non-model organisms that fill the huge evolutionary gaps among the few model organisms. In a recent issue of Nature, Brawand et al (2011) reported a large RNA-seq data set from six organs (cerebral cortex or whole brain without cerebellum, cerebellum, heart, kidney, liver, and testis) of nine mammals (human, chimpanzee, bonobo, gorilla, orangutan, macaque, mouse, opossum, and platypus), and one bird (chicken), including both males and females. This data set, largest of its kind in the literature, promises to provide a grand view of gene expression evolution in mammals.

Microinjection of Medaka Embryos for use as a Model Genetic Organism

In this video, we demonstrate the technique of microinjection into one-cell stage medaka embryos. Medaka is a small egg-laying freshwater fish that allows both genetic and embryological analyses and is one of the vertebrate model organisms in which genome-wide phenotype-driven mutant screens were carried out (1), as in zebrafish and the mouse. Divergence of functional overlap of related genes between medaka and zebrafish allows identification of novel phenotypes that are unidentifiable in a single species (2), thus medaka and zebrafish are complementary for genetic dissection of vertebrate genome functions. To take advantage of medaka fish whose embryos are transparent and develop externally, microinjection is an essential technique to inject cell-tracers for labeling cells, mRNAs or anti-sense oligonucleotides for over-expressing and knocking-down genes of interest, and DNAs for making transgenic lines.

Microinjection of Medaka Embryos for use as a Model Genetic Organism

In this video, we demonstrate the technique of microinjection into one-cell stage medaka embryos. Medaka is a small egg-laying freshwater fish that allows both genetic and embryological analyses and is one of the vertebrate model organisms in which genome-wide phenotype-driven mutant screens were carried out 1, as in zebrafish and the mouse. Divergence of functional overlap of related genes between medaka and zebrafish allows identification of novel phenotypes that are unidentifiable in a single species 2, thus medaka and zebrafish are complementary for genetic dissection of vertebrate genome functions. To take advantage of medaka fish whose embryos are transparent and develop externally, microinjection is an essential technique to inject cell-tracers for labeling cells, mRNAs or anti-sense oligonucleotides for over-expressing and knocking-down genes of interest, and DNAs for making transgenic lines.

The developmental transcriptome of Drosophila melanogaster

Drosophila melanogaster is one of the most well studied genetic model organisms, nonetheless its genome still contains unannotated coding and non-coding genes, transcripts, exons, and RNA editing sites. Full discovery and annotation are prerequisites for understanding how the regulation of transcription, splicing, and RNA editing directs development of this complex organism. We used RNA-Seq, tiling microarrays, and cDNA sequencing to explore the transcriptome in 30 distinct developmental stages. We identified 111,195 new elements, including thousands of genes, coding and non-coding transcripts, exons, splicing and editing events and inferred protein isoforms that previously eluded discovery using established experimental, prediction and conservation-based approaches. Together, these data substantially expand the number of known transcribed elements in the Drosophila genome and provide a high-resolution view of transcriptome dynamics throughout development.

Cellular Stress Responses, Mitostress and Carnitine Insufficiencies as Critical Determinants in Aging and Neurodegenerative Disorders: Role of Hormesis and Vitagenes

The widely accepted oxidative stress theory of aging postulates that aging results from accumulation of oxidative damage. A prediction of this theory is that, among species, differential rates of aging may be apparent on the basis of intrinsic differences in oxidative damage accrual. Although widely accepted, there is a growing number of exceptions to this theory, most contingently related to genetic model organism investigations. Proteins are one of the prime targets for oxidative damage and cysteine residues are particularly sensitive to reversible and irreversible oxidation. The adaptation and survival of cells and organisms requires the ability to sense proteotoxic insults and to coordinate protective cellular stress response pathways and chaperone networks related to protein quality control and stability. The toxic effects that stem from the misassembly or aggregation of proteins or peptides, in any cell type, are collectively termed proteotoxicity. Despite the abundance and apparent capacity of chaperones and other components of homeostasis to restore folding equilibrium, the cell appears poorly adapted for chronic proteotoxic stress which increases in cancer, metabolic and neurodegenerative diseases. Pharmacological modulation of cellular stress response pathways has emerging implications for the treatment of human diseases, including neurodegenerative disorders, cardiovascular disease, and cancer. A critical key to successful medical intervention is getting the dose right. Achieving this goal can be extremely challenging due to human inter-individual variation as affected by age, gender, diet, exercise, genetic factors and health status. The nature of the dose response in and adjacent to the therapeutic zones, over the past decade has received considerable advances. The hormetic dose-response, challenging long-standing beliefs about the nature of the dose-response in a lowdose zone, has the potential to affect significantly the design of pre-clinical studies and clinical trials as well as strategies for optimal patient dosing in the treatment of numerous diseases. Given the broad cytoprotective properties of the heat shock response there is now strong interest in discovering and developing pharmacological agents capable of inducing stress responses, including carnitines. This paper describes in mechanistic detail how hormetic dose responses are mediated for endogenous cellular defense pathways, including the possible signaling mechanisms by which the carnitine system, by interplaying metabolism, mitochondrial energetics and activation of critical vitagenes, modulates signal transduction cascades that confer cytoprotection against chronic degenerative damage associated to aging and neurodegenerative disorders.

Glial solute carrier transporters in drosophila and mice

Glia regulate brain physiology primarily by regulating the movement and concentration of substances in the extracellular fluid. Therefore, one approach to understanding the role of glia in brain physiology is to study what happens when glial transporters are removed or modified. The largest and most highly conserved class of transporter is solute carrier (SLC) proteins. SLC proteins are highly expressed in brain, and many are found in glia. The function of many SLC proteins in the brain--particularly in glia--is very poorly understood. SLC proteins can be relatively easily knocked out or modified in genetic model organisms to better understand glial function. Drosophila are popular genetic model organisms that offer a nice balance between genetic malleability and brain complexity. They are ideal for such an endeavor. This article lists and discusses SLC transporter family members that are expressed in both mouse and Drosophila glia, in an effort to provide a foundation for studies of glial SLC transporters using Drosophila as a model.

The natural history of Caenorhabditis elegans

Summary In the laboratory, the nematode Caenorhabditis elegans lives on the surface of nutrient agar in Petri dishes, feeding on a lawn of the uracil auxotroph strain OP50, an Escherichia coli mutant strain. This sentence sums up the fundamentals of C. elegans ecology, as most of us know it. While over 15,000 articles on diverse biological aspects of C. elegans attest to the worm's undisputable virtues as a major model organism, its biology in the wild remains mysterious. To properly interpret and fully understand the available wealth of genetic, molecular and other biological observations made in the laboratory, it will be important to know its natural history and to place the species in its ecological and evolutionary context. With the aim of connecting the discoveries that have been made about C. elegans biology to its ‘real life', we shall discuss recent studies on the worm's natural habitat and population biology, and outline key issues in attaining a modern natural history of C. elegans.

Day and Night: Circadian Rhythms in Worms

Day and Night: Circadian Rhythms in Worms

Reactive Oxygen Species and Aging inCaenorhabditis elegans: Causal or Casual Relationship?

The free radical theory of aging proposes a causal relationship between reactive oxygen species (ROS) and aging. While it is clear that oxidative damage increases with age, its role in the aging process is uncertain. Testing the free radical theory of aging requires experimentally manipulating ROS production or detoxification and examining the resulting effects on lifespan. In this review, we examine the relationship between ROS and aging in the genetic model organism Caenorhabditis elegans, summarizing experiments using long-lived mutants, mutants with altered mitochondrial function, mutants with decreased antioxidant defenses, worms treated with antioxidant compounds, and worms exposed to different environmental conditions. While there is frequently a negative correlation between oxidative damage and lifespan, there are many examples in which they are uncoupled. Neither is resistance to oxidative stress sufficient for a long life nor are all long-lived mutants more resistant to oxidative stress. Similarly, sensitivity to oxidative stress does not necessarily shorten lifespan and is in fact compatible with long life. Overall, the data in C. elegans indicate that oxidative damage can be dissociated from aging in experimental situations.

FLOUR BEETLES JOIN THE GAL4/UAS CLUB

Being able to control gene expression in defined parts of an organism's body and at distinct developmental stages allows researchers to determine the functional significance of gene expression patterns. Unfortunately, the ability to do this has been limited to a few genetic model organisms. Johannes Schinko and colleagues at the Georg-August-Universität Göttingen recently tackled the task of adding a new species, the red flour beetle (Tribolium castaneum, which, as a beetle, represents the most species-rich metazoan taxon) to the list of model organisms in which spatial and temporal control of gene expression is possible. The team published their work in BMC Developmental Biology.The team focused on adapting existing technology to their animal. The GAL4/UAS system is a widely used technique for controlling gene expression in fruit flies. It is composed of a yeast protein (GAL4) that specifically binds to a sequence of DNA known as the upstream activator sequence (UAS), triggering the expression of any gene inserted immediately behind the UAS. The choice of gene is up to the researcher. By themselves, GAL4 and UAS do nothing in a non-yeast cell, but bring them together in the same cell and together they function as an ‘on’ switch that activates expression of the chosen gene. Researchers then control GAL4 expression so that they can selectively switch their chosen gene on and off in specific locations and at specific times.First, Schinko and his colleagues tried to directly translate all of these Drosophila techniques into beetles. They incorporated GAL4 (under the control of fruit fly promoters) into flour beetle tissues. They then put ‘reporter genes’ under UAS control in the same tissues. Reporter genes encode proteins (e.g. green fluorescent protein) that allow researchers to visualise when and where GAL4 successfully initiates gene expression. But all of this turned out to be a dead end. Under conditions that lead to robust GAL4 activity in flies, the researchers saw no evidence of reporter gene expression in beetles. Fruit-fly-based GAL4/UAS appeared not to work in beetles.The team reasoned that the use of native beetle promoters might solve the problem, so they swapped a fruit fly heat shock promoter, which switches on the insect's heat shock response, for an equivalent beetle promoter. When these beetles were heat shocked, they showed robust reporter gene expression in multiple body areas, indicating that the GAL4/UAS system was functioning. The team then went on to assay how quickly they could induce this reporter gene expression in embryos. They found evidence of reporter gene activity within 2–4 hours of heat shock. This response time is only a fraction of the time needed for embryogenesis (72 h), allowing researchers to use the GAL4/UAS system for fine analyses of gene function in T. castaneum embryos during development. Finally, the team tested a second beetle promoter ‘Tc-hairy’ and found reporter gene expression confined primarily to the central nervous system of the animal. This suggests that GAL4/UAS can also be used to spatially restrict the activity of selected genes in beetles.Overall, the work of Schinko and colleagues lays the methodological foundations for a new era of powerful developmental and genetic experiments in flour beetles. The experiments presented are not flashy, but they are carefully done with an eye on the long-term future. Unfortunately, the importance of (and the amount of work that goes into) this type of methods publication is often under-appreciated. Ironically though, in the long run, these types of understated ‘tool-building’ efforts often end up being the real engines driving innovation in biological research.