Genetic Model Organism Research Articles

The role of ubiquitin‐mediated pathways in regulating synaptic development, axonal degeneration and regeneration: insights from fly and worm

The covalent attachment of the 76 amino acid peptide ubiquitin to target proteins is a rapid and reversible modification that regulates protein stability, activity and localization. As such, it is a potent mechanism for sculpting the synapse. Recent studies from two genetic model organisms, Caenorhabditis elegans and Drosophila, have provided mounting evidence that ubiquitin-mediated pathways play important roles in controlling the presynaptic size, synaptic elimination and stabilization, synaptic transmission, postsynaptic receptor abundance, axonal degeneration and regeneration. While the data supporting the requirement of ubiquitination/deubiquitination for normal synaptic development and repair are compelling, detailed analyses of signalling events up- and downstream of these ubiquitin modifications are often challenging. This article summarizes the related research conducted in worms and flies and provides insight into the fundamental questions facing this field.

Kinesin-1 acts with netrin and DCC to maintain sensory neuron position in Caenorhabditis elegans.

The organization of neurons and the maintenance of that arrangement are critical to brain function. Failure of these processes in humans can lead to severe birth defects, mental retardation, and epilepsy. Several kinesins have been shown to play important roles in cell migration in vertebrate systems, but few upstream and downstream pathway members have been identified. Here, we utilize the genetic model organism Caenorhabditis elegans to elucidate the pathway by which the C. elegans Kinesin-1 Heavy Chain (KHC)/KIF5 ortholog UNC-116 functions to maintain neuronal cell body position in the PHB sensory neurons. We find that UNC-116/KHC acts in part with the cell and axon migration molecules UNC-6/Netrin and UNC-40/DCC in this process, but in parallel to SAX-3/Robo. We have also identified several potential adaptor, cargo, and regulatory proteins that may provide insight into the mechanism of UNC-116/KHC's function in this process. These include the cargo receptor UNC-33/CRMP2, the cargo adaptor protein UNC-76/FEZ and its regulator UNC-51/ULK, the cargo molecule UNC-69/SCOCO, and the actin regulators UNC-44/Ankyrin and UNC-34/Enabled. These genes also act in cell migration and axon outgrowth; however, many proteins that function in these processes do not affect PHB position. Our findings suggest an active posterior cell migration mediated by UNC-116/KHC occurs throughout development to maintain proper PHB cell body position and define a new pathway that mediates maintenance of neuronal cell body position.

Arbuscular mycorrhizal fungi reduce growth and infect roots of the non‐host plant Arabidopsis thaliana

The arbuscular mycorrhizal (AM) symbiosis is widespread throughout the plant kingdom and important for plant nutrition and ecosystem functioning. Nonetheless, most terrestrial ecosystems also contain a considerable number of non-mycorrhizal plants. The interaction of such non-host plants with AM fungi (AMF) is still poorly understood. Here, in three complementary experiments, we investigated whether the non-mycorrhizal plant Arabidopsis thaliana, the model organism for plant molecular biology and genetics, interacts with AMF. We grew A. thaliana alone or together with a mycorrhizal host species (either Trifolium pratense or Lolium multiflorum) in the presence or absence of the AMF Rhizophagus irregularis. Plants were grown in a dual-compartment system with a hyphal mesh separating roots of A. thaliana from roots of the host species, avoiding direct root competition. The host plants in the system ensured the presence of an active AM fungal network. AM fungal networks caused growth depressions in A. thaliana of more than 50% which were not observed in the absence of host plants. Microscopy analyses revealed that R. irregularis supported by a host plant was capable of infecting A. thaliana root tissues (up to 43% of root length colonized), but no arbuscules were observed. The results reveal high susceptibility of A. thaliana to R. irregularis, suggesting that A. thaliana is a suitable model plant to study non-host/AMF interactions and the biological basis of AM incompatibility.

Principles of transcriptome analysis and gene expression quantification: an RNA‐seq tutorial

Genome-wide analyses and high-throughput screening was long reserved for biomedical applications and genetic model organisms. With the rapid development of massively parallel sequencing nanotechnology (or next-generation sequencing) and simultaneous maturation of bioinformatic tools, this situation has dramatically changed. Genome-wide thinking is forging its way into disciplines like evolutionary biology or molecular ecology that were historically confined to small-scale genetic approaches. Accessibility to genome-scale information is transforming these fields, as it allows us to answer long-standing questions like the genetic basis of local adaptation and speciation or the evolution of gene expression profiles that until recently were out of reach. Many in the eco-evolutionary sciences will be working with large-scale genomic data sets, and a basic understanding of the concepts and underlying methods is necessary to judge the work of others. Here, I briefly introduce next-generation sequencing and then focus on transcriptome shotgun sequencing (RNA-seq). This article gives a broad overview and provides practical guidance for the many steps involved in a typical RNA-seq work flow from sampling, to RNA extraction, library preparation and data analysis. I focus on principles, present useful tools where appropriate and point out where caution is needed or progress to be expected. This tutorial is mostly targeted at beginners, but also contains potentially useful reflections for the more experienced.

Stable Gene Silencing in Zebrafish with Spatiotemporally Targetable RNA Interference

The ability to regulate gene activity in a spatiotemporally controllable manner is vital for biological discovery that will impact disease diagnosis and treatment. While conditional gene silencing is possible in other genetic model organisms, this technology is largely unavailable in zebrafish, an important vertebrate model organism for functional gene discovery. Here, using short hairpin RNAs (shRNAs) designed in the microRNA-30 backbone, which have been shown to mimic natural microRNA primary transcripts and be more effective than simple shRNAs, we report stable RNA interference-mediated gene silencing in zebrafish employing the yeast Gal4-UAS system. Using this approach, we reveal at single-cell resolution the role of atypical protein kinase Cλ (aPKCλ) in regulating neural progenitor/stem cell division. We also show effective silencing of the one-eyed-pinhead and no-tail/brachyury genes. Furthermore, we demonstrate stable integration and germ-line transmission of the UAS-miR-shRNAs for aPKCλ, the expressivity of which is controllable by the strength and expression of Gal4. This technology shall significantly advance the utility of zebrafish for understanding fundamental vertebrate biology and for the identification and evaluation of important therapeutic targets.

What do human parasites with a chloroplast anyway?

Apicomplexan parasites are a major threat to human health causing malaria and a variety of AIDS associated opportunistic infections. One of the most promising targets for chemotherapeutic intervention for this group of pathogens is the apicoplast. The apicoplast is a unique parasite organelle that was derived from a red algal endosymbiont. Genome analyses suggest that the apicoplast is engaged in a variety of biosynthetic activities that could be targeted for drug development. Using Toxoplasma gondii as a genetic model organism we are dissecting the apicoplast metabolism to identify the most effective choke points. Taking a broader biological view we are interested to understand which of the endosymbiont's functions is most critical and the reason for the continued presence of a plastid long after the loss of photosynthesis. Our current work is focused on the mechanisms and importance of parasite lipid synthesis in particular that of fatty acids and isoprenoids.

TALEN-Based Gene Disruption in the Dengue Vector Aedes aegypti

In addition to its role as the primary vector for dengue viruses, Aedes aegypti has a long history as a genetic model organism for other bloodfeeding mosquitoes, due to its ease of colonization, maintenance and reproductive productivity. Though its genome has been sequenced, functional characterization of many Ae. aegypti genes, pathways and behaviors has been slow. TALE nucleases (TALENs) have been used with great success in a number of organisms to generate site-specific DNA lesions. We evaluated the ability of a TALEN pair to target the Ae. aegypti kmo gene, whose protein product is essential in the production of eye pigmentation. Following injection into pre-blastoderm embryos, 20–40% of fertile survivors produced kmo alleles that failed to complement an existing khw mutation. Most of these individuals produced more than 20% white-eyed progeny, with some producing up to 75%. Mutant alleles were associated with lesions of 1–7 bp specifically at the selected target site. White-eyed individuals could also be recovered following a blind intercross of G1 progeny, yielding several new white-eyed strains in the genetic background of the sequenced Liverpool strain. We conclude that TALENs are highly active in the Ae. aegypti germline, and have the potential to transform how reverse genetic experiments are performed in this important disease vector.

Primary Neuron Culture for Nerve Growth and Axon Guidance Studies in Zebrafish (Danio rerio)

Zebrafish (Danio rerio) is a widely used model organism in genetics and developmental biology research. Genetic screens have proven useful for studying embryonic development of the nervous system in vivo, but in vitro studies utilizing zebrafish have been limited. Here, we introduce a robust zebrafish primary neuron culture system for functional nerve growth and guidance assays. Distinct classes of central nervous system neurons from the spinal cord, hindbrain, forebrain, and retina from wild type zebrafish, and fluorescent motor neurons from transgenic reporter zebrafish lines, were dissociated and plated onto various biological and synthetic substrates to optimize conditions for axon outgrowth. Time-lapse microscopy revealed dynamically moving growth cones at the tips of extending axons. The mean rate of axon extension in vitro was 21.4±1.2 µm hr−1 s.e.m. for spinal cord neurons, which corresponds to the typical ∼0.5 mm day−1 growth rate of nerves in vivo. Fluorescence labeling and confocal microscopy demonstrated that bundled microtubules project along axons to the growth cone central domain, with filamentous actin enriched in the growth cone peripheral domain. Importantly, the growth cone surface membrane expresses receptors for chemotropic factors, as detected by immunofluorescence microscopy. Live-cell functional assays of axon extension and directional guidance demonstrated mammalian brain-derived neurotrophic factor (BDNF)-dependent stimulation of outgrowth and growth cone chemoattraction, whereas mammalian myelin-associated glycoprotein inhibited outgrowth. High-resolution live-cell Ca2+-imaging revealed local elevation of cytoplasmic Ca2+ concentration in the growth cone induced by BDNF application. Moreover, BDNF-induced axon outgrowth, but not basal outgrowth, was blocked by treatments to suppress cytoplasmic Ca2+ signals. Thus, this primary neuron culture model system may be useful for studies of neuronal development, chemotropic axon guidance, and mechanisms underlying inhibition of neural regeneration in vitro, and complement observations made in vivo.

Reverse genetics tools in zebrafish: A forward dive into endocrinology

The zebrafish is a powerful genetic model organism. In recent years, zebrafish has been increasingly used to model human diseases. Due to a number of recent technological advancements, the genetic tool box is now also stocked with sophisticated transgenic and reverse genetic tools. Here, we focus on both commonly used and recently established reverse genetic and transgenic tools available in zebrafish. These new developments make the zebrafish an even more attractive animal model in comparative endocrinology.

Dynamic microtubules and endomembrane cycling contribute to polarity establishment and early development of Ectocarpus mitospores.

Many zygotes and spores of brown algae are photosensitive and establish a developmental axis in accordance with directional light cues. Ectocarpus siliculosus is being advanced as a genetic and genomic model organism for investigating brown alga development, and this report investigates photopolarization of the growth axis of mitospores. When exposed to unidirectional light, mitospores photopolarized and established a growth axis such that germination was preferentially localized to the shaded hemisphere of the spore body. The roles of the microtubule cytoskeleton and endomembrane cycling in the photopolarization process were investigated using pharmacological agents. Disruption of microtubule dynamics progressively reduced the percentage of mitospores that photopolarized, while inhibition of vesicle secretion blocked photopolarization nearly completely. Chronic treatment with these pharmacological agents severely affected algal morphogenesis. Microtubules in mitospores and algal filaments were imaged by confocal microscopy. Mitospores contained a radial microtubule array, emanating from a centrosome associated with the nuclear envelope. At germination, the radial array gradually transitioned into a longitudinal array with microtubules extending into the emerging apex. At mitosis, spindles were aligned with the growth axis of cylindrical cells in the filament, and the division plane bisected the spindle axis. These studies demonstrate that dynamic membrane cycling and microtubule assembly play fundamental roles in photopolarization and provide a foundation for future genetic and genomic investigations of this important developmental process.

A cell-based fascin bioassay identifies compounds with potential anti-metastasis or cognition-enhancing functions

SUMMARY The actin-bundling protein fascin is a key mediator of tumor invasion and metastasis and its activity drives filopodia formation, cell-shape changes and cell migration. Small-molecule inhibitors of fascin block tumor metastasis in animal models. Conversely, fascin deficiency might underlie the pathogenesis of some developmental brain disorders. To identify fascin-pathway modulators we devised a cell-based assay for fascin function and used it in a bidirectional drug screen. The screen utilized cultured fascin-deficient mutant Drosophila neurons, whose neurite arbors manifest the 'filagree' phenotype. Taking a repurposing approach, we screened a library of 1040 known compounds, many of them FDA-approved drugs, for filagree modifiers. Based on scaffold distribution, molecular-fingerprint similarities, and chemical-space distribution, this library has high structural diversity, supporting its utility as a screening tool. We identified 34 fascin-pathway blockers (with potential anti-metastasis activity) and 48 fascin- pathway enhancers (with potential cognitive-enhancer activity). The structural diversity of the active compounds suggests multiple molecular targets. Comparisons of active and inactive compounds provided preliminary structure-activity relationship information. The screen also revealed diverse neurotoxic effects of other drugs, notably the 'beads-on-a-string' defect, which is induced solely by statins. Statin-induced neurotoxicity is enhanced by fascin deficiency. In summary, we provide evidence that primary neuron culture using a genetic model organism can be valuable for early-stage drug discovery and developmental neurotoxicity testing. Furthermore, we propose that, given an appropriate assay for target-pathway function, bidirectional screening for brain-development disorders and invasive cancers represents an efficient, multipurpose strategy for drug discovery.

Using fly genetics to dissect the cytoskeletal machinery of neurons during axonal growth and maintenance

The extension of long slender axons is a key process of neuronal circuit formation, both during brain development and regeneration. For this, growth cones at the tips of axons are guided towards their correct target cells by signals. Growth cone behaviour downstream of these signals is implemented by their actin and microtubule cytoskeleton. In the first part of this Commentary, we discuss the fundamental roles of the cytoskeleton during axon growth. We present the various classes of actin- and microtubule-binding proteins that regulate the cytoskeleton, and highlight the important gaps in our understanding of how these proteins functionally integrate into the complex machinery that implements growth cone behaviour. Deciphering such machinery requires multidisciplinary approaches, including genetics and the use of simple model organisms. In the second part of this Commentary, we discuss how the application of combinatorial genetics in the versatile genetic model organism Drosophila melanogaster has started to contribute to the understanding of actin and microtubule regulation during axon growth. Using the example of dystonin-linked neuron degeneration, we explain how knowledge acquired by studying axonal growth in flies can also deliver new understanding in other aspects of neuron biology, such as axon maintenance in higher animals and humans.

Sarcopenia and piscines: the case for indeterminate-growing fish as unique genetic model organisms in aging and longevity research

Sarcopenia and dynapenia pose significant problems for the aged, especially as life expectancy rises in developed countries. Current therapies are marginally efficacious at best, and barriers to breakthroughs in treatment may result from currently employed model organisms. Here, we argue that the use of indeterminate-growing teleost fish in skeletal muscle aging research may lead to therapeutic advancements not possible with current mammalian models. Evidence from a comparative approach utilizing the subfamily Danioninae suggests that the indeterminate growth paradigm of many teleosts arises from adult muscle stem cells with greater proliferative capacity, even in spite of smaller progenitor populations. We hypothesize that paired-box transcription factors, Pax3/7, are involved with this enhanced self-renewal and that prolonged expression of these factors may allow some fish species to escape, or at least forestall, sarcopenia/dynapenia. Future research efforts should focus on the experimental validation of these genes as key factors in indeterminate growth, both in the context of muscle stem cell proliferation and in prevention of skeletal muscle senescence.

A new day dawning: Hemerocallis (daylily) as a future model organism

Genetic model organisms have revolutionized science, and today, with the rapid advances in technology, there is significant potential to launch many more plant species towards model status. However, these new model organisms will have to be carefully selected. Here, we argue that Hemerocallis (daylily) satisfies multiple criteria for selection and deserves serious consideration as a subject of intensive biological investigation. Several attributes of the genus are of great biological interest. These include the strict control of flower opening and, within a short period, the precisely regulated floral death by a programmed cell death system. The self-incompatibility system in Hemerocallis is also noteworthy and deserves more attention. Importantly, the genus is widely cultivated for food, medicinal value and ornamental interest. Hemerocallis has considerable potential as a 'nutraceutical' food plant and the source of new compounds with biomedical activity. The genus has also been embraced by ornamental plant breeders and the extraordinary morphological diversity of hybrid cultivars, produced within a relatively short time by amateur enthusiasts, is an exceptional resource for botanical and genetic studies. We explore these points in detail, explaining the reasons why this genus has considerable value-both academic and socio-economic-and deserves new resources devoted to its exploration as a model. Its impact as a future model will be enhanced by its amenability to cultivation in laboratory and field conditions. In addition, established methods for various tissue and cell culture systems as well as transformation will permit maximum exploitation of this genus by science.

Drosophila modifier screens to identify novel neuropsychiatric drugs including aminergic agents for the possible treatment of Parkinson’s disease and depression

Small molecules that increase the presynaptic function of aminergic cells may provide neuroprotection in Parkinson’s disease as well as treatments for attention deficit hyperactivity disorder (ADHD) and depression. Model genetic organisms such as Drosophila melanogaster may enhance the detection of new drugs via modifier or “enhancer/suppressor” screens, but this technique has not been applied to processes relevant to psychiatry. To identify new aminergic drugs in vivo, we used a mutation in the Drosophila vesicular monoamine transporter (dVMAT) as a sensitized genetic background, and performed a suppressor screen. We fed dVMAT mutant larvae ~1000 known drugs and quantitated rescue (suppression) of an amine-dependent locomotor deficit in the larva. To determine which drugs might specifically potentiate neurotransmitter release, we performed an additional secondary screen for drugs that require presynaptic amine storage to rescue larval locomotion. Using additional larval locomotion and adult fertility assays, we validated that at least one compound previously used clinically as an antineoplastic agent potentiates the presynaptic function of aminergic circuits. We suggest that structurally similar agents might be used to development treatments for Parkinson’s disease, depression and ADHD and that modifier screens in Drosophila provide a new strategy to screen for neuropsychiatric drugs. More generally, our findings demonstrate the power of physiologically based screens for identifying bioactive agents for select neurotransmitter systems.

Transcriptomics Research in Chicken

The chicken (Gallus gallus) is an important model organism in genetics, developmental biology, immunology and evolutionary research. Moreover, besides being an important model organism the chicken is also a very important agricultural species and an important source of food (eggs and meat). The availability of the draft chicken genome sequence provided many possibilities to in detail study a variety of genomic changes during evolution using a comparison between chicken and mammals. For example, compared to mammals, the use of a Z/W sex determination system is a special aspect of the avian genome where the female is the heterogametic sex (ZW) and the male is the homogametic (ZZ) sex. A comparison of the genomic sequences of platypus, chicken and human showed that sex chromosomes evolved separately in birds and mammals

The Epigenetics of Emerging and Nonmodel Organisms

The Epigenetics of Emerging and Nonmodel Organisms

Forward Chemical Genetics in Yeast for Discovery of Chemical Probes Targeting Metabolism

The many virtues that made the yeast Saccharomyces cerevisiae a dominant model organism for genetics and molecular biology, are now establishing its role in chemical genetics. Its experimental tractability (i.e., rapid doubling time, simple culture conditions) and the availability of powerful tools for drug-target identification, make yeast an ideal organism for high-throughput phenotypic screening. It may be especially applicable for the discovery of chemical probes targeting highly conserved cellular processes, such as metabolism and bioenergetics, because these probes would likely inhibit the same processes in higher eukaryotes (including man). Importantly, changes in normal cellular metabolism are associated with a variety of diseased states (including neurological disorders and cancer), and exploiting these changes for therapeutic purposes has accordingly gained considerable attention. Here, we review progress and challenges associated with forward chemical genetic screening in yeast. We also discuss evidence supporting these screens as a useful strategy for discovery of new chemical probes and new druggable targets related to cellular metabolism.

Diversity in global maize germplasm: Characterization and utilization

Maize (Zea mays L.) is not only of worldwide importance as a food, feed and as a source of diverse industrially important products, but is also a model genetic organism with immense genetic diversity. Although it was first domesticated in Mexico, maize landraces are widely found across the continents. Several studies in Mexico and other countries highlighted the genetic variability in the maize germplasm. Applications of molecular markers, particularly in the last two decades, have led to new insights into the patterns of genetic diversity in maize globally, including landraces as well as wild relatives (especially teosintes) in Latin America, helping in tracking the migration routes of maize from the centers of origin, and understanding the fate of genetic diversity during maize domestication. The genome sequencing of B73 (a highly popular US Corn Belt inbred) and Palomero (a popcorn landrace in Mexico) in the recent years are important landmarks in maize research, with significant implications to our understanding of the maize genome organization and evolution. Next-generation sequencing and high-throughput genotyping platforms promise to further revolutionize our understanding of genetic diversity and for designing strategies to utilize the genomic information for maize improvement. However, the major limiting factor to exploit the genetic diversity in crops like maize is no longer genotyping, but high-throughput and precision phenotyping. There is an urgent need to establish a global phenotyping network for comprehensive and efficient characterization of maize germplasm for an array of target traits, particularly for biotic and abiotic stress tolerance and nutritional quality. 'Seeds of Discovery' (SeeD), a novel initiative by CIMMYT with financial support from the Mexican Government for generating international public goods, has initiated intensive exploration of phenotypic and molecular diversity of maize germplasm conserved in the CIMMYT Gene Bank; this is expected to aid in effective identification and use of novel alleles and haplotypes for maize improvement. Multi-institutional efforts are required at the global level to systematically explore the maize germplasm to diversify the genetic base of elite breeding materials, create novel varieties and counter the effects of global climate changes.

Changes in the dielectric properties of medaka fish embryos during development, studied by electrorotation

The Japanese medaka fish, Oryzias latipes, has become a powerful vertebrate model organism in developmental biology and genetics. The present study explores the dielectric properties of medaka embryos during pre-hatching development by means of the electrorotation (ROT) technique. Due to their layered structure, medaka eggs exhibited up to three ROT peaks in the kHz–MHz frequency range. During development from blastula to early somite stage, ROT spectra varied only slightly. But as the embryo progressed to the late-somite stage, the ROT peaks underwent significant changes in frequency and amplitude. Using morphological data obtained by light and electron microscopy, we analyzed the ROT spectra with a three-shell dielectric model that accounted for the major embryonic compartments. The analysis yielded a very high value for the ionic conductivity of the egg shell (chorion), which was confirmed by independent osmotic experiments. A relatively low capacitance of the yolk envelope was consistent with its double-membrane structure revealed by transmission electron microscopy. Yolk-free dead eggs exhibited only one co-field ROT peak, shifted markedly to lower frequencies with respect to the corresponding peak of live embryos. The dielectric data may be useful for monitoring the development and changes in fish embryos’ viability/conditions in basic research and industrial aquaculture.