Genetic Model System Research Articles

The Intestine of Drosophila melanogaster: An Emerging Versatile Model System to Study Intestinal Epithelial Homeostasis and Host-Microbial Interactions in Humans.

In all metazoans, the intestinal tract is an essential organ to integrate nutritional signaling, hormonal cues and immunometabolic networks. The dysregulation of intestinal epithelium functions can impact organism physiology and, in humans, leads to devastating and complex diseases, such as inflammatory bowel diseases, intestinal cancers, and obesity. Two decades ago, the discovery of an immune response in the intestine of the genetic model system, Drosophila melanogaster, sparked interest in using this model organism to dissect the mechanisms that govern gut (patho) physiology in humans. In 2007, the finding of the intestinal stem cell lineage, followed by the development of tools available for its manipulation in vivo, helped to elucidate the structural organization and functions of the fly intestine and its similarity with mammalian gastrointestinal systems. To date, studies of the Drosophila gut have already helped to shed light on a broad range of biological questions regarding stem cells and their niches, interorgan communication, immunity and immunometabolism, making the Drosophila a promising model organism for human enteric studies. This review summarizes our current knowledge of the structure and functions of the Drosophila melanogaster intestine, asserting its validity as an emerging model system to study gut physiology, regeneration, immune defenses and host-microbiota interactions.

Cohesin Impedes Heterochromatin Assembly in Fission Yeast Cells Lacking Pds5.

The fission yeast Schizosaccharomyces pombe is a powerful genetic model system for uncovering fundamental principles of heterochromatin assembly and epigenetic inheritance of chromatin states. Heterochromatin defined by histone H3 lysine 9 methylation and HP1 proteins coats large chromosomal domains at centromeres, telomeres, and the mating-type (mat) locus. Although genetic and biochemical studies have provided valuable insights into heterochromatin assembly, many key mechanistic details remain unclear. Here, we use a sensitized reporter system at the mat locus to screen for factors affecting heterochromatic silencing. In addition to known components of heterochromatin assembly pathways, our screen identified eight new factors including the cohesin-associated protein Pds5. We find that Pds5 enriched throughout heterochromatin domains is required for proper maintenance of heterochromatin. This function of Pds5 requires its associated Eso1 acetyltransferase, which is implicated in the acetylation of cohesin. Indeed, introducing an acetylation-mimicking mutation in a cohesin subunit suppresses defects in heterochromatin assembly in pds5∆ and eso1∆ cells. Our results show that in cells lacking Pds5, cohesin interferes with heterochromatin assembly. Supporting this, eliminating cohesin from the mat locus in the pds5∆ mutant restores both heterochromatin assembly and gene silencing. These analyses highlight an unexpected requirement for Pds5 in ensuring proper coordination between cohesin and heterochromatin factors to effectively maintain gene silencing.

Autism Spectrum Disorder-Related Syndromes: Modeling with Drosophila and Rodents.

Whole exome analyses have identified a number of genes associated with autism spectrum disorder (ASD) and ASD-related syndromes. These genes encode key regulators of synaptogenesis, synaptic plasticity, cytoskeleton dynamics, protein synthesis and degradation, chromatin remodeling, transcription, and lipid homeostasis. Furthermore, in silico studies suggest complex regulatory networks among these genes. Drosophila is a useful genetic model system for studies of ASD and ASD-related syndromes to clarify the in vivo roles of ASD-associated genes and the complex gene regulatory networks operating in the pathogenesis of ASD and ASD-related syndromes. In this review, we discuss what we have learned from studies with vertebrate models, mostly mouse models. We then highlight studies with Drosophila models. We also discuss future developments in the related field.

A sugarcane mosaic virusvector for gene expression in maize.

Zea mays L. ssp. mays (maize) is an important crop plant as well as model system for genetics and plant biology. The ability to select among different virus‐based platforms for transient gene silencing or protein expression experiments is expected to facilitate studies of gene function in maize and complement experiments with stable transgenes. Here, we describe the development of a sugarcane mosaic virus (SCMV) vector for the purpose of protein expression in maize. An infectious SCMV cDNA clone was constructed, and heterologous genetic elements were placed between the protein 1 (P1) and helper component‐proteinase (HC‐Pro) cistrons in the SCMV genome. Recombinant SCMV clones engineered to express green fluorescent protein (GFP), β‐glucuronidase (GUS), or bialaphos resistance (BAR) protein were introduced into sweet corn (Golden × Bantam) plants. Documentation of developmental time courses spanning maize growth from seedling to tasseling showed that the SCMV genome tolerates insertion of foreign sequences of at least 1,809 nucleotides at the P1/HC‐Pro junction. Analysis of insert stability showed that the integrity of GFP and BAR coding sequences was maintained longer than that of the much larger GUS coding sequence. The SCMV isolate from which the expression vector is derived is able to infect several important maize inbred lines, suggesting that this SCMV vector has potential to be a valuable tool for gene functional analysis in a broad range of experimentally important maize genotypes.

A NOVEL DROSOPHILA MODEL OF ALZHEIMER’S DISEASE

Alzheimer's disease (AD) is a progressive neurodegenerative disease and is the main cause of senile dementia. AD is characterized by two pathological hallmarks, senile plaques composed by amyloid β peptides (Aβ) and neurofibrillary tangles composed by hyper-phosphorylated tau proteins. The accumulation of Aβ42 in the brain is thought to be the primary event in AD pathogenesis. To understand pathological roles of Aβ42 in vivo, we previously established a Drosophila model of AD expressing Aβ42 peptides in the secretory pathway of neurons (Iijima, K. et al. 2004, PNAS). This fly model developed several key pathological phenotypes in an age-dependent manner. However, substantial amount of Aβ42 was accumulated intracellularly and caused chronic ER stress, which may not be common pathologies in AD patients. To overcome this issue, we generated novel transgenic flies that efficiently secrete Aβ42 peptides from neurons to extracellular space in the brains. To examine the effects of and metabolism of Aβ42 peptides in the extracellular space of fly brains, we generated transgenic fly lines carrying engineered Aβ42 construct that efficiently secrete Aβ42 from neurons in the brains. We then systematically characterized their phenotypes including Aβ42 accumulation, locomotor functions, neurodegeneration and life span during aging and compared those in our previous Aβ42 fly model. As reported, our previous Aβ42 fly model showed age-dependent Aβ42 accumulation, locomotor defects, neurodegeneration and premature death. In contrast, despite high expression levels of Aβ42 in the brains, the new Aβ42 fly model did not show any significant phenotypes including Aβ42 deposits, suggesting that secreted Aβ42 did not accumulate and aggregate in the extracellular space even in aged fly brains. Our data indicate that overexpression of Aβ42 in the extracellular space of fly brain is not sufficient to produce Aβ42 deposits and neurotoxicity in flies. This result might be similar to recent observation in App knock-in mouse of AD carrying only Swedish mutation (Salas, IH. et al. 2018, J Alzheimers Dis. & Sakakibara, Y. et al. 2019, BMC Neurosci.). Our new Aβ42 fly model can be used as an efficient genetic model system to provide significant insights into pathomechanisms of an early phase of AD.

Clove Oil and AQUI-S Efficacy for Zebrafish Embryo, Larva, and Adult Anesthesia

Since the use of the zebrafish Danio rerio genetic model organism within the scientific research community continues to grow rapidly, continued procedural refinement to support high-quality, reproducible research and improve animal welfare remains an important focus. As such, anesthesia remains one of the most frequent procedures conducted. Here, we compared the effectiveness of clove oil (active ingredient eugenol) and AQUI-S (active ingredient iso-eugenol) with the currently most commonly used tricaine/MS-222 (ethyl 3-aminobenzoate methanesulfonate) and benzocaine anesthesia. We focused on embryos (1 day postfertilization), larvae (5 days postfertilization), and adults (9-11 months) and for the first time used exposure times that are the most relevant in research settings by using zebrafish as a genetic model system. For each age, tricaine and benzocaine achieved the most reproducible, robust anesthesia with the quickest induction and recovery. For some experimental procedures, specific clove oil concentrations in embryos and larvae may represent suitable alternatives. Although different aquatic species at specific ages respond differentially to these agents, the systematic study of comparable effective dosages for procedures most commonly employed represent an important step toward refinement.

Selective elimination of cancer cells by the adenovirus E4orf4 protein in a Drosophila cancer model: a new paradigm for cancer therapy

The adenovirus (Ad) E4orf4 protein contributes to efficient progression of virus infection. When expressed alone E4orf4 induces p53- and caspase-independent cell-death, which is more effective in cancer cells than in normal cells in tissue culture. Cancer selectivity of E4orf4-induced cell-death may result from interference with various regulatory pathways that cancer cells are more dependent on, including DNA damage signaling and proliferation control. E4orf4 signaling is conserved in several organisms, including yeast, Drosophila, and mammalian cells, indicating that E4orf4-induced cell-death can be investigated in these model organisms. The Drosophila genetic model system has contributed significantly to the study of cancer and to identification of novel cancer therapeutics. Here, we used the fly model to investigate the ability of E4orf4 to eliminate cancer tissues in a whole organism with minimal damage to normal tissues. We show that E4orf4 dramatically inhibited tumorigenesis and rescued survival of flies carrying a variety of tumors, including highly aggressive and metastatic tumors in the fly brain and eye discs. Moreover, E4orf4 rescued the morphology of adult eyes containing scrib− cancer clones even when expressed at a much later stage than scrib elimination. The E4orf4 partner protein phosphatase 2A (PP2A) was required for inhibition of tumorigenesis by E4orf4 in the system described here, whereas another E4orf4 partner, Src kinase, provided only minimal contribution to this process. Our results suggest that E4orf4 is an effective anticancer agent and reveal a promising potential for E4orf4-based cancer treatments.

Stable transgenesis in Astyanax mexicanus using the Tol2 transposase system.

Astyanax mexicanus is a well-established fish model system for evolutionary and developmental biology research. These fish exist as surface forms that inhabit rivers and 30 different populations of cavefish. Despite important progress in the deployment of new technologies, deep mechanistic insights into the genetic basis of evolution, development, and behavior have been limited by a lack of transgenic lines commonly used in genetic model systems. Here, we expand the toolkit of transgenesis by characterizing two novel stable transgenic lines that were generated using the highly efficient Tol2 system, commonly used to generate transgenic zebrafish. A stable transgenic line consisting of the zebrafish ubiquitin promoter expresses enhanced green fluorescent protein ubiquitously throughout development in a surface population of Astyanax. To define specific cell-types, a Cntnap2-mCherry construct labels lateral line mechanosensory neurons in zebrafish. Strikingly, both constructs appear to label the predicted cell types, suggesting many genetic tools and defined promoter regions in zebrafish are directly transferrable to cavefish. The lines provide proof-of-principle for the application of Tol2 transgenic technology in A. mexicanus. Expansion on these initial transgenic lines will provide a platform to address broadly important problems in the quest to bridge the genotype-phenotype gap.

A chromosome-scale genome assembly and dense genetic map for Xenopus tropicalis

The Western clawed frog Xenopus tropicalis is a diploid model system for both frog genetics and developmental biology, complementary to the paleotetraploid X. laevis. Here we report a chromosome-scale assembly of the X. tropicalis genome, improving the previously published draft genome assembly through the use of new assembly algorithms, additional sequence data, and the addition of a dense genetic map. The improved genome enables the mapping of specific traits (e.g., the sex locus or Mendelian mutants) and the characterization of chromosome-scale synteny with other tetrapods. We also report an improved annotation of the genome that integrates deep transcriptome sequence from diverse tissues and stages. The exon-intron structures of these genes are highly conserved relative to both X. laevis and human, as are chromosomal linkages (“synteny”) and local gene order. A network analysis of developmental gene expression will aid future studies.

Circadian Clocks in Fish-What Have We Learned so far?

Zebrafish represent the one alternative vertebrate, genetic model system to mice that can be easily manipulated in a laboratory setting. With the teleost Medaka (Oryzias latipes), which now has a significant following, and over 30,000 other fish species worldwide, there is great potential to study the biology of environmental adaptation using teleosts. Zebrafish are primarily used for research on developmental biology, for obvious reasons. However, fish in general have also contributed to our understanding of circadian clock biology in the broadest sense. In this review, we will discuss selected areas where this contribution seems most unique. This will include a discussion of the issue of central versus peripheral clocks, in which zebrafish played an early role; the global nature of light sensitivity; and the critical role played by light in regulating cell biology. In addition, we also discuss the importance of the clock in controlling the timing of fundamental aspects of cell biology, such as the temporal control of the cell cycle. Many of these findings are applicable to the majority of vertebrate species. However, some reflect the unique manner in which “fish” can solve biological problems, in an evolutionary context. Genome duplication events simply mean that many fish species have more gene copies to “throw at a problem”, and evolution seems to have taken advantage of this “gene abundance”. How this relates to their poor cousins, the mammals, remains to be seen.

Developmental Control of the Cell Cycle: Insights from Caenorhabditis elegans.

During animal development, a single fertilized egg forms a complete organism with tens to trillions of cells that encompass a large variety of cell types. Cell cycle regulation is therefore at the center of development and needs to be carried out in close coordination with cell differentiation, migration, and death, as well as tissue formation, morphogenesis, and homeostasis. The timing and frequency of cell divisions are controlled by complex combinations of external and cell-intrinsic signals that vary throughout development. Insight into how such controls determine in vivo cell division patterns has come from studies in various genetic model systems. The nematode Caenorhabditis elegans has only about 1000 somatic cells and approximately twice as many germ cells in the adult hermaphrodite. Despite the relatively small number of cells, C. elegans has diverse tissues, including intestine, nerves, striated and smooth muscle, and skin. C. elegans is unique as a model organism for studies of the cell cycle because the somatic cell lineage is invariant. Somatic cells divide at set times during development to produce daughter cells that adopt reproducible developmental fates. Studies in C. elegans have allowed the identification of conserved cell cycle regulators and provided insights into how cell cycle regulation varies between tissues. In this review, we focus on the regulation of the cell cycle in the context of C. elegans development, with reference to other systems, with the goal of better understanding how cell cycle regulation is linked to animal development in general.

Conserved functions of RNA-binding proteins in muscle.

Animals require different types of muscle for survival, for example for circulation, motility, reproduction and digestion. Much emphasis in the muscle field has been placed on understanding how transcriptional regulation generates diverse types of muscle during development. Recent work indicates that alternative splicing and RNA regulation are as critical to muscle development, and altered function of RNA-binding proteins causes muscle disease. Although hundreds of genes predicted to bind RNA are expressed in muscles, many fewer have been functionally characterized. We present a cross-species view summarizing what is known about RNA-binding protein function in muscle, from worms and flies to zebrafish, mice and humans. In particular, we focus on alternative splicing regulated by the CELF, MBNL and RBFOX families of proteins. We discuss the systemic nature of diseases associated with loss of RNA-binding proteins in muscle, focusing on mis-regulation of CELF and MBNL in myotonic dystrophy. These examples illustrate the conservation of RNA-binding protein function and the marked utility of genetic model systems in understanding mechanisms of RNA regulation.

Ephrin-B3 controls excitatory synapse density through cell-cell competition for EphBs.

Cortical networks are characterized by sparse connectivity, with synapses found at only a subset of axo-dendritic contacts. Yet within these networks, neurons can exhibit high connection probabilities, suggesting that cell-intrinsic factors, not proximity, determine connectivity. Here, we identify ephrin-B3 (eB3) as a factor that determines synapse density by mediating a cell-cell competition that requires ephrin-B-EphB signaling. In a microisland culture system designed to isolate cell-cell competition, we find that eB3 determines winning and losing neurons in a contest for synapses. In a Mosaic Analysis with Double Markers (MADM) genetic mouse model system in vivo the relative levels of eB3 control spine density in layer 5 and 6 neurons. MADM cortical neurons in vitro reveal that eB3 controls synapse density independently of action potential-driven activity. Our findings illustrate a new class of competitive mechanism mediated by trans-synaptic organizing proteins which control the number of synapses neurons receive relative to neighboring neurons.

VICKZ1 enhances tumor progression and metastasis in lung adenocarcinomas in mice.

The VICKZ (Igf2bp) family of RNA binding proteins regulate RNA function at many levels, including intracellular RNA localization, RNA stability, and translational control. One or more of the three VICKZ paralogs are upregulated in many different types of cancers. Here, we show how VICKZ1 enhances, and dominant negative VICKZ1 inhibits, cell migration, growth in soft agar, and wound healing in a mouse lung adenocarcinoma cell line containing a constitutively active, mutant Kras. Similarly, modulation of VICKZ1 activity promotes or inhibits metastases upon implantation of these cells into syngeneic mice. To test these effects in a genetic model system, we generated a mouse with an inducible VICKZ1 transgene and found that isolated overexpression of VICKZ1 in the lungs had no noticeable effect on morphology. Although directed overexpression of mutant Kras in the lungs led to the formation of small adenomas, concurrent overexpression of VICKZ1 remarkably accelerated tumor growth and formation of pulmonary adenocarcinomas. VICKZ1-containing ribonucleoprotein complexes are highly enriched in Kras mRNA in lung adenocarcinoma cells, and Kras signaling is enhanced in these cells by overexpression of VICKZ1. Analysis of lung carcinoma patients reveals that elevated VICKZ1 expression correlates with lower overall survival; this reduction is dramatically enhanced in those patients bearing a mutant Kras gene. Our study reveals that RNA binding proteins of the VICKZ family can synergize with Kras to influence signaling and oncogenic activity.

The Cricket Gryllus bimaculatus: Techniques for Quantitative and Functional Genetic Analyses of Cricket Biology.

All extant species are an outcome of nature's "experiments" during evolution, and hence multiple species need to be studied and compared to gain a thorough understanding of evolutionary processes. The field of evolutionary developmental biology (evo-devo) aspires to expand the number of species studied, because most functional genetic studies in animalshave been limited to a small number of "traditional" model organisms, many of which belong to the same phylum (Chordata). The phylum Arthropoda, and particularly its component class Insecta, possesses many important characteristics that are considered favorable and attractive for evo-devo research, including an astonishing diversity of extant species and a wide disparity in body plans. The development of the most thoroughly investigated insect genetic model system to date, the fruit fly Drosophila melanogaster (a holometabolous insect), appears highly derived with respect to other insects and indeed with respect to most arthropods. In comparison, crickets (a basally branching hemimetabolous insect lineage compared to the Holometabola) are thought to embody many developmental features that make them more representative of insects. Here we focus on crickets as emerging models to study problems in a wide range of biological areas and summarize the currently available molecular, genomic, forward and reverse genetic, imaging and computational tool kit that has been established or adapted for cricket research. With an emphasis on the cricket species Gryllus bimaculatus, we highlight recent efforts made by the scientific community in establishing this species as a laboratory model for cellular biology and developmental genetics. This broad toolkit has the potential to accelerate many traditional areas of cricket research, including studies of adaptation, evolution, neuroethology, physiology, endocrinology, regeneration, and reproductive behavior. It may also help to establish newer areas, for example, the use of crickets as animal infection model systems and human food sources.

Linking genes with ecological strategies in Arabidopsis thaliana.

Arabidopsis thaliana is the most prominent model system in plant molecular biology and genetics. Although its ecology was initially neglected, collections of various genotypes revealed a complex population structure, with high levels of genetic diversity and substantial levels of phenotypic variation. This helped identify the genes and gene pathways mediating phenotypic change. Population genetics studies further demonstrated that this variation generally contributes to local adaptation. Here, we review evidence showing that traits affecting plant life history, growth rate, and stress reactions are not only locally adapted, they also often co-vary. Co-variation between these traits indicates that they evolve as trait syndromes, and reveals the ecological diversification that took place within A. thaliana. We argue that examining traits and the gene that control them within the context of global summary schemes that describe major ecological strategies will contribute to resolve important questions in both molecular biology and ecology.

Sweet fish: Fish models for the study of hyperglycemia and diabetes.

Fish are good for your health in more ways than you may expect. For one, eating fish is a common dietary recommendation for a healthy diet. However, fish have much more to provide than omega-3 fatty acids to your circulatory system. Some fish species now serve as important and innovative model systems for diabetes research, providing novel and unique advantages compared with classical research models. Not surprisingly, the largest share of diabetes research in fish occurs in the laboratory workhorse among fish, the zebrafish (Danio rerio). Established as a genetic model system to study development, these small cyprinid fish have eventually conquered almost every scientific discipline and, over the past decade, have emerged as an important model system for metabolic diseases, including diabetes mellitus. In this review we highlight the practicability of using zebrafish to study diabetes and hyperglycemia, and summarize some of the recent research and breakthroughs made using this model. Equally exciting is the appearance of another emerging discipline, one that is taking advantage of evolution by studying cases of naturally occurring insulin resistance in fish species. We briefly discuss two such models in this review, namely the rainbow trout (Oncorhynchus mykiss) and the cavefish (Astyanax mexicanus).

A phylogenetic examination of host use evolution in the quinaria and testacea groups of Drosophila

Adaptive radiations provide an opportunity to examine complex evolutionary processes such as ecological specialization and speciation. While a well-resolved phylogenetic hypothesis is critical to completing such studies, the rapid rates of evolution in these groups can impede phylogenetic studies. Here we study the quinaria and testacea species groups of the immigrans-tripunctata radiation of Drosophila, which represent a recent adaptive radiation and are a developing model system for ecological genetics. We were especially interested in understanding host use evolution in these species. In order to infer a phylogenetic hypothesis for this group we sampled loci from both the nuclear genome and the mitochondrial DNA to develop a dataset of 43 protein-coding loci for these two groups along with their close relatives in the immigrans-tripunctata radiation. We used this dataset to examine their evolutionary relationships along with the evolution of feeding behavior. Our analysis recovers strong support for the monophyly of the testacea but not the quinaria group. Results from our ancestral state reconstruction analysis suggests that the ancestor of the testacea and quinaria groups exhibited mushroom-feeding. Within the quinaria group, we infer that transition to vegetative feeding occurred twice, and that this transition did not coincide with a genome-wide change in the rate of protein evolution.

Genetic Dissection of Hybrid Male Sterility Across Stages of Spermatogenesis.

Hybrid sterility is a common form of reproductive isolation between nascent species. Although hybrid sterility is routinely documented and genetically dissected in speciation studies, its developmental basis is rarely examined, especially in generations beyond the F1 generation. To identify phenotypic and genetic determinants of hybrid male sterility from a developmental perspective, we characterized testis histology in 312 F2 hybrids generated by intercrossing inbred strains of Mus musculus domesticus and M. m. musculus, two subspecies of house mice. Hybrids display a range of histologic abnormalities that indicate defective spermatogenesis. Among these abnormalities, we quantified decreased testis size, reductions in spermatocyte and spermatid number, increased apoptosis of meiosis I spermatocytes, and more multinucleated syncytia. Collectively, our phenotypic data point to defects in meiosis I as a primary barrier to reproduction. We identified seven quantitative trait loci (QTL) controlling five histologic traits. A region of chromosome 17 that contains Prdm9, a gene known to confer F1 hybrid male sterility, affects multinucleated syncytia and round spermatids, potentially extending the phenotypic outcomes of this incompatibility. The X chromosome also plays a key role, with loci affecting multinucleated syncytia, apoptosis of round spermatids, and round spermatid numbers. We detected an epistatic interaction between QTL on chromosomes 17 and X for multinucleated syncytia. Our results refine the developmental basis of a key reproductive barrier in a classic model system for speciation genetics.

The genomic basis of environmental adaptation in house mice.

House mice (Mus musculus) arrived in the Americas only recently in association with European colonization (~400–600 generations), but have spread rapidly and show evidence of local adaptation. Here, we take advantage of this genetic model system to investigate the genomic basis of environmental adaptation in house mice. First, we documented clinal patterns of phenotypic variation in 50 wild-caught mice from a latitudinal transect in Eastern North America. Next, we found that progeny of mice from different latitudes, raised in a common laboratory environment, displayed differences in a number of complex traits related to fitness. Consistent with Bergmann’s rule, mice from higher latitudes were larger and fatter than mice from lower latitudes. They also built bigger nests and differed in aspects of blood chemistry related to metabolism. Then, combining exomic, genomic, and transcriptomic data, we identified specific candidate genes underlying adaptive variation. In particular, we defined a short list of genes with cis-eQTL that were identified as candidates in exomic and genomic analyses, all of which have known ties to phenotypes that vary among the studied populations. Thus, wild mice and the newly developed strains represent a valuable resource for future study of the links between genetic variation, phenotypic variation, and climate.