Abstract
The fruit fly, Drosophila melanogaster (Meigen, 1830) has been established as a cornerstone for research into a wide array of subjects including diseases, development, physiology, and genetics. Thanks to an abundance of genetic tools, publicly available fly stocks, and databases, as well as their considerable biological similarity to mammalian systems, Drosophila has been solidified as a key model organism for elucidating many aspects of human disease. Herein is presented an overview of what makes Drosophila such an appealing model organism. In Part I of this chapter, basic Drosophila biology is reviewed and the most relevant genetic tools available to Drosophila researchers are covered. Then in part II, we outline the use of Drosophila as a model organism to study a wide array of pathologies in which Drosophila has been used, along with key advances made in the specific field using the fly as a model organism.
Keywords
- animal model
- cancer
- diseases
- Drosophila
- genetic techniques
- heart
- immunology
- kidney
- metabolic disorders
- neurodegeneration
1. Introduction
Searching PubMed with the key words “
It is little more than 100 years since Thomas Hunt Morgan and his colleagues, including his pupil Calvin Bridge and his wife Lillian Vaughan Morgan, redefined important concepts of
In 1946 to Hermann Joseph Muller for the use of X-ray irradiation to produce in vivo mutations.
In 1995 to Edward B. Lewis, Christine Nuesslein-Volhard and Eric F. Wieschaus for their contribution to the discovery of the genetic control of early embryonic development.
In 2011 to Bruce A. Beutler and Jules A. Hoffmann, for their success in defining innate immunity.
In 2017 to Jeffrey C. Hall, Micheal Rosbash and Michael W. Young for their contributions to the molecular mechanisms that control the circadian rhythm.
Fly work has also benefitted from the strong commitment of
Finally,
In this two-part chapter, some of the many aspects that make
2. Basic biology/life cycle
As a holometabolous insect,
2.1. Life cycle and regulation of development
The
2.2. Regulation of body size the interplay between hormones and growth factors
Larval growth is regulated by the interplay of the function of different organs (
Figure 4
), among which the fat body works as a hub to regulate several important processes. First, by sensing the amino acid concentration in the hemolymph, the fat body remotely controls the release of Dilps, in particular dilp2, from the Insulin Producing Cells (IPCs) in the brain [17]. This mechanism depends on the release into the hemolymph of secreted factors, like the Growth-Blocking Peptide-1 (GBP1) and CG11395 (GBP2) [18] and Stunted [19] with a mechanism that is dependent on the activation of the Target of Rapamycin (TOR) pathway in the fat cells. Second, the fat body controls animal survival with the activation of autophagy, consuming the fats and sugars that accumulated during the feeding phase. Third, the fat body responds to reduced ecdysone signaling from the brain by restraining metabolism and protein synthesis cell-autonomously before each molt by controlling the expression of the growth regulator Myc [20], which was shown to also regulate growth and Dilp2 secretion [21] constituting a regulatory loop that controls animal growth. Insulin signaling is the foremost important growth signal that in flies controls both growth/development and metabolism, with a unique and conserved pathway [22]. Dilps are produced by different organs and activate the Insulin Receptor (InR). Among Dilps (1-8), Dilp2, 3, 5 are produced by the IPCs in the brain and control animal growth and development [22, 23] while Dilp6, produced by the fat body and regulated by FOXO, functions to indirectly restrain Dilp2 secretion from the IPCs and to regulate longevity in the adult flies, a function similar to mammalian InR in aging [24]. A novel and exciting function was recently identified for dilp8, the last member of the Dilp family, to indirectly control ecdysone levels [25, 26].
3. Fly genetics
3.1. Generation of transgenes
3.1.1. P-element transposons
Several commonly used techniques exist to integrate DNA into the genome. Transposon mediated integration, first utilized by Rubin and Spradling in 1982 [29] is one of the most commonly employed methods [28]. This technique capitalizes on the action of the P-element transposon. Transposons are pieces of DNA with specific sequence characteristics that have the ability to cut themselves out of the genome and reintegrate in another location through the action of the transposase enzyme [30]. The transposons can be modified though cloning to contain a desired piece of DNA Plasmids containing the modified P-element constructs are injected into the
3.1.2. Homologous recombination
P-element transposon mediated transgenesis has several drawbacks, including that the location of the insertion cannot be selected and sometimes the transgene may be inserted within the regulatory or coding region of another gene and disrupt its function [32]. Rong and Golic in 2000 pioneered a procedure that can target specific genes in the
Several other methods that generate double stranded breaks to trigger homologous recombination have been developed [28]. These include using zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALEN), which are enzymes that can target specific DNA sequences and cause double stranded breaks, however each gene requires generating a new specific enzyme and can be challenging [36].
3.1.3. phiC31 integrase: site-specific integrase insertion
Another method to target specific locations in the genome uses the bacteriophage ɸC31 integrase which can insert a transgene at a specific recognition site in the genome [37]. Bacteriophages are viruses that target bacteria. The ɸC31 integrase is an enzyme that recognizes specific attachment sites in both the bacteriophage genome (designated
3.1.4. Bacterial artificial chromosomes
Bacterial artificial chromosomes (BACs) and recombination engineering (recombineering) are gaining traction in the
3.1.5. CRISPR/Cas 9
The CRISPR/
3.2. Generation of mutants
In addition to ectopically expressing or reducing the function of a gene using the classic binary system (see last section in methods), another useful way to study gene function is to generate mutations in the genome and observe the resulting phenotypes, and then work backwards to figure out what gene was modified. The function of this gene can be inferred by the phenotype that occurred when the gene was destroyed. These studies involve mutating a large number of genes in many flies, then screening though the phenotypes and determining what genes were altered. There are a number of ways to generate mutants, including using P-element transposons and chemical mutagens like Ethyl Methanesulfonate (EMS).
3.2.1. P-element mutagenesis
This technique utilizes P-elements (usually containing gene markers as described above), or other transposable elements, to move around in the genome to disrupt gene function. This is possible either by inserting themselves in a new position that could interfere with a gene or removing it from a gene and degrading a little bit more of the DNA sequence from where it was removed [51, 52]. Though P-elements show certain preferences for where they reinsert, they cannot be directed to a specific location and have no precise recognition sequence [30]. It is therefore necessary after P-element mobilization to screen the flies that show an altered phenotype to determine which gene or genes were disrupted and use PCR to identify where the insertion occurred [51].
3.2.2. EMS mutagenesis
This method uses the chemical EMS to generate random mutations in the genome [53]. EMS produces a form of guanine, O6-methylguanine, that incorrectly base pairs with thymine during DNA replication, usually generating GC to AT transitions that potentially alter codons or destroy splice sites, which will damage the function of a gene product. These mutations are generated at random and while some create visible phenotypes, or even lethality, others show no obvious changes, so extensive screening is needed to determine which mutation or mutations caused the observed phenotypes [53].
3.3. Genetic screens
The large use of
A genetic screen can follow two main strategies: Forward or Reverse Genetics [54]. A Forward Genetics approach is based on random, genome-wide mutagenesis to generate a large progeny with aberrant phenotypes and allows the identification of individual genes involved in any given process. Traditional forward genetic screens in
3.4. Most common techniques in Drosophila
A huge step forward on the feasibility of genetic screens was improved by the generation of the UAS/GAL4 system [57] that that allowed the expression of transgenes within specific tissues of interest.
3.4.1. The UAS/GAL4 system and its modifications
This system requires the use of lines that are generated and maintained in separate stocks and targeted gene expression will be visible only in the progeny of the cross. Using the yeast transcription factor GAL4 cloned into a P-element vector, a tissue specific promoter is cloned upstream of the GAL4 gene. In parallel, a line is generated that includes a P-element vector containing the upstream activating sequences (UAS) to which GAL4 protein can bind [58]. This binary expression system is used to drive the expression of a gene of interest in any tissues where the promoter GAL4 is expressed (
Figure 6
). Because experimental design may demand expression in a more limited time window (i.e. in adult only or if the expression of the gene of interest is detrimental), the UAS/GAL4 system is often accompanied by the use of the yeast
Nowadays, the number of GAL4 lines available is constantly growing. There are UAS lines both for overexpression or RNAi interference targeting almost for all the genes in the fly. UAS- lines with more applied specific modifications, like the enhancer-trap GFP vectors, include those from the Janelia Farm Fly light project that created more than 7000 driver lines with an intergene overlapping sequence of 3 kb fragment near the gene of interest. These lines have been characterized for their expression pattern in embryos, larvae brain and adult CNS [64] and in the larval imaginal discs [65], available at the BDRC stock center. The use of binary systems is continually evolving to provide even more inter-exchangeable systems. Indeed the recent design of the LexA/lexAop [66] and the Q system [67], both inducible systems that can be used in combination with GAL4/UAS gene expression, allows researchers to perform screens in a tissue using the UAS/GAL4, with the specific patterns of expression determined by the LexA/LexAop or Q system. They can be used simultaneously in the same animal because neither of these systems cross-react to each-other.
3.4.2. The FLP/FRT system and Mosaic Analysis with a Repressible Cell Marker
To characterize the role of a gene in a small group of cells and not in the whole compartment, or to analyze the role of a mutation, it is possible to create mosaics that have homozygous mutant cells (clones) in an otherwise heterozygous animal via mitotic recombination. These studies were made possible with the combined used of the UAS/GAL4 system with the
4. Conclusions
As illustrated in Part I of this chapter,
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