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Home , Drosophila melanogaster

as a Model 1 Masamitsu Yamaguchi and Hideki Yoshida

Abstract lished by studying Drosophila, which advanced Drosophila has been widely our understanding of , , and used in classical and modern for more the inheritance of genetic information (Ashburner than 100 years. The history of the Drosophila and Bergman 2017). techniques model in the study of various aspects of life using radiation and chemicals were also devel- sciences will be summarized in this chapter. oped with , yeast, and Furthermore, commonly used techniques and Drosophila, allowing scientists to clarify tools with Drosophila models will be briefly functions by studying the induced by described, with a special emphasis on the . advantages of Drosophila models in the study After 1970, various molecular, developmental, of various diseases. and biological techniques began to be applied to Drosophila, such as gene cloning, hybridization, Keywords P-element-based transformation, and clonal anal- Drosophila · History · · yses. These techniques allowed scientists to per- · · GAL4-UAS form analytical rather than descriptive studies on the development and behavior of Drosophila. In 1994, the Nobel Prize in or Medicine was awarded to Dr. Lewis for his studies with 1.1 History of Studies Drosophila to elucidate gene structures, as well as with Drosophila Drs. Weischaus and Nusslein-Volhard for their pioneering work on embryogenesis and the Drosophila melanogaster is one of the most identification of a large number of genes involved commonly used experimental and in all aspects of Drosophila development, was first studied experimentally by Dr. Castle including segmentation. Most of the mammalian (Castle 1906) and used by Dr. Morgan for genetic homologues of these genes were then found to experiments from 1909 (Sturtevant 1959). be essential for mammalian development. In During the following 30 years, a number of the addition, many tumor suppressor genes were ini- main principles of classical genetics were estab- tially identified inDrosophila , and their human homologues were subsequently detected and M. Yamaguchi (*) · H. Yoshida proven to play important roles in oncogenesis. Department of Applied Biology, Kyoto Institute of The of Drosophila melano- Technology, Kyoto, Japan gaster (D. melanogaster) was completed in 2000. e-mail: [email protected]; [email protected]

© Springer Nature Singapore Pte Ltd. 2018 1 M. Yamaguchi (ed.), Drosophila Models for Human Diseases, Advances in Experimental Medicine and Biology 1076, https://doi.org/10.1007/978-981-13-0529-0_1 2 M. Yamaguchi and H. Yoshida

A few years later, the project was 1.2 Biology of Drosophila also finished, and comparisons of both genome sequences revealed high homologies between the Drosophila has a relatively rapid life . A Drosophila and human , thereby con- single fertile mating pair may produce hundreds firming the importance of Drosophila as a model of genetically identical offspring in approxi- to study human diseases (Adams et al. 2000; mately 10 days at 25 °C. This is markedly faster Myers et al. 2000). Nearly 75% of human disease-­ than the commonly used rodent models. related genes have been estimated to have func- Drosophila may also be regarded as a model tional orthologues in Drosophila (Pandy and organism defined by its developmental stage: the Nichols 2011; Yamamoto et al. 2014). Overall , , , and adult (Pandy and identity at the nucleotide or sequence Nichols 2011). between Drosophila and mammals is approxi- After fertilization, undergo highly mately 40% between homologues. Regarding the synchronized nuclear division cycles. These conserved functional domains of proteins, iden- cycles, which are composed of only G and S tity may be more than 80%. The completion of phases, proceed very rapidly, requiring approxi- the genome also promoted the study of tran- mately 10 min for one cycle to form a multinu- scription, protein binding to specific DNA clear syncytial blastoderm. After nine nuclear sequences, and genetic variations at the molecu- division cycles, most nuclei move to the surface lar level. Based on genome information, we may of the embryo and undergo four successive perform RNA-sequence analyses or microarrays nuclear divisions at the surface of the embryo. for expression profiling, targeted to all known or These nuclei then simultaneously cellularize to predicted coding regions or against the entire form a cellular blastoderm. After , Drosophila genome including noncoding regions. they undergo segmentation processes. Early We may also perform the genome-wide mapping embryos store large amounts of DNA replication of binding sites for chromatin-associated proteins enzymes that are enzymatically and cytologically at a high resolution using DNA adenine methyl- characterized by the embryo (Yamaguchi et al. transferase identification (DamID) (Sun et al. 1991). The embryo may be used in studies on 2003; Bianchi-Frias et al. 2004), chromatin fundamental by examin- immunoprecipitation assays (ChIP assays), or a ing , cell fate determination, ChIP-sequence analysis (MacAlpine et al. 2004; organogenesis, central/peripheral neuronal devel- Birch-Machin et al. 2005). Genome-wide sur- opment, and axon pathfinding. veys for polymorphisms using high-throughput The larva, particularly the wandering third PCR strategies are now also available (Glinka instar larva, is commonly used to study develop- et al. 2003). Thus, Drosophila is always at the mental and physiological processes as well as forefront of modern biology, in which genes, less complex behaviors such as foraging. A group gene engineering, and other new findings are of cells called imaginal discs produce the future often achieved first inDrosophila and then gener- adult external structures of Drosophila and are alized to other organisms including . It is contained within the larva. They are primarily important to note that the Nobel Prize in composed of an undifferentiated epithelium. In Physiology or Medicine in 2017 was awarded to the late third instar larval stage and subsequent Drs. Hall, Rosbash, and Young for their discovery pupal stage, imaginal discs undergo morphologi- of the molecular mechanisms controlling the cir- cal changes that produce adult external structures cadian rhythm in Drosophila. such as the antenna, compound , wings, and 1 Drosophila as a 3 legs. cells undergo a typical G1, S, tract. The adult fly brain contains more than G2, and M phase cycle. In terms of 100,000 that form discrete circuits and studies, eye imaginal discs in the third instar lar- neuropils, which mediate complex behaviors vae are particularly useful. Cells in the anterior including wake and sleep circadian rhythms, region to the morphogenetic furrow undergo ran- and memory, feeding, , court- dom cell division, and those at the morphogenetic ship, and grooming. More significantly, the furrow are arrested at the G1 phase and then syn- responses of Drosophila to various drugs that act chronously undergo the S, G2, and M phases to on the central nervous system are similar to the double the cell number. Then all cells fall into the effects observed in mammals (Rothenfluh and G1/G0 and undergo differentiation. Thus, the eye Heberlein 2002; Satta et al. 2003; Wolf and imaginal disc provides a naturally occurring syn- Heberlein 2003; Nichols 2006; Andretic et al. chronized cell system that is very useful for char- 2008). Therefore, Drosophila provides a useful acterizing the genes involved in the regulation of model for screening therapeutic drugs for various the cell cycle and DNA replication (Yamaguchi human neuropathies. et al. 1999). The differentiation processes of eight (R1–R8) photoreceptor cells have also been stud- ied in detail. Clarification of the mechanisms 1.3 Chromosomes of Drosophila responsible for the developmental processes of imaginal discs has provided significant insights 1.3.1 Overview into Drosophila and human biologies. Furthermore, learning and memory assays are D. melanogaster has four sets of chromosomes, possible with larvae. the X and Y chromosomes, two autosomal In the pupal stage, Drosophila undergoes chromosomes 2 and 3, and the very small chro- , and during metamorphosis, mosome 4 (Metz 1914; Deng et al. 2007). Female imaginal discs undergo cell proliferation, differ- carry two X chromosomes and males carry a entiation, and organogenesis to produce various single X and . Females and males adult external structures, while most larval tis- carry two sets of the autosomal second, third, and sues undergo autophagy and cell death (Aguila fourth chromosomes. The is et al. 2007). Cells undergo these processes in acrocentric and may be divided into two arms by response to the hormone 20-hydroxyecdysone the centromere, a large left arm and a markedly (), which initiates larval-prepupal and smaller right arm. The Y chromosome is also prepupal-­pupal transitions (Baehrecke 1996). acrocentric with a slightly longer long arm and Consequently, Drosophila undergoes morpho- shorter arm. In contrast, chromosomes 2 and 3 logical changes with the tight regulation of vari- are metacentric with the centromere located in ous biological pathways. During metamorphosis, nearly the center of two left and right arms, the metabolic rate of Drosophila follows a named 2L, 2R, 3L, and 3R, respectively. The U-shaped curve in which energy consumption is fourth chromosome is also acrocentric, carrying a high during the first stages, declines toward the small left arm and larger right arm. mid-pupal stage, and increases again toward the Drosophila chromosomes may be function- last phases of the larval-adult transformation ally and structurally divided into heterochromatic (Merkey et al. 2011). Further details on the meta- and euchromatic regions. is bolic changes that occur during the development designated as the darkly staining regions in of Drosophila are described in Chap. 14. karyotyping. The heterochromatic region is also The Drosophila adult provides a complex known to be late replicating in the S phase of the model organism that is somewhat similar to cell cycle and is enriched with highly repetitive mammals in many aspects. The adult fly has nucleotide sequences and transposable elements organs that are functionally similar to the mam- (Dimitri 1997). The X, second, and third chromo- malian heart, lung, kidney, gut, and reproductive somal regions adjacent to the centromeres are 4 M. Yamaguchi and H. Yoshida darkly staining and are designated as pericentric reached by the euchromatic regions of the heterochromatin. The Y and fourth chromosomes genome because the heterochromatic regions are are also darkly staining and entirely heterochro- under-replicated. Furthermore, homologous matic, although the fourth chromosome has a chromosomes undergo pairing in the small euchromatic right arm. The gene densities . Thus, the combination of of the heterochromatic regions of the genome are polyploidy and pairing may produce 1024 DNA lower than those of euchromatic regions. The Y strands for each euchromatic chromosome arm. chromosome is not necessary for the viability of All chromosome arms corresponding to X, 2L, Drosophila; however, XO males lacking the Y 2R, 3L, 3R, and the small 4 expand from a chromosome are sterile. Furthermore, XXY flies central region called the heterochromatic are female, indicating that the Y chromosome chromocenter (Fig. 1.1). The heterochromatic plays no role in sex determination in Drosophila. chromocenter is composed of pericentric hetero- Sex is determined by the balance between the X chromatin and, in the case of males, the Y chro- chromosome and in Drosophila: mosome. Polytene chromosomes are sufficiently X:A = 1 is female and X:A = 0.5 is male. large to be easily observed using a standard light microscope. Each of the euchromatic arms shows a unique banding pattern caused by the differen- 1.3.2 Polytene Chromosomes tial condensation of chromatin to form darkly stained bands and less stained interbands In Drosophila, after differentiation, most cells (Fig. 1.1). undergo endoreplication in which the S and G In Drosophila, the band pattern of salivary phases are repeated without any M phase. The gland polytene chromosomes is highly most typical endoreplicating tissue in Drosophila ­reproducible among individuals (Bridges 1935). is the larval salivary glands. In the case of the Each large arm is cytologically divided into 20 third instar larval salivary gland, the level roughly equal numbered segments (X = 1–20; reaches 1024 (Rodman 1967; Hammond and 2L = 21–40; 2R = 41–60; 3L = 61–80; Laird 1985). The levels of polyploidy are mainly 3R = 81–100; 4 = 101–102). Each of these num-

Fig. 1.1 In situ hybridization of the gene on salivary gland polytene chromosomes. The arrowhead indicates the white gene locus (3B6) 1 Drosophila as a Model Organism 5 bered segments is further divided into six roughly ancer in crossing schemes. Many balancer chro- equal lettered segments, A to F, and the bands in mosomes also carry a set of recessive visible each lettered segment are numbered. Therefore, mutations that are useful for designing screens each band has a unique address, and its position and distinguishing complex genotypes. is easily discernible from the address. Moreover, Transgenic flies carrying a set of new and useful the positions of genes may now be mapped on visible markers to the balancer have been devel- these addresses. oped as follows. expressing visible Various research tools have been developed to markers, such as LacZ, GFP, or other fluoro- mark functional regions on polytene chromo- phores, in various spatial and temporal patterns somes. Anti-phosphorylated RNA polymerase II is have been inserted into different balancers as new used to mark the transcriptionally active domain of and useful dominant markers. These transgenic polytene chromosomes, and anti-heterochromatin­ flies may be used to easily distinguish the marked protein 1 (HP-1) marks heterochromatic and het- balancer flies from non-balancer flies at various erochromatin-like regions on chromosomes (Kato developmental stages (Kaufman 2017). A list of et al. 2007, 2008). By immunostaining chromatin- balancers may be found at the BDSC site (http:// binding proteins with specific antibodies in combi- flystocks.bio.indiana.edu/Browse/balancers/bal- nation with various markers, it is possible to ancer_main.htm). identify the protein of interest binding to the rele- vant functional region of chromosomes, such as euchromatic or heterochromatic and - 1.3.4 Drosophila Genome ally active or inactive regions. The genome size of D. melanogaster is approxi- mately 180 Mb, with 2/3 (120 Mb) representing 1.3.3 Balancer Chromosomes the euchromatic region and 1/3 (60 Mb) the het- erochromatic region. After the first report of the Balancer chromosomes are an extremely valu- D. melanogaster genome by the consortium of able tool in studies on Drosophila. They contain the Berkley Drosophila Genome Project and extensive inversions through the entire chromo- Celera (Adams et al. 2000; Myers some that prevent the recovery of chromosome et al. 2000), the annotation of the genome has exchange events, thereby isolating and maintain- been revised several times by incorporating data ing the sequences in the balancer and balanced from genome-wide RNA sequencing analyses chromosome. They do not prevent crossing over and those on heterochromatin (modENCODE but inhibit the recovery of exchanged chromatids et al. 2010; Graveley et al. 2011; Boley et al. (Kaufman 2017). Balancer chromosomes are 2014; Brown et al. 2014; Chen et al. 2014; used to stably maintain lethal and sterile muta- Kaufman 2017). Based on the current release, the tions in the Drosophila stock without the selec- total sequence length is 143,726,002 bp with a tion process. Balancer chromosomes are also total gap length mainly in heterochromatin, useful for effectively screening for mutations by including major and minor scaffolds of maintaining the linear integrity of a mutagenized 1,152,978 bp (Kaufman 2017). The sequence is homologue (Kaufman 2017). These processes assembled into 1870 scaffolds with the majority are very difficult to perform in other model of the sequence, 137.6 Mbp, residing on the organisms, such as the mouse without balancers. seven chromosome arms (X, Y, 2L, 2R, 3L, 3R, Balancer chromosomes carry a recessive and 4) and the entire mitochondrial genome. The lethal that is not related to the lesion sequence includes contiguous portions of the being balanced and, thus, may efficiently balance pericentric heterochromatin of X, 2, 3, and 4. lethal and sterile mutations. Balancer chromo- Some may be mapped to the highly repetitive somes also carry dominant visible mutations, and rRNA-encoding genes in the nucleolus organizer scientists may easily follow flies carrying the bal- of X and Y (He et al. 2012). 6 M. Yamaguchi and H. Yoshida

Annotation of the genome currently identifies called GAL4-UAS targeted expression system 17,726 genes, 13,907 of which are protein coding (Brand and Perrimon 1993). GAL4 is a yeast that 21,953 unique polypeptides. The transcription factor that is used to control the spa- remaining 3821 identified loci are genes encod- tial and temporal expression of target genes, ing various types of noncoding RNA, 147 for which consequently directs gene activity at a spe- rRNA, 313 for tRNA, 31 for snRNA, 288 for cific developmental stage and specific cells and snoRNA, 256 for miRNA, 2470 for lncRNA, and tissues. In one parental , promoter regions 315 for (Kaufman 2017). The for a particular gene are designed to drive the importance of many of these genes is now being expression of GAL4 in some tissues. In another recognized, particularly in relation to human dis- strain, the GAL4-binding upstream-activating eases. Further details on noncoding RNA related sequence (UAS) is placed in front of the trans- to human diseases will be described in Chap. 8. gene. When these two strains are genetically crossed, their progenies express the in specific tissues driven by the GAL4-UAS system. 1.4 Strategies and Techniques In combination with RNA interference (RNAi), it to Study Human Diseases is also possible to knockdown specific genes by Using Drosophila expressing double-stranded RNAs targeted to specific mRNAs using the GAL4-UAS system. A There are two main strategies to study human useful resource for this purpose is the collection diseases using the Drosophila model: forward of UAS-RNAi responder strains of the Vienna and reverse genetics. Drosophila Stock Center (VDRC) (http://stock- center.vdrc.at/control/main). These RNAi knock- down strains cover nearly 90% of all Drosophila 1.4.1 Studies with Forward Genetics protein-coding genes and are available to the research community from VDRC (Dietzl et al. In forward genetics, mutations are induced at 2007). The basic GAL4-UAS targeted expression random, and flies are screened for a of system has been modified to further refine cell interest. Mutations may be generated by ethyl and tissue specificities as well as temporal methanesulfonate (EMS) or the insertion of expression specificities (Roman et al. 2001: transposons, such as P-element and piggyback McGuire et al. 2004). (Venken and Bellen 2014). Mutants may also be isolated by screen RNAi libraries or chromosome deficiency kits that cover 95% of the euchromatic 1.4.3 Studies with Reverse Genetics region of the Drosophila genome (Ida et al. 2009; Cook et al. 2012). These strategies are useful for In reverse genetics, mutations are generated in identifying uncharacterized mutations in already Drosophila homologues of human genes to char- known disease-related genes as well as genes that acterize their phenotypes in vivo. There are sev- have not yet been linked to disease. Therefore, eral approaches to knockdown or knockout genes this represents a useful strategy for identifying in Drosophila. One strategy is targeted gene dis- previously unknown genes and clarifying various ruption using clustered regularly interspaced biological events. short palindromic repeats/Cas9 (CRISPR/Cas9) (Beumer and Carroll 2014), transposon-mediated mutagenesis, and the excision of pre-existing 1.4.2 GAL4-UAS Targeted transposable elements. The other is gene silenc- Expression System ing with RNAi combined with the GAL4-UAS system or CRISPR (Mohr 2014). In addition to A commonly used approach to express or knock- these loss-of-function studies, a wild-type or down specific genes in Drosophila is the so-­ mutant form of a human disease-causing gene 1 Drosophila as a Model Organism 7 may be introduced and overexpressed in is small, easy to handle, and inexpensive to main- Drosophila in order to examine its effects in spe- tain and manipulate in the laboratory. Drosophila cific tissues and organs (Feany and Bender 2000). has a short life span and produces a large number Drosophila is also useful for studying the of offspring, which facilitates statistical analyses pathogenesis of rare variants that are linked to of the data obtained. Drosophila development is human diseases (Ugur et al. 2016). There are external, and, thus, it is very easy to follow using US-based initiatives to identify human disease-­ various microscopes. Many mutants and trans- causing genes by deep sequencing of the whole genic fly lines may be obtained from stock cen- exomes or genomes of patients and their families, ters such as the Bloomington Drosophila Stock which are coordinated by the Centers for Center (https://bdsc.indiana.edu/), Kyoto Stock Mendelian Genomics (http://www.mendelian. Center (http://www.dgrc.kit.ac.jp/), and org/) and Undiagnosed Diseases Network (UDN; VDRC. A plethora of information from previous http://undisgnosed.hms.harvard.edu/). Similar experiments and discoveries is available. strategies have been performed in other coun- Sequencing of the genome is nearly complete, as tries, such as the UK (http://www.uk10k.org/) described above. Homologues for nearly 75% of and China (Guangzhou Drosophila Resource human disease-related genes have been identified Center and the Center for Genomic Sciences in (Pandy and Nichols 2011; Yamamoto et al. 2014). the University of Hong Kong). These strategies Drosophila shows complex behaviors including are sometimes not sufficient for identifying the social activity. In addition, there are fewer ethical disease-causing gene if only a few individuals are concerns because the is outside assessed. In these cases, the Drosophila ortho- laws in many countries. In combination with logue may be knocked out or down in order to genome-wide genetic screening, genome-wide examine phenotypes. If the observed phenotype analyses with deep sequencers, such as RNA-seq is rescued by the expression of wild-type UAS-­ and ChIP-seq, and metabolomics analyses, human-­cDNA, but not by its human variant, dis- Drosophila is now commonly used as a model to ease causality may be confirmed (Bellen and study human diseases with the aim of identifying Yamamoto 2015; Wangler et al. 2015). novel biomarkers or therapeutic targets for human diseases together with the screening of candidate substances for their treatment (Pandy 1.5 Advantages of Drosophila and Nichols 2011). Drosophila is now used in the in the Study of Human study of various human diseases related to the Diseases central and peripheral nervous systems such as neurodegeneration, Alzheimer’s disease (Chap. Several model organisms are intensively studied 3), Parkinson’s disease (Chap. 4), triplet repeat in life sciences, such as the mouse, , expansion disease (Chap. 5), sleep disorders , Arabidopsis, Drosophila, (Pandy and Nichols 2011), seizure disorders , yeast, and E. coli. These (Pandy and Nichols 2011), cognitive and psycho- are specific species that are extensively examined sis disorders (Pandy and Nichols 2011), amyo- in research laboratories. Studies using these trophic lateral sclerosis (Chap. 6), and model organisms will advance our understanding Charcot-Marie-Tooth disease (Chap. 7). It is also of cellular functions, development, and human used as a model including tumor forma- diseases. The knowledge obtained from these tion and metastasis (Chaps. 10 and 11). model organisms may also be applied to other Drosophila may also be employed in the study of organisms, which will result in the generalization cardiovascular diseases (Pandy and Nichols of findings. 2011; Ugur et al. 2016). Although the fly heart The following characteristics of Drosophila has only one cardiac chamber, it may still be used demonstrate that it is a good model organism. It to study some steps of heart development and its 8 M. Yamaguchi and H. Yoshida defects. Drosophila also provides a model for Drosophila may be summarized as follows. A inflammation/infectious diseases, metabolic dis- number of genes related to human diseases have orders, and (Chaps. 13 and 14). already been discovered and various useful tech- There are some limitations to Drosophila niques developed. Powerful tools for studying models. Drosophila does not possess hemoglobin developmental/neurological disorders and cancer (Adams et al. 2000; Myers et al. 2000), and, thus, are now available. Therefore, Drosophila is a Drosophila models cannot be generated for very effective model with more simplicity than human diseases related to hemoglobin. Smaller mammalian models and greater complexity than organisms, such as yeast and E. coli, which have yeast and bacterial models. shorter generation times, smaller genomes, and produce more offspring than Drosophila, are pre- ferred for the study of cell autonomous functions, 1.6 Commonly Used Websites such as DNA replication and repair. Therefore, for Drosophila Studies an ideal study of human diseases will be a paral- lel analysis with relevant models. For example, Commonly used online databases for Drosophila cell autonomous effects will be studied in yeast, studies are now available to support experimental while multicellular or inductive events mediated design, the identification of relevant fly stocks, by genes will be examined using Drosophila. A research tools, reagents such as antibodies, and more accurate disease model needs to be estab- related human diseases (Table 1.1). These data- lished in the mouse. In any case, the benefits of bases are particularly useful for beginners. More

Table 1.1 A list of websites providing information about Drosophila or human diseases Website URL FlyBase http://flybase.org modENCODE http://modencode.sciencemag.org/drosophila/introduction Drosophila Genomics Resource Center https://dgrc.bio.indiana.edu/Home Berkeley Drosophila Genome Project http://www.fruitfly.org/ Drosophila Genomics and Genetic Resources http://www.dgrc.kit.ac.jp/ Bloomington Drosophila Stock Center https://bdsc.indiana.edu/ Vienna Drosophila Resource Center http://stockcenter.vdrc.at/control/main NIG-FLY https://shigen.nig.ac.jp/fly/nigfly/ The Exelixis Collection at Harvard Medical School https://drosophila.med.harvard.edu/ DRSC/TRiP Functional Genomics Resources https://fgr.hms.harvard.edu/fly-in-vivo-rnai FlyORF http://flyorf.ch/index.php/orf-collection FlyExpress 7 http://www.flyexpress.net/ FlyBook http://www.genetics.org/content/flybook FlyMove http://flymove.uni-muenster.de/ Fly-FISH http://fly-fish.ccbr.utoronto.ca/ Flygut http://flygut.epfl.ch/ FlyMine http://www.flymine.org/ Gene Disruption Project http://flypush.imgen.bcm.tmc.edu/pscreen/index.php The Interactive Fly http://www.sdbonline.org/sites/fly/aimain/1aahome.htm Textpresso for Fly http://www.textpresso.org/fly/ BruinFly http://www.bruinfly.ucla.edu/index.php Virtual Fly Brain https://www.virtualflybrain.org/site/vfb_site/home.htm Fruit Fly Brain Observatory http://fruitflybrain.org/ DroID – The Drosophila Interactions Database http://flygut.epfl.ch/ J-FLY http://jfly.iam.u-tokyo.ac.jp/index.html Neuromuscular Disease Center http://neuromuscular.wustl.edu/ 1 Drosophila as a Model Organism 9 specialized bioinformatics resources for age and subdivision of the Drosophila melanogaster genome. 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