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1.  The Little Fly that Could: Wizardry and Artistry of Drosophila Genomics 
Genes  2014;5(2):385-414.
For more than 100 years now, the fruit fly Drosophila melanogaster has been at the forefront of our endeavors to unlock the secrets of the genome. From the pioneering studies of chromosomes and heredity by Morgan and his colleagues, to the generation of fly models for human disease, Drosophila research has been at the forefront of genetics and genomics. We present a broad overview of some of the most powerful genomics tools that keep Drosophila research at the cutting edge of modern biomedical research.
doi:10.3390/genes5020385
PMCID: PMC4094939  PMID: 24827974
Drosophila; genetics; genomics
2.  Regulation of branching dynamics by axon-intrinsic asymmetries in Tyrosine Kinase Receptor signaling 
eLife  2014;3:e01699.
Axonal branching allows a neuron to connect to several targets, increasing neuronal circuit complexity. While axonal branching is well described, the mechanisms that control it remain largely unknown. We find that in the Drosophila CNS branches develop through a process of excessive growth followed by pruning. In vivo high-resolution live imaging of developing brains as well as loss and gain of function experiments show that activation of Epidermal Growth Factor Receptor (EGFR) is necessary for branch dynamics and the final branching pattern. Live imaging also reveals that intrinsic asymmetry in EGFR localization regulates the balance between dynamic and static filopodia. Elimination of signaling asymmetry by either loss or gain of EGFR function results in reduced dynamics leading to excessive branch formation. In summary, we propose that the dynamic process of axon branch development is mediated by differential local distribution of signaling receptors.
DOI: http://dx.doi.org/10.7554/eLife.01699.001
eLife digest
In the human brain, 100 billion neurons form 100 trillion connections. Each neuron consists of a cell body with numerous small branch-like projections known as dendrites (from the Greek word for ‘tree’), plus a long cable-like structure called the axon. Neurons receive electrical inputs from neighboring cells via their dendrites, and then relay these signals onto other cells in their network via their axons.
The development of the brain relies on new neurons integrating successfully into existing networks. Axon branching helps with this by enabling a single neuron to establish connections with several cells, but it is unclear how individual neurons decide when and where to form branches. Now, Zschätzsch et al. have revealed the mechanism behind this process in the fruit fly, Drosophila.
Mutant flies that lack a protein called EGFR produce abnormal numbers of axon branches, suggesting that this molecule regulates branch formation. Indeed in fruit flies, just as in mammals, the developing brain initially produces excessive numbers of branches, which are subsequently pruned to leave only those that have formed appropriate connections. In Drosophila, an uneven distribution of EGFR between branches belonging to the same axon acts as a signal to regulate this pruning process.
To examine this mechanism in more detail, high-resolution four-dimensional imaging was used to study brains that had been removed from Drosophila pupae and kept alive in special culture chambers. Axon branching and loss could now be followed in real time, and were found to occur more slowly in brains that lacked EGFR. The receptor controlled the branching of axons by influencing the distribution of another protein called actin, which is a key component of the internal skeleton that gives cells their structure.
In addition to providing new insights into a fundamental aspect of brain development, the work of Zschätzsch et al. also highlights the importance of stochastic events in shaping the network of connections within the developing brain. These findings may well be relevant to ongoing efforts to map the human brain ‘connectome’.
DOI: http://dx.doi.org/10.7554/eLife.01699.002
doi:10.7554/eLife.01699
PMCID: PMC3990184  PMID: 24755286
axonal branching; brain development; signaling; D. melanogaster
3.  Proper connectivity of Drosophila motion detector neurons requires Atonal function in progenitor cells 
Neural Development  2014;9:4.
Background
Vertebrates and invertebrates obtain visual motion information by channeling moving visual cues perceived by the retina through specific motion sensitive synaptic relays in the brain. In Drosophila, the series of synaptic relays forming the optic lobe are known as the lamina, medulla, lobula and lobula plate neuropiles. The fly’s motion detection output neurons, called the T4 and T5 cells, reside in the lobula plate. Adult optic lobe neurons are derived from larval neural progenitors in two proliferating compartments known as the outer and inner proliferation centers (OPC and IPC). Important insight has been gained into molecular mechanisms involved in the development of the lamina and medulla from the OPC, though less is known about the development of the lobula and lobula plate.
Results
Here we show that the proneural gene Atonal is expressed in a subset of IPC progenitors that give rise to the higher order motion detection neurons, T4 and T5, of the lobula plate. We also show that Atonal does not act as a proneural gene in this context. Rather, it is required specifically in IPC neural progenitors to regulate neurite outgrowth in the neuronal progeny.
Conclusions
Our findings reveal that a proneural gene is expressed in progenitors but is required for neurite development of their progeny neurons. This suggests that transcriptional programs initiated specifically in progenitors are necessary for subsequent neuronal morphogenesis.
doi:10.1186/1749-8104-9-4
PMCID: PMC3941608  PMID: 24571981
neural progenitor; Drosophila; atonal; neurite guidance
5.  Ubiquitin Ligase HUWE1 Regulates Axon Branching through the Wnt/β-Catenin Pathway in a Drosophila Model for Intellectual Disability 
PLoS ONE  2013;8(11):e81791.
We recently reported that duplication of the E3 ubiquitin ligase HUWE1 results in intellectual disability (ID) in male patients. However, the underlying molecular mechanism remains unknown. We used Drosophila melanogaster as a model to investigate the effect of increased HUWE1 levels on the developing nervous system. Similar to the observed levels in patients we overexpressed the HUWE1 mRNA about 2-fold in the fly. The development of the mushroom body and neuromuscular junctions were not altered, and basal neurotransmission was unaffected. These data are in agreement with normal learning and memory in the courtship conditioning paradigm. However, a disturbed branching phenotype at the axon terminals of the dorsal cluster neurons (DCN) was detected. Interestingly, overexpression of HUWE1 was found to decrease the protein levels of dishevelled (dsh) by 50%. As dsh as well as Fz2 mutant flies showed the same disturbed DCN branching phenotype, and the constitutive active homolog of β-catenin, armadillo, could partially rescue this phenotype, our data strongly suggest that increased dosage of HUWE1 compromises the Wnt/β-catenin pathway possibly by enhancing the degradation of dsh.
doi:10.1371/journal.pone.0081791
PMCID: PMC3841167  PMID: 24303071
6.  The Drosophila Homologue of the Amyloid Precursor Protein Is a Conserved Modulator of Wnt PCP Signaling 
PLoS Biology  2013;11(5):e1001562.
The Drosophila homolog of the Alzheimer's disease protein APP, known as APPL, regulates axon growth during brain development.
Wnt Planar Cell Polarity (PCP) signaling is a universal regulator of polarity in epithelial cells, but it regulates axon outgrowth in neurons, suggesting the existence of axonal modulators of Wnt-PCP activity. The Amyloid precursor proteins (APPs) are intensely investigated because of their link to Alzheimer's disease (AD). APP's in vivo function in the brain and the mechanisms underlying it remain unclear and controversial. Drosophila possesses a single APP homologue called APP Like, or APPL. APPL is expressed in all neurons throughout development, but has no established function in neuronal development. We therefore investigated the role of Drosophila APPL during brain development. We find that APPL is involved in the development of the Mushroom Body αβ neurons and, in particular, is required cell-autonomously for the β-axons and non-cell autonomously for the α-axons growth. Moreover, we find that APPL is a modulator of the Wnt-PCP pathway required for axonal outgrowth, but not cell polarity. Molecularly, both human APP and fly APPL form complexes with PCP receptors, thus suggesting that APPs are part of the membrane protein complex upstream of PCP signaling. Moreover, we show that APPL regulates PCP pathway activation by modulating the phosphorylation of the Wnt adaptor protein Dishevelled (Dsh) by Abelson kinase (Abl). Taken together our data suggest that APPL is the first example of a modulator of the Wnt-PCP pathway specifically required for axon outgrowth.
Author Summary
Wnt Planar Cell Polarity (PCP) signaling is a universal regulator of polarity in epithelial cells, but in neurons it regulates axon outgrowth, suggesting the existence of axonal modulators of Wnt-PCP activity. The Amyloid Precursor Proteins (APPs) are intensely investigated because of their link to Alzheimer's disease (AD). APP's in vivo function in the brain and the mechanisms underlying it remain unclear and controversial. In the present work we investigate the role of the Drosophila neuron-specific APP homologue, called APPL, during brain development. We find that APPL is required for the development of αβ neurons in the mushroom body, a structure critical for learning and memory. We find that APPL is a modulator of the Wnt-PCP pathway required for axonal outgrowth, but not for cell polarity. Molecularly, both human APP and fly APPL are found in membrane complexes with PCP receptors. Moreover, we show that APPL regulates PCP pathway activation through its downstream effector Abelson kinase (Abl), which modulates the phosphorylation of the Wnt adaptor protein Dishevelled (Dsh) and the subsequent activation of Wnt-PCP signaling. Taken together our data suggest that APPL is the first example of a neuron-specific modulator of the Wnt-PCP pathway.
doi:10.1371/journal.pbio.1001562
PMCID: PMC3653798  PMID: 23690751
7.  Mutual inhibition among postmitotic neurons regulates robustness of brain wiring in Drosophila 
eLife  2013;2:e00337.
Brain connectivity maps display a delicate balance between individual variation and stereotypy, suggesting the existence of dedicated mechanisms that simultaneously permit and limit individual variation. We show that during the development of the Drosophila central nervous system, mutual inhibition among groups of neighboring postmitotic neurons during development regulates the robustness of axon target choice in a nondeterministic neuronal circuit. Specifically, neighboring postmitotic neurons communicate through Notch signaling during axonal targeting, to ensure balanced alternative axon target choices without a corresponding change in cell fate. Loss of Notch in postmitotic neurons modulates an axon's target choice. However, because neighboring axons respond by choosing the complementary target, the stereotyped connectivity pattern is preserved. In contrast, loss of Notch in clones of neighboring postmitotic neurons results in erroneous coinnervation by multiple axons. Our observations establish mutual inhibition of axonal target choice as a robustness mechanism for brain wiring and unveil a novel cell fate independent function for canonical Notch signaling.
DOI: http://dx.doi.org/10.7554/eLife.00337.001
eLife digest
The brains of all members of a species are similar, but not identical, and these differences are partly responsible for the range of behaviors displayed by individuals. The development of the nervous system is known to depend on the Notch signaling pathway, but the mechanisms that regulate the balance between fixed patterns of neuronal connectivity vs individual variability are largely unknown.
Notch proteins are transmembrane proteins, which means that they have one part inside the cell membrane and another outside the cell. When a ligand protein—such as a Delta ligand—binds to the part that is outside the cell, the Notch protein breaks in two and the part inside the cell travels to the nucleus, where it can influence the expression of genes.
Cells are selected to become neurons through a process known as mutual, or lateral, inhibition. When a Delta ligand belonging to one cell binds to the Notch receptor on a neighboring cell, the production of Delta ligands in the second cell is reduced. This amplifies any initial differences in the amount of Delta produced by each cell, and leads ultimately to them becoming distinct cell types.
Now, Langen et al. show that this same mechanism is reactivated at a later stage of development during wiring up of the visual system. They used the fruit fly (Drosophila)—a model organism with a fully sequenced genome and short intergeneration time—to study a group of brain cells known as dorsal cluster neurons. At the end of the fruit fly larval stage, these neurons extend long axons across the brain to the opposite hemisphere: however, it is unclear how the neurons decide which cells to form connections with.
Using genetically modified flies, Langen et al. showed that inhibiting Notch in a single dorsal cluster neuron caused that neuron to target a different cell: however, other neurons adjusted their choices accordingly so that the overall pattern of connections remained unchanged. Inhibiting Notch in a cluster of dorsal cluster neurons, on the other hand, disrupted the entire network, suggesting that Notch-mediated communication between neurons (via mutual inhibition) is needed to establish a robust wiring map.
Langen et al. suggest that evolution has favored a mechanism that ensures that the overall pattern of connections within a circuit is preserved, while individual connections differ from one species member to the next.
DOI: http://dx.doi.org/10.7554/eLife.00337.002
doi:10.7554/eLife.00337
PMCID: PMC3589824  PMID: 23471010
Neurobiology; Neural Circuit; Robustness; Variability; Notch Signaling; Axonal targeting; D. melanogaster
8.  Drosophila syndecan regulates tracheal cell migration by stabilizing Robo levels 
EMBO Reports  2011;12(10):1039-1046.
Drosophila syndecan regulates tracheal cell migration by stabilizing Robo levels
Syndecans have crucial roles in cell adhesion, polarization and migration through their interaction with a range of extracellular ligands. The authors show in this report that Drosophila syndecan is required for the extension and fusion of the dorsal branches of the tracheal system, by reducing Slit/Robo signalling levels.
Here we identify a new role for Syndecan (Sdc), the only transmembrane heparan sulphate proteoglycan in Drosophila, in tracheal development. Sdc is required cell autonomously for efficient directed migration and fusion of dorsal branch cells, but not for dorsal branch formation per se. The cytoplasmic domain of Sdc is dispensable, indicating that Sdc does not transduce a signal by itself. Although the branch-specific phenotype of sdc mutants resembles those seen in the absence of Slit/Robo2 signalling, genetic interaction experiments indicate that Sdc also helps to suppress Slit/Robo2 signalling. We conclude that Sdc cell autonomously regulates Slit/Robo2 signalling in tracheal cells to guarantee ordered directional migration and branch fusion.
doi:10.1038/embor.2011.153
PMCID: PMC3185339  PMID: 21836636
cell migration; Drosophila; heparan sulphate; Robo; tracheal system
9.  Robust Target Gene Discovery through Transcriptome Perturbations and Genome-Wide Enhancer Predictions in Drosophila Uncovers a Regulatory Basis for Sensory Specification 
PLoS Biology  2010;8(7):e1000435.
CisTarget X is a novel computational method that accurately predicts Atonal governed regulatory networks in the retina of the fruit fly.
A comprehensive systems-level understanding of developmental programs requires the mapping of the underlying gene regulatory networks. While significant progress has been made in mapping a few such networks, almost all gene regulatory networks underlying cell-fate specification remain unknown and their discovery is significantly hampered by the paucity of generalized, in vivo validated tools of target gene and functional enhancer discovery. We combined genetic transcriptome perturbations and comprehensive computational analyses to identify a large cohort of target genes of the proneural and tumor suppressor factor Atonal, which specifies the switch from undifferentiated pluripotent cells to R8 photoreceptor neurons during larval development. Extensive in vivo validations of the predicted targets for the proneural factor Atonal demonstrate a 50% success rate of bona fide targets. Furthermore we show that these enhancers are functionally conserved by cloning orthologous enhancers from Drosophila ananassae and D. virilis in D. melanogaster. Finally, to investigate cis-regulatory cross-talk between Ato and other retinal differentiation transcription factors (TFs), we performed motif analyses and independent target predictions for Eyeless, Senseless, Suppressor of Hairless, Rough, and Glass. Our analyses show that cisTargetX identifies the correct motif from a set of coexpressed genes and accurately predicts target genes of individual TFs. The validated set of novel Ato targets exhibit functional enrichment of signaling molecules and a subset is predicted to be coregulated by other TFs within the retinal gene regulatory network.
Author Summary
Tens of thousands of regulatory elements determine the spatiotemporal expression pattern of protein-coding genes in the metazoan genome. Each regulatory element, when bound by the appropriate transcription factors, can affect the temporal transcription of a nearby target gene in a particular cell type. Annotating the genome for regulatory elements, as well as determining the input transcription factors for each element, is a key challenge in genome biology. In this study, we introduce a computational method, cisTargetX, that predicts transcription factor binding motifs and their target genes through the integration of gene expression data and comparative genomics. We first validate this method in silico using public gene expression data and, then, apply cisTargetX to the developmental program governing photoreceptor neuron specification in the retina of Drosophila melanogaster. Particularly, we perturbed predicted key transcription factors during the initial steps of neurogenesis; measure gene expression by microarrays; identify motifs and predict target genes; validate the predictions in vivo using transgenic animals; and study several functional and evolutionary aspects of the validated regulatory elements for the proneural factor Atonal. Overall, we show that cisTargetX efficiently predicts genetic regulatory interactions and provides mechanistic insight into gene regulatory networks of postembryonic developmental systems.
doi:10.1371/journal.pbio.1000435
PMCID: PMC2910651  PMID: 20668662
10.  Intestinal stem cells lacking the Math1 tumour suppressor are refractory to Notch inhibitors 
Nature Communications  2010;1(2):1-5.
Intestinal cells are constantly produced from a stem cell reservoir that gives rise to proliferating transient amplifying cells, which subsequently differentiate into one of the four principal cell types. Signalling pathways, including the Notch signalling pathway, coordinate these differentiation processes and their deregulation may cause cancer. Pharmacological inhibition through γ-secretase inhibitors or genetic inactivation of the Notch signalling pathway results in the complete loss of proliferating crypt progenitors due to their conversion into post-mitotic goblet cells. The basic helix–loop–helix transcription factor Math1 is essential for intestinal secretory cell differentiation. Because of the critical roles of both Math1 and Notch signalling in intestinal homeostasis and neoplastic transformation, we sought to determine the genetic hierarchy regulating the differentiation of intestinal stem cells into secretory cells. In this paper, we demonstrate that the conversion of intestinal stem cells into goblet cells upon inhibition of the Notch signalling pathway requires Math1.
Notch inhibitors result in the differentiation of intestinal crypt progenitors into goblet cells, suggesting that they could be of use in treating intestinal neoplasia. Here van Es et al. show that Math1 is required for intestinal cell differentiation induced by Notch inhibition.
doi:10.1038/ncomms1017
PMCID: PMC2895507  PMID: 20975679
11.  Epidermal progenitors give rise to Merkel cells during embryonic development and adult homeostasis 
The Journal of Cell Biology  2009;187(1):91-100.
Lineage-tracing experiments show that the origin of specialized mechanosensory Merkel cells in the skin is epidermal progenitors, not the neural crest.
Merkel cells (MCs) are located in the touch-sensitive area of the epidermis and mediate mechanotransduction in the skin. Whether MCs originate from embryonic epidermal or neural crest progenitors has been a matter of intense controversy since their discovery >130 yr ago. In addition, how MCs are maintained during adulthood is currently unknown. In this study, using lineage-tracing experiments, we show that MCs arise through the differentiation of epidermal progenitors during embryonic development. In adults, MCs undergo slow turnover and are replaced by cells originating from epidermal stem cells, not through the proliferation of differentiated MCs. Conditional deletion of the Atoh1/Math1 transcription factor in epidermal progenitors results in the absence of MCs in all body locations, including the whisker region. Our study demonstrates that MCs arise from the epidermis by an Atoh1-dependent mechanism and opens new avenues for study of MC functions in sensory perception, neuroendocrine signaling, and MC carcinoma.
doi:10.1083/jcb.200907080
PMCID: PMC2762088  PMID: 19786578
12.  Axonal injury and regeneration in the adult brain of Drosophila 
Drosophila melanogaster is a leading genetic model system in nervous system development and disease research. Using the power of fly genetics in traumatic axonal injury research will significantly speed up the characterization of molecular processes that control axonal regeneration in the Central Nervous System (CNS). We developed a versatile and physiologically robust preparation for the long-term culture of the whole Drosophila brain. We use this method to develop a novel Drosophila model for CNS axonal injury and regeneration. We first show that, similar to mammalian CNS axons, injured adult wild type fly CNS axons fail to regenerate, whereas adult-specific enhancement of Protein Kinase A activity increases the regenerative capacity of lesioned neurons. Combined, these observations suggest conservation of neuronal regeneration mechanisms following injury. We next exploit this model to explore pathways that induce robust regeneration and find that adult-specific activation of JNK signalling is sufficient for de novo CNS axonal regeneration after injury, including the growth of new axons past the lesion site and into the normal target area.
doi:10.1523/JNEUROSCI.0101-08.2008
PMCID: PMC2693324  PMID: 18524906
Axon; Drosophila; Explant; Injury; Regeneration; Signal transduction
13.  A novel method for tissue-specific RNAi rescue in Drosophila 
Nucleic Acids Research  2009;37(13):e93.
Targeted gene silencing by RNA interference allows the study of gene function in plants and animals. In cell culture and small animal models, genetic screens can be performed—even tissue-specifically in Drosophila—with genome-wide RNAi libraries. However, a major problem with the use of RNAi approaches is the unavoidable false-positive error caused by off-target effects. Until now, this is minimized by computational RNAi design, comparing RNAi to the mutant phenotype if known, and rescue with a presumed ortholog. The ultimate proof of specificity would be to restore expression of the same gene product in vivo. Here, we present a simple and efficient method to rescue the RNAi-mediated knockdown of two independent genes in Drosophila. By exploiting the degenerate genetic code, we generated Drosophila RNAi Escape Strategy Construct (RESC) rescue proteins containing frequent silent mismatches in the complete RNAi target sequence. RESC products were no longer efficiently silenced by RNAi in cell culture and in vivo. As a proof of principle, we rescue the RNAi-induced loss of function phenotype of the eye color gene white and tracheal defects caused by the knockdown of the heparan sulfate proteoglycan syndecan. Our data suggest that RESC is widely applicable to rescue and validate ubiquitous or tissue-specific RNAi and to perform protein structure–function analysis.
doi:10.1093/nar/gkp450
PMCID: PMC2715260  PMID: 19483100
14.  The Atonal Proneural Transcription Factor Links Differentiation and Tumor Formation in Drosophila 
PLoS Biology  2009;7(2):e1000040.
The acquisition of terminal cell fate and onset of differentiation are instructed by cell type–specific master control genes. Loss of differentiation is frequently observed during cancer progression, but the underlying causes and mechanisms remain poorly understood. We tested the hypothesis that master regulators of differentiation may be key regulators of tumor formation. Using loss- and gain-of-function analyses in Drosophila, we describe a critical anti-oncogenic function for the atonal transcription factor in the fly retina, where atonal instructs tissue differentiation. In the tumor context, atonal acts by regulating cell proliferation and death via the JNK stress response pathway. Combined with evidence that atonal's mammalian homolog, ATOH1, is a tumor suppressor gene, our data support a critical, evolutionarily conserved, function for ato in oncogenesis.
Author Summary
During embryonic development, cells become more and more specialized, and this process is referred to as differentiation. In contrast to normal adult cells, cancer cells—like embryonic cells—display fewer differentiated properties. It has been postulated that the acquisition of terminal differentiation helps inhibit tumor formation; however, no direct evidence for this hypothesis was available. The development of the eye in the fruit fly, Drosophila melanogaster, has long been used as a model for studying genetic factors controlling differentiation. More recently, eye development has also been used to study how tumors can form and progress. In this study, we used this model to show that genes, such as atonal, that instruct the differentiation of specific tissues can act as tumor suppressers and inhibit the formation and progression of tumors in those tissues. Losing such genes can generate tumors, whereas activating them can strongly inhibit these tumors.
We establish a direct genetic link between cancer and the initiation of differentiation in theDrosophila eye.
doi:10.1371/journal.pbio.1000040
PMCID: PMC2652389  PMID: 19243220
15.  Atonal homolog 1 Is a Tumor Suppressor Gene 
PLoS Biology  2009;7(2):e1000039.
Colon cancer accounts for more than 10% of all cancer deaths annually. Our genetic evidence from Drosophila and previous in vitro studies of mammalian Atonal homolog 1 (Atoh1, also called Math1 or Hath1) suggest an anti-oncogenic function for the Atonal group of proneural basic helix-loop-helix transcription factors. We asked whether mouse Atoh1 and human ATOH1 act as tumor suppressor genes in vivo. Genetic knockouts in mouse and molecular analyses in the mouse and in human cancer cell lines support a tumor suppressor function for ATOH1. ATOH1 antagonizes tumor formation and growth by regulating proliferation and apoptosis, likely via activation of the Jun N-terminal kinase signaling pathway. Furthermore, colorectal cancer and Merkel cell carcinoma patients show genetic and epigenetic ATOH1 loss-of-function mutations. Our data indicate that ATOH1 may be an early target for oncogenic mutations in tissues where it instructs cellular differentiation.
Author Summary
Like most cancers, colon cancer displays a loss of differentiation, and the stronger this property, the more aggressive the cancer. This suggests that the loss of the capacity to differentiate may be a critical and possibly early event during the formation of these tumors. The key gene instructing secretory cell fate differentiation in the epithelium of the colon, namely Atonal homolog 1 (ATOH1), is highly conserved in flies, mice, and humans. We asked whether ATOH1 could be a pivotal factor in causing colon cancer in mice and humans. Our studies show that colon-specific loss of ATOH1 in mice is sufficient to trigger colon cancer and that the majority of human colon cancers also have an inactivated ATOH1. Reactivating ATOH1 in cultured human colon cancer cells causes these cells to stop dividing and to commit suicide. Since reactivation of this epigenetically silenced gene can be achieved using small chemical compounds, studying how ATOH1 acts may offer therapeutic avenues in the future.
A master regulator of differentiation in the colon is a tumor suppressor gene for colon cancer.
doi:10.1371/journal.pbio.1000039
PMCID: PMC2652388  PMID: 19243219
16.  Integrating Computational Biology and Forward Genetics in Drosophila 
PLoS Genetics  2009;5(1):e1000351.
Genetic screens are powerful methods for the discovery of gene–phenotype associations. However, a systems biology approach to genetics must leverage the massive amount of “omics” data to enhance the power and speed of functional gene discovery in vivo. Thus far, few computational methods for gene function prediction have been rigorously tested for their performance on a genome-wide scale in vivo. In this work, we demonstrate that integrating genome-wide computational gene prioritization with large-scale genetic screening is a powerful tool for functional gene discovery. To discover genes involved in neural development in Drosophila, we extend our strategy for the prioritization of human candidate disease genes to functional prioritization in Drosophila. We then integrate this prioritization strategy with a large-scale genetic screen for interactors of the proneural transcription factor Atonal using genomic deficiencies and mutant and RNAi collections. Using the prioritized genes validated in our genetic screen, we describe a novel genetic interaction network for Atonal. Lastly, we prioritize the whole Drosophila genome and identify candidate gene associations for ten receptor-signaling pathways. This novel database of prioritized pathway candidates, as well as a web application for functional prioritization in Drosophila, called Endeavour-HighFly, and the Atonal network, are publicly available resources. A systems genetics approach that combines the power of computational predictions with in vivo genetic screens strongly enhances the process of gene function and gene–gene association discovery.
Author Summary
Genome sequencing and annotation, combined with large-scale molecular experiments to query gene expression and molecular interactions, collectively known as Systems Biology, have resulted in an enormous wealth in biological databases. Yet, it remains a daunting task to use these data to decipher the rules that govern biological systems. One of the most trusted approaches in biology is genetic analysis because of its emphasis on gene function in living organisms. Genetics, however, proceeds slowly and unravels small-scale interactions. Turning genetics into an effective tool of Systems Biology requires harnessing the large-scale molecular data for the design and execution of genetic screens. In this work, we test the idea of exploiting a computational approach known as gene prioritization to pre-rank genes for the likelihood of their involvement in a process of interest. By carrying out a gene prioritization–supported genetic screen, we greatly enhance the speed and output of in vivo genetic screens without compromising their sensitivity. These results mean that future genetic screens can be custom-catered for any process of interest and carried out with a speed and efficiency that is comparable to other large-scale molecular experiments. We refer to this combined approach as Systems Genetics.
doi:10.1371/journal.pgen.1000351
PMCID: PMC2628282  PMID: 19165344
17.  Discovery of functional elements in 12 Drosophila genomes using evolutionary signatures 
Nature  2007;450(7167):219-232.
Sequencing of multiple related species followed by comparative genomics analysis constitutes a powerful approach for the systematic understanding of any genome. Here, we use the genomes of 12 Drosophila species for the de novo discovery of functional elements in the fly. Each type of functional element shows characteristic patterns of change, or ‘evolutionary signatures’, dictated by its precise selective constraints. Such signatures enable recognition of new protein-coding genes and exons, spurious and incorrect gene annotations, and numerous unusual gene structures, including abundant stop-codon readthrough. Similarly, we predict non-protein-coding RNA genes and structures, and new microRNA (miRNA) genes. We provide evidence of miRNA processing and functionality from both hairpin arms and both DNA strands. We identify several classes of pre- and post-transcriptional regulatory motifs, and predict individual motif instances with high confidence. We also study how discovery power scales with the divergence and number of species compared, and we provide general guidelines for comparative studies.
doi:10.1038/nature06340
PMCID: PMC2474711  PMID: 17994088
18.  Recombineering-mediated tagging of Drosophila genomic constructs for in vivo localization and acute protein inactivation 
Nucleic Acids Research  2008;36(18):e114.
Studying gene function in the post-genome era requires methods to localize and inactivate proteins in a standardized fashion in model organisms. While genome-wide gene disruption and over-expression efforts are well on their way to vastly expand the repertoire of Drosophila tools, a complementary method to efficiently and quickly tag proteins expressed under endogenous control does not exist for fruit flies. Here, we describe the development of an efficient procedure to generate protein fusions at either terminus in an endogenous genomic context using recombineering. We demonstrate that the fluorescent protein tagged constructs, expressed under the proper control of regulatory elements, can rescue the respective mutations and enable the detection of proteins in vivo. Furthermore, we also adapted our method for use of the tetracysteine tag that tightly binds the fluorescent membrane-permeable FlAsH ligand. This technology allows us to acutely inactivate any tagged protein expressed under native control using fluorescein-assisted light inactivation and we provide proof of concept by demonstrating that acute loss of clathrin heavy chain function in the fly eye leads to synaptic transmission defects in photoreceptors. Our tagging technology is efficient and versatile, adaptable to any tag desired and paves the way to genome-wide gene tagging in Drosophila.
doi:10.1093/nar/gkn486
PMCID: PMC2566861  PMID: 18676454
19.  Fine-Tuning Enhancer Models to Predict Transcriptional Targets across Multiple Genomes 
PLoS ONE  2007;2(11):e1115.
Networks of regulatory relations between transcription factors (TF) and their target genes (TG)- implemented through TF binding sites (TFBS)- are key features of biology. An idealized approach to solving such networks consists of starting from a consensus TFBS or a position weight matrix (PWM) to generate a high accuracy list of candidate TGs for biological validation. Developing and evaluating such approaches remains a formidable challenge in regulatory bioinformatics. We perform a benchmark study on 34 Drosophila TFs to assess existing TFBS and cis-regulatory module (CRM) detection methods, with a strong focus on the use of multiple genomes. Particularly, for CRM-modelling we investigate the addition of orthologous sites to a known PWM to construct phyloPWMs and we assess the added value of phylogenentic footprinting to predict contextual motifs around known TFBSs. For CRM-prediction, we compare motif conservation with network-level conservation approaches across multiple genomes. Choosing the optimal training and scoring strategies strongly enhances the performance of TG prediction for more than half of the tested TFs. Finally, we analyse a 35th TF, namely Eyeless, and find a significant overlap between predicted TGs and candidate TGs identified by microarray expression studies. In summary we identify several ways to optimize TF-specific TG predictions, some of which can be applied to all TFs, and others that can be applied only to particular TFs. The ability to model known TF-TG relations, together with the use of multiple genomes, results in a significant step forward in solving the architecture of gene regulatory networks.
doi:10.1371/journal.pone.0001115
PMCID: PMC2047340  PMID: 17973026
21.  A Signaling Network for Patterning of Neuronal Connectivity in the Drosophila Brain 
PLoS Biology  2006;4(11):e348.
The precise number and pattern of axonal connections generated during brain development regulates animal behavior. Therefore, understanding how developmental signals interact to regulate axonal extension and retraction to achieve precise neuronal connectivity is a fundamental goal of neurobiology. We investigated this question in the developing adult brain of Drosophila and find that it is regulated by crosstalk between Wnt, fibroblast growth factor (FGF) receptor, and Jun N-terminal kinase (JNK) signaling, but independent of neuronal activity. The Rac1 GTPase integrates a Wnt-Frizzled-Disheveled axon-stabilizing signal and a Branchless (FGF)-Breathless (FGF receptor) axon-retracting signal to modulate JNK activity. JNK activity is necessary and sufficient for axon extension, whereas the antagonistic Wnt and FGF signals act to balance the extension and retraction required for the generation of the precise wiring pattern.
This neurodevelopment study dissects how development of axonal wiring in theDrosophila optic lobe is regulated by crosstalk between Wnt, fibroblast growth factor receptor (FGFR), and Jun N-terminal kinase (JNK) signaling.
doi:10.1371/journal.pbio.0040348
PMCID: PMC1592317  PMID: 17032066

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