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1.  Multiple Redundant Medulla Projection Neurons Mediate Color Vision in Drosophila 
Journal of neurogenetics  2014;28(0):374-388.
The receptor mechanism for color vision has been extensively studied. In contrast, the circuit(s) that transform(s) photoreceptor signals into color percepts to guide behavior remain(s) poorly characterized. Using intersectional genetics to inactivate identified subsets of neurons, we have uncovered the first-order interneurons that are functionally required for hue discrimination in Drosophila. We developed a novel aversive operant conditioning assay for intensity independent color discrimination (true color vision) in Drosophila. Single flying flies are magnetically tethered in an arena surrounded by blue and green LEDs. The flies’ optomotor response is used to determine the blue-green isoluminant intensity. Flies are then conditioned to discriminate between equiluminant blue or green stimuli. Wild-type flies are successfully trained in this paradigm when conditioned to avoid either blue or green. Functional color entrainment requires the function of the narrow spectrum photoreceptors R8 and/or R7, and is within a limited range, intensity independent, suggesting that it is mediated by a color vision system. The medulla projection neurons, Tm5a/b/c and Tm20, receive direct inputs from R7 or R8 photoreceptors and indirect input from the broad spectrum photoreceptors R1-R6 via the lamina neuron L3. Genetically inactivating these four classes of medulla projection neurons abolished color learning. However, inactivation of subsets of these neurons is insufficient to block color learning, suggesting that true color vision is mediated by multiple redundant pathways. We hypothesize that flies represent color along multiple axes at the first synapse in the fly visual system. The apparent redundancy in learned color discrimination sharply contrasts with innate UV spectral preference, which is dominated by a single pathway from the amacrine neuron Dm8 to the Tm5c projection neurons.
doi:10.3109/01677063.2014.891590
PMCID: PMC4245076  PMID: 24766346
color discrimination; medulla projection neurons; neural substrate; visual behavior
2.  Photoreceptor-derived Activin Promotes Dendritic Termination and Restricts the Receptive Fields of First-order Interneurons in Drosophila 
Neuron  2014;81(4):830-846.
SUMMARY
How CNS neurons form appropriately sized dendritic fields to encounter their presynaptic partners is poorly understood. The Drosophila medulla is organized in layers and columns, and innervated by medulla neurons dendrites and photoreceptor axons. Here we show that three types of medulla projection (Tm) neurons extend their dendrites in stereotyped directions and to distinct layers within a single column for processing retinotopic information. In contrast, the Dm8 amacrine neurons form a wide dendritic field to receive ~16 R7 photoreceptor inputs. R7- and R8-derived Activin/TGF-β selectively restricts the dendritic fields of their respective postsynaptic partners, Dm8 and Tm20, to the size appropriate for their functions. Canonical Activin signaling promotes dendritic termination without affecting dendritic routing direction or layer. Tm20 neurons lacking Activin signaling expanded their dendritic fields and aberrantly synapsed with neighboring photoreceptors. We suggest that afferent-derived Activin regulates the dendritic field size of their postsynaptic partners to ensure appropriate synaptic partnership.
doi:10.1016/j.neuron.2013.12.012
PMCID: PMC4004379  PMID: 24462039
3.  A Hard-wired Glutamatergic Circuit Pools and Relays UV Signals to Mediate Spectral Preference in Drosophila 
Neuron  2014;81(3):603-615.
SUMMARY
Many visual animals have innate preferences for particular wavelengths of light, which can be modified by learning. Drosophila’s preference for UV over visible light requires UV-sensing R7 photoreceptors and specific wide-field amacrine neurons, called Dm8. Here we identify three types of medulla projection neurons downstream of R7 and Dm8, and show that selectively inactivating one of them (Tm5c) abolishes UV preference. Using a modified GRASP method to probe synaptic connections at the single-cell level, we reveal that each Dm8 neuron forms multiple synaptic contacts with Tm5c in the center of Dm8’s dendritic field, but sparse connections in the periphery. By single-cell transcript profiling and RNAi-mediated knockdown, we determine that Tm5c uses the kainate receptor Clumsy to receive excitatory glutamate input from Dm8. We conclude that R7s->Dm8->Tm5c form a hard-wired glutamatergic circuit that mediates UV preference by pooling ~16 R7 signals for transfer to the lobula, a higher visual center.
doi:10.1016/j.neuron.2013.12.010
PMCID: PMC3920195  PMID: 24507194
4.  Visual Circuit Assembly in Drosophila 
Developmental neurobiology  2011;71(12):1286-1296.
Both insect and vertebrate visual circuits are organized into orderly arrays of columnar and layered synaptic units that correspond to the array of photoreceptors in the eye. Recent genetic studies in Drosophila have yielded insights into the molecular and cellular mechanisms that pattern the layers and columns and establish specific connections within the synaptic units. A sequence of inductive events and complex cellular interactions coordinates the assembly of visual circuits. Photoreceptor-derived ligands, such as hedgehog and Jelly-Belly, induce target development and expression of specific adhesion molecules, which in turn serve as guidance cues for photoreceptor axons. Afferents are directed to specific layers by adhesive afferent-target interactions mediated by leucine-rich repeat proteins and cadherins, which are restricted spatially and/or modulated dynamically. Afferents are restricted to their topographically appropriate columns by repulsive interactions between afferents and by autocrine Activin signalling. Finally, Dscam-mediated repulsive interactions between target neuron dendrites ensure appropriate combinations of post-synaptic elements at synapses. Essentially all of these Drosophila molecules have vertebrate homologs, some of which are known to carry out analogous functions. Thus, the studies of Drosophila visual circuit development would shed light on neural circuit assembly in general.
doi:10.1002/dneu.20894
PMCID: PMC4245071  PMID: 21538922
5.  Birth order dependent growth cone segregation determines synaptic layer identity in the Drosophila visual system 
eLife  null;5:e13715.
The precise recognition of appropriate synaptic partner neurons is a critical step during neural circuit assembly. However, little is known about the developmental context in which recognition specificity is important to establish synaptic contacts. We show that in the Drosophila visual system, sequential segregation of photoreceptor afferents, reflecting their birth order, lead to differential positioning of their growth cones in the early target region. By combining loss- and gain-of-function analyses we demonstrate that relative differences in the expression of the transcription factor Sequoia regulate R cell growth cone segregation. This initial growth cone positioning is consolidated via cell-adhesion molecule Capricious in R8 axons. Further, we show that the initial growth cone positioning determines synaptic layer selection through proximity-based axon-target interactions. Taken together, we demonstrate that birth order dependent pre-patterning of afferent growth cones is an essential pre-requisite for the identification of synaptic partner neurons during visual map formation in Drosophila.
DOI: http://dx.doi.org/10.7554/eLife.13715.001
eLife digest
A nervous system requires a precise network of connections between cells called neurons to work properly. Within the brain, the fiber-like connections between pairs of neurons are not running crisscross like a pile of spaghetti. Instead, connected partner neurons are organized into distinct layers and columns.
Many questions remain about how these partner neurons find each other and how the layers of fiber-like connections form. To answer these questions, scientists often study the part of the fruit fly nervous system that controls the insect’s vision. This brain-like structure is simple and can be easily manipulated with genetic engineering. Fruit fly studies have helped identify some molecules that play a role in helping partner cells find one another and connect. These studies have also shown that the timing of brain cell development appears to play a role. But the role that layer formation plays in the process is still a mystery.
Now, Kulkarni et al. show that the birthdate of neurons in the fruit fly visual system helps organize them into layers. These neurons are generated early in the development of the fly. Shortly after birth, a molecular clock under the control of a protein called Sequoia starts within each newly generated neuron. The Sequoia protein is a transcription factor and controls the activity of many genes, and the molecular clock provides the growing neuron fibers with information about where and when to look for its partner neurons.
By manipulating the amount and time that Sequoia is produced in the fly visual system, Kulkarni et al. show that this clock helps arrange the growing cells into layers. Cells with similar birthdates connect and are arranged into layers. How much and when Sequoia is produced dictates where each new layer begins. The next steps for the research will be to learn more about how the clock works and identify any intermediaries between the clock and cell growth patterns.
DOI: http://dx.doi.org/10.7554/eLife.13715.002
doi:10.7554/eLife.13715
PMCID: PMC4846375  PMID: 26987017
neural circuit; synapse; visual system; cell recognition; D. melanogaster
6.  A gustatory second-order neuron that connects sucrose-sensitive primary neurons and a distinct region of the gnathal ganglion in the Drosophila brain 
Journal of neurogenetics  2015;29(0):144-155.
Although the gustatory system provides animals with sensory cues important for food choice and other critical behaviors, little is known about neural circuitry immediately following gustatory sensory neurons (GSNs). Here, we identify and characterize a bilateral pair of gustatory second-order neurons in Drosophila. Previous studies identified GSNs that relay taste information to distinct subregions of the primary gustatory center (PGC) in the gnathal ganglia (GNG). To identify candidate gustatory second-order neurons (G2Ns) we screened ~5,000 GAL4 driver strains for lines that label neural fibers innervating the PGC. We then combined GRASP (GFP reconstitution across synaptic partners) with presynaptic labeling to visualize potential synaptic contacts between the dendrites of the candidate G2Ns and the axonal terminals of Gr5a-expressing GSNs, which are known to respond to sucrose. Results of the GRASP analysis, followed by a single cell analysis by FLPout recombination, revealed a pair of neurons that contact Gr5a axon terminals in both brain hemispheres, and send axonal arborizations to a distinct region outside the PGC but within the GNG. To characterize the input and output branches, respectively, we expressed fluorescence-tagged acetylcholine receptor subunit (Dα7) and active-zone marker (Brp) in the G2Ns. We found that G2N input sites overlaid GRASP-labeled synaptic contacts to Gr5a neurons, while presynaptic sites were broadly distributed throughout the neurons’ arborizations. GRASP analysis and further tests with the Syb-GRASP method suggested that the identified G2Ns receive synaptic inputs from Gr5a-expressing GSNs, but not Gr66a-expressing GSNs, which respond to caffeine. The identified G2Ns relay information from Gr5a-expressing GSNs to distinct regions in the GNG, and are distinct from other, recently identified gustatory projection neurons, which relay information about sugars to a brain region called the antennal mechanosensory and motor center (AMMC). Our findings suggest unexpected complexity for taste information processing in the first relay of the gustatory system.
doi:10.3109/01677063.2015.1054993
PMCID: PMC4750650  PMID: 26004543
Taste; interneuron; subesophageal zone; higher-order circuits
7.  The Neural Substrate of Spectral Preference in Drosophila 
Neuron  2008;60(2):328-342.
Drosophila vision is mediated by inputs from three types of photoreceptor neurons: R1–R6 mediate achromatic motion detection while R7 and R8 constitute two chromatic channels. Neural circuits for processing chromatic information are not known. Here we identified the first-order interneurons downstream of the chromatic channels. Serial-EM revealed that small-field projection neurons Tm5 and Tm9 receive direct synaptic input from R7 and R8, respectively, and indirect input from R1–R6, qualifying them to function as color-opponent neurons. Wide-field Dm8 amacrine neurons receive input from 13–16 UV-sensing R7s and provide output to projection neurons. Using a combinatorial expression system to manipulate activity in different neuron subtypes, we determined that Dm8 neurons are both necessary and sufficient for phototaxis to ultraviolet in preference to green light. We propose that Dm8 sacrifices spatial resolution for sensitivity by relaying signals from multiple R7s to projection neurons, which then provide output to higher visual centers.
doi:10.1016/j.neuron.2008.08.010
PMCID: PMC2665173  PMID: 18957224
8.  Tiling of R7 Axons in the Drosophila Visual System is Mediated Both by Transduction of an Activin Signal to the Nucleus and by Mutual Repulsion 
Neuron  2007;56(5):793-806.
Summary
The organization of neuronal wiring into layers and columns is a common feature of both vertebrate and invertebrate brains. In the Drosophila visual system, each R7 photoreceptor axon projects within a single column to a specific layer of the optic lobe. We refer to the restriction of terminals to single columns as tiling. In a genetic screen based on an R7-dependent behavior, we identified the Activin receptor Baboon and the nuclear import adaptor Importin-α3 as being required to prevent R7 axon terminals from overlapping with the terminals of R7s in neighboring columns. This tiling function requires the Baboon ligand, dActivin, the transcription factor, dSmad2, and retrograde transport from the growth cone to the R7 nucleus. We propose that dActivin is an autocrine signal that restricts R7 growth cone motility, and we demonstrate that it acts in parallel with a paracrine signal that mediates repulsion between R7 terminals.
doi:10.1016/j.neuron.2007.09.033
PMCID: PMC2693211  PMID: 18054857
9.  Adhesive but not Signaling Activity of Drosophila N-cadherin is Essential for Target Selection of Photoreceptor Afferents 
Developmental biology  2007;304(2):759-770.
Drosophila N-cadherin (CadN) is an evolutionarily conserved, atypical classical cadherin, which has a large complex extracellular domain and a catenin-binding cytoplasmic domain. We have previously shown that CadN regulates target selection of R7 photoreceptor axons. To determine the functional domains of CadN, we conducted a structure-function analysis focusing on its in vitro adhesive activity and in vivo function in R7 growth cones. We found that the cytoplasmic domain of CadN is largely dispensable for the targeting of R7 growth cones, and it is not essential for mediating homophilic interaction in cultured cells. Instead, the cytoplasmic domain of CadN is required for maintaining proper growth cone morphology. Domain swapping with the extracellular domain of CadN2, a related but non-adhesive cadherin, revealed that the CadN extracellular domain is required for both adhesive activity and R7 targeting. Using a target-mosaic system, we generated CadN mutant clones in the optic lobe and examined the target-selection of genetically wild-type R7 growth cones to CadN mutant target neurons. We found that CadN, but neither LAR nor Liprin-α, is required in the medulla neurons for R7 growth cones to select sthe correct medulla layer. Together, these data suggest that CadN mediates homophilic adhesive interactions between R7 growth cones and medulla neurons to regulate layer-specific target selection.
doi:10.1016/j.ydbio.2007.01.030
PMCID: PMC1959568  PMID: 17320070
N-cadherin; neural target selection; layer-specific targeting; visual system development; adhesion molecule
10.  Dynamic labelling of neural connections in multiple colours by trans-synaptic fluorescence complementation 
Nature Communications  2015;6:10024.
Determining the pattern of activity of individual connections within a neural circuit could provide insights into the computational processes that underlie brain function. Here, we develop new strategies to label active synapses by trans-synaptic fluorescence complementation in Drosophila. First, we demonstrate that a synaptobrevin-GRASP chimera functions as a powerful activity-dependent marker for synapses in vivo. Next, we create cyan and yellow variants, achieving activity-dependent, multi-colour fluorescence reconstitution across synapses (X-RASP). Our system allows for the first time retrospective labelling of synapses (rather than whole neurons) based on their activity, in multiple colours, in the same animal. As individual synapses often act as computational units in the brain, our method will promote the design of experiments that are not possible using existing techniques. Moreover, our strategies are easily adaptable to circuit mapping in any genetic system.
The ability to map the activity of synapses within a circuit will help in elucidating the neural basis of behaviour. Here, Macpherson et al. report new strategies to specifically label active synapses in Drosophila with multi-colour fluorescence tags.
doi:10.1038/ncomms10024
PMCID: PMC4686661  PMID: 26635273
11.  Candidate Neural Substrates for Off-Edge Motion Detection in Drosophila 
Current biology : CB  2014;24(10):1062-1070.
Summary
Background
In the fly’s visual motion pathways, two cell types - T4 and T5 - are the first known relay neurons to signal small-field direction-selective motion responses [1]. These cells then feed into large tangential cells that signal wide-field motion. Recent studies identified two types of columnar neurons in the second neuropil, or medulla, that relay input to T4 from L1, the ON-channel neuron in the first neuropil, or lamina, thus providing a candidate substrate for the elementary motion detector (EMD) [2]. Interneurons relaying the OFF-channel from L1’s partner, L2, to T5 are so far not known however.
Results
Here we report that multiple types of transmedulla (Tm) neurons provide unexpectedly complex inputs to T5 at their terminals in the third neuropil, or lobula. From the L2 pathway, single-column input comes from Tm1 and Tm2 and multiple-column input from Tm4 cells. Additional input to T5 comes from Tm9, the medulla target of a third lamina interneuron L3, providing a candidate substrate for L3’s combinatorial action with L2 [3]. Most numerous, Tm2 and Tm9’s input synapses are spatially segregated on T5’s dendritic arbor, providing candidate anatomical substrates for the two arms of a T5 EMD circuit; Tm1 and Tm2 provide a second. Transcript profiling indicates that T5 expresses both nicotinic and muscarinic cholinoceptors, qualifying T5 to receive cholinergic inputs from Tm9 and Tm2, which both express ChAT.
Conclusions
We hypothesize that T5 computes small-field motion signals by integrating multiple cholinergic Tm inputs using nicotinic and muscarinic cholinoceptors.
doi:10.1016/j.cub.2014.03.051
PMCID: PMC4031294  PMID: 24768048
fly; photoreceptors; visual pathway; column; motion sensitivity; directional selectivity; ‘off’ edge response; elementary motion detector
12.  'The genetic analysis of functional connectomics in Drosophila' 
Advances in genetics  2012;80:99-151.
Fly and vertebrate nervous systems share many organization characteristics, such as layers, columns and glomeruli, and utilize similar synaptic components, such ion channels and receptors. Both also exhibit similar network features. Recent technological advances, especially in electron microscopy, now allow us to determine synaptic circuits and identify pathways cell-by-cell, as part of the fly’s connectome. Genetic tools provide the means to identify synaptic components, as well as to record and manipulate neuronal activity, adding function to the connectome. This review discusses technical advances in these emerging areas of functional connectomics, offering prognoses in each and identifying the challenges in bridging structural connectomics to molecular biology and synaptic physiology, thereby determining fundamental computation mechanisms that underlie behaviour.
doi:10.1016/B978-0-12-404742-6.00003-X
PMCID: PMC4251806  PMID: 23084874
connectome; synapse; serial-section EM; neurotransmitter; receptor; transporter
13.  The split view of motion 
Nature  2010;468(7321):178-179.
In fruitflies and vertebrates, signals from photoreceptor cells are immediately split into two opposing channels in the downstream neurons. This might facilitate the computation of visual motion.
doi:10.1038/468178a
PMCID: PMC4243609  PMID: 21068820
14.  cis-Regulatory Complexity within a Large Non-Coding Region in the Drosophila Genome 
PLoS ONE  2013;8(4):e60137.
Analysis of cis-regulatory enhancers has revealed that they consist of clustered blocks of highly conserved sequences. Although most characterized enhancers reside near their target genes, a growing number of studies have shown that enhancers located over 50 kb from their minimal promoter(s) are required for appropriate gene expression and many of these ‘long-range’ enhancers are found in genomic regions that are devoid of identified exons. To gain insight into the complexity of Drosophila cis-regulatory sequences within exon-poor regions, we have undertaken an evolutionary analysis of 39 of these regions located throughout the genome. This survey revealed that within these genomic expanses, clusters of conserved sequence blocks (CSBs) are positioned once every 1.1 kb, on average, and that a typical cluster contains multiple (5 to 30 or more) CSBs that have been maintained for at least 190 My of evolutionary divergence. As an initial step toward assessing the cis-regulatory activity of conserved clusters within gene-free genomic expanses, we have tested the in-vivo enhancer activity of 19 consecutive CSB clusters located in the middle of a 115 kb gene-poor region on the 3rd chromosome. Our studies revealed that each cluster functions independently as a specific spatial/temporal enhancer. In total, the enhancers possess a diversity of regulatory functions, including dynamically activating expression in defined patterns within subsets of cells in discrete regions of the embryo, larvae and/or adult. We also observed that many of the enhancers are multifunctional–that is, they activate expression during multiple developmental stages. By extending these results to the rest of the Drosophila genome, which contains over 70,000 non-coding CSB clusters, we suggest that most function as enhancers.
doi:10.1371/journal.pone.0060137
PMCID: PMC3632565  PMID: 23613719
15.  Use of a Drosophila Genome-Wide Conserved Sequence Database to Identify Functionally Related cis-Regulatory Enhancers 
Phylogenetic footprinting has revealed that cis-regulatory modules consist of clusters of conserved DNA sequences. We have generated a Drosophila melanogaster genomic database of conserved sequence clusters (CSCs) to facilitate enhancer discovery and analysis of their sub-structure. The database consists of >100,000 CSCs gleaned from EvoPrints spanning over 90% of the genome. To identify related enhancers based on shared conserved sequence elements, we have developed database search and alignment algorithms, collectively known as cis-Decoder. These web accessible tools initially identify conserved repeat elements within an EvoPrinted input enhancer and then search the database for CSCs that score highly against the input enhancer. To demonstrate the utility of these tools, a temporally-restricted CNS neuroblast enhancer was used to identify other functionally related enhancers and analyze their structural organization. The genome-wide CSC database and cis-Decoder algorithms can be used to discover and analyze cis-regulatory DNA involved in any developmental process.
doi:10.1002/dvdy.22728
PMCID: PMC3243966  PMID: 22174086
16.  Cholinergic circuits integrate neighboring visual signals in a Drosophila motion detection pathway 
Current Biology  2011;21(24):2077-2084.
Detecting motion is a feature of all advanced visual systems [1], nowhere more so than in flying animals, such as insects [2,3]. In flies, an influential auto-correlation model for motion detection, the elementary motion detector circuit (EMD; [4,5]), compares visual signals from neighboring photoreceptors to derive information on motion direction and velocity. This information is fed by two types of interneuron, L1 and L2, in the first optic neuropile, or lamina, to downstream local motion detectors in columns of the second neuropile, the medulla. Despite receiving carefully matched photoreceptor inputs, L1 and L2 drive distinct, separable pathways responding preferentially to moving ‘on’- and ‘off’-edges, respectively [6,7]. Our serial EM identifies two types of transmedulla (Tm) target neurons, Tm1 and Tm2, that receive apparently matched synaptic inputs from L2. Tm2 neurons also receive inputs from two retinotopically posterior neighboring columns via L4, a third type of lamina neuron. Light microscopy reveals that the connections in these L2/L4/Tm2 circuits are highly determinate. Single-cell transcript profiling suggests that nicotinic acetylcholine receptors mediate transmission within the L2/L4/Tm2 circuits while L1 is apparently glutamatergic. We propose that Tm2 integrates sign-conserving inputs from neighboring columns to mediate the detection of front-to-back motion generated during forward motion.
doi:10.1016/j.cub.2011.10.053
PMCID: PMC3265035  PMID: 22137471
fly; photoreceptors; visual pathway; column; tetrad; ‘off’ response
17.  A Presynaptic Gain Control Mechanism Fine-Tunes Olfactory Behavior 
Neuron  2008;59(2):311-321.
Early sensory processing can play a critical role in sensing environmental cues. We have investigated the physiological and behavioral function of gain control at the first synapse of olfactory processing in Drosophila. We report that olfactory receptor neurons (ORNs) express the GABAB receptor (GABABR) and its expression expands the dynamic range of ORN synaptic transmission that is preserved in projection neuron responses. Strikingly, we find that different ORN channels have unique baseline levels of GABABR expression. ORNs that sense the aversive odorant CO2 do not express GABABRs nor exhibit any presynaptic inhibition. In contrast, pheromone-sensing ORNs express a high level of GABABRs and exhibit strong presynaptic inhibition. Furthermore, a behavioral significance of presynaptic inhibition was revealed by a courtship behavior in which pheromone-dependent mate localization is impaired in flies that lack GABABRs in specific ORNs. Together, these findings indicate that different olfactory receptor channels may employ heterogeneous presynaptic gain control as a mechanism to allow an animal’s innate behavioral responses to match its ecological needs.
doi:10.1016/j.neuron.2008.07.003
PMCID: PMC2539065  PMID: 18667158
Drosophila; olfaction; GABAB; presynaptic inhibition; gain control; dynamic range; two-photon imaging
18.  Conserved Alternative Splicing and Expression Patterns of Arthropod N-Cadherin 
PLoS Genetics  2009;5(4):e1000441.
Metazoan development requires complex mechanisms to generate cells with diverse function. Alternative splicing of pre-mRNA not only expands proteomic diversity but also provides a means to regulate tissue-specific molecular expression. The N-Cadherin gene in Drosophila contains three pairs of mutually-exclusive alternatively-spliced exons (MEs). However, no significant differences among the resulting protein isoforms have been successfully demonstrated in vivo. Furthermore, while the N-Cadherin gene products exhibit a complex spatiotemporal expression pattern within embryos, its underlying mechanisms and significance remain unknown. Here, we present results that suggest a critical role for alternative splicing in producing a crucial and reproducible complexity in the expression pattern of arthropod N-Cadherin. We demonstrate that the arthropod N-Cadherin gene has maintained the three sets of MEs for over 400 million years using in silico and in vivo approaches. Expression of isoforms derived from these MEs receives precise spatiotemporal control critical during development. Both Drosophila and Tribolium use ME-13a and ME-13b in “neural” and “mesodermal” splice variants, respectively. As proteins, either ME-13a- or ME-13b-containing isoform can cell-autonomously rescue the embryonic lethality caused by genetic loss of N-Cadherin. Ectopic muscle expression of either isoform beyond the time it normally ceases leads to paralysis and lethality. Together, our results offer an example of well-conserved alternative splicing increasing cellular diversity in metazoans.
Author Summary
Animal development requires complex mechanisms to generate many different types of cells. Alternative splicing is a process by which a single gene could produce several protein variants under particular circumstances. It is a useful means to generate a diversified set of proteins in different cell types. In this report, we showed that the alternative splicing of the arthropod N-Cadherin gene has been maintained for over 400 million years. The switch of expression patterns of two distinct variants is also well conserved in arthropods. As proteins, these two N-Cadherin splice variants have similar ability to rescue the embryonic lethality caused by genetic loss of N-Cadherin. However, when the expression of either isoforms was prolonged in muscles where the endogenous expression ceased beyond certain stages, it leads to larval lethality, suggesting the importance of precise spatiotemporal regulation of N-Cadherin splice-variant expression. This finding is particularly important because it offers an example of well-conserved alternative splicing increasing cellular diversity in animals.
doi:10.1371/journal.pgen.1000441
PMCID: PMC2655722  PMID: 19343204
19.  The Variable Transmembrane Domain of Drosophila N-Cadherin Regulates Adhesive Activity†  
Molecular and Cellular Biology  2006;26(17):6598-6608.
Drosophila N-cadherin (CadN) is an evolutionarily conserved classic cadherin which has a large, complex extracellular domain and a catenin-binding cytoplasmic domain. The CadN locus contains three modules of alternative exons (7a/b, 13a/b, and 18a/b) and undergoes alternative splicing to generate multiple isoforms. Using quantitative transcript analyses and green fluorescent protein-based cell sorting, we found that during development CadN alternative splicing is regulated in a temporal but not cell-type-specific fashion. In particular, exon 18b is predominantly expressed during early developmental stages, while exon 18a is prevalent at the late developmental and adult stages. All CadN isoforms share the same molecular architecture but have different sequences in their extracellular and transmembrane domains, suggesting functional diversity. In vitro quantitative cell aggregation assays revealed that all CadN isoforms mediate homophilic interactions, but the isoforms encoded by exon 18b have a higher adhesive activity than those by its alternative, 18a. Domain-swapping experiments further revealed that the different sequences in the transmembrane domains of isoforms are responsible for their differential adhesive activities. CadN alternative splicing might provide a novel mechanism to fine-tune its adhesive activity at different developmental stages or to restrict the use of high-affinity 18b-type isoforms at the adult stage.
doi:10.1128/MCB.00241-06
PMCID: PMC1592838  PMID: 16914742
20.  Mouse Disabled 1 Regulates the Nuclear Position of Neurons in a Drosophila Eye Model 
Molecular and Cellular Biology  2006;26(4):1510-1517.
Nucleokinesis has recently been suggested as a critical regulator of neuronal migration. Here we show that Disabled 1 (Dab1), which is required for neuronal positioning in mammals, regulates the nuclear position of postmitotic neurons in a phosphorylation-site dependent manner. Dab1 expression in the Drosophila visual system partially rescues nuclear position defects caused by a mutation in the Dynactin subunit Glued. Furthermore, we observed that a loss-of-function allele of amyloid precursor protein (APP)-like, a kinesin cargo receptor, enhanced the severity of a Dab1 overexpression phenotype characterized by misplaced nuclei in the adult retina. In mammalian neurons, overexpression of APP reduced the ability of Reelin to induce Dab1 tyrosine phosphorylation, suggesting an antagonistic relationship between APP family members and Dab1 function. This is the first evidence that signaling which regulates Dab1 tyrosine phosphorylation determines nuclear positioning through Dab1-mediated influences on microtubule motor proteins in a subset of neurons.
doi:10.1128/MCB.26.4.1510-1517.2006
PMCID: PMC1367204  PMID: 16449660
21.  Use of a Drosophila Genome-Wide Conserved Sequence Database to Identify Functionally Related cis-Regulatory Enhancers 
Developmental Dynamics  2011;241(1):169-189.
Background: Phylogenetic footprinting has revealed that cis-regulatory enhancers consist of conserved DNA sequence clusters (CSCs). Currently, there is no systematic approach for enhancer discovery and analysis that takes full-advantage of the sequence information within enhancer CSCs. Results: We have generated a Drosophila genome-wide database of conserved DNA consisting of >100,000 CSCs derived from EvoPrints spanning over 90% of the genome. cis-Decoder database search and alignment algorithms enable the discovery of functionally related enhancers. The program first identifies conserved repeat elements within an input enhancer and then searches the database for CSCs that score highly against the input CSC. Scoring is based on shared repeats as well as uniquely shared matches, and includes measures of the balance of shared elements, a diagnostic that has proven to be useful in predicting cis-regulatory function. To demonstrate the utility of these tools, a temporally-restricted CNS neuroblast enhancer was used to identify other functionally related enhancers and analyze their structural organization. Conclusions: cis-Decoder reveals that co-regulating enhancers consist of combinations of overlapping shared sequence elements, providing insights into the mode of integration of multiple regulating transcription factors. The database and accompanying algorithms should prove useful in the discovery and analysis of enhancers involved in any developmental process. Developmental Dynamics 241:169–189, 2012. © 2011 Wiley Periodicals, Inc.
Key findings
A genome-wide catalog of Drosophila conserved DNA sequence clusters.cis-Decoder discovers functionally related enhancers.Functionally related enhancers share balanced sequence element copy numbers.Many enhancers function during multiple phases of development.
doi:10.1002/dvdy.22728
PMCID: PMC3243966  PMID: 22174086
cis-regulatory enhancers; conserved sequence clusters (CSCs); Drosophila melanogaster

Results 1-21 (21)