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ABSTRACT: We describe LOLLIbow, a Brainbow-based live imaging system with applications in developmental biology and neurobiology. The development of an animal, including the environmentally sensitive adaptation of its brain, is thought to proceed through continual orchestration among diverse cell types as they divide, migrate, transform and interact with one another within the body. To facilitate direct visualization of such dynamic morphogenesis by individual cells in vivo, we have modified the original Brainbow for Drosophila in which live imaging is practical during much of its development. Our system offers permanent fluorescent labels that reveal fine morphological details of individual cells without requiring dissection or fixation of the samples. It also features a non-invasive means to control the timing of stochastic tricolor cell labeling with a light pulse. We demonstrate applicability of the new system in a variety of settings that could benefit from direct imaging of the developing multicellular organism with single-cell resolution.
Development 04/2013; 140(7):1605-1613. · 6.60 Impact Factor
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ABSTRACT: Type-specific dendritic arborization patterns dictate synaptic connectivity and are fundamental determinants of neuronal function. We exploit the morphological stereotypy and relative simplicity of the Drosophila nervous system to model the diverse dendritic morphologies of individual motor neurons (MNs) to understand underlying principles of synaptic connectivity in a motor circuit. The genetic tractability of Drosophila allows us to label single MNs with green fluorescent protein (GFP) and serially reconstruct identifiable MNs in 3D with confocal microscopy. Our computational approach aims at the robust segmentation of the MN volumes and the simultaneous partitioning into their compartments, namely the soma, axon and dendrites. We use the idea of co-segmentation, where every image along the z-axis (depth) is clustered using information from 'neighboring' depths. As appearance we use a 3D extension of Haar features and for the shape we define an implicit representation of the segmentation domain.
Conference proceedings: ... Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE Engineering in Medicine and Biology Society. Conference 08/2011; 2011:7755-8.
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IEEE 1st International Conference on Computational Advances in Bio and Medical Sciences, ICCABS 2011, Orlando, FL, USA, February 3-5, 2011; 01/2011
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ABSTRACT: Tissue-specific alternative splicing is a key mechanism for generating tissue-specific proteomic diversity in eukaryotes. Splicing regulatory elements (SREs) in pre-mature messenger RNA play a very important role in regulating alternative splicing. In this article, we use mouse RNA-Seq data to determine a positive data set where SREs are over-represented and a reliable negative data set where the same SREs are most likely under-represented for a specific tissue and then employ a powerful discriminative approach to identify SREs. We identified 456 putative splicing enhancers or silencers, of which 221 were predicted to be tissue-specific. Most of our tissue-specific SREs are likely different from constitutive SREs, since only 18% of our exonic splicing enhancers (ESEs) are contained in constitutive RESCUE-ESEs. A relatively small portion (20%) of our SREs is included in tissue-specific SREs in human identified in two recent studies. In the analysis of position distribution of SREs, we found that a dozen of SREs were biased to a specific region. We also identified two very interesting SREs that can function as an enhancer in one tissue but a silencer in another tissue from the same intronic region. These findings provide insight into the mechanism of tissue-specific alternative splicing and give a set of valuable putative SREs for further experimental investigations.
Nucleic Acids Research 12/2010; 38(22):7895-907. · 8.03 Impact Factor
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ABSTRACT: Knowing when and where a given protein is activated within intact animals assists in elucidating its in vivo function. With the use of a genetically encoded A-probe (activation bioprobe), we revealed that Cdc42 guanosine triphosphatase (GTPase) remains inactive within Drosophila embryos during the first two-thirds of embryogenesis. Within the central nervous system where Cdc42 activity first becomes up-regulated, individual neurons display patterns restricted to specific subcellular compartments. At both organismal and cellular levels, Cdc42's endogenous activation patterns in the wild type allow predictions of where loss-of-function phenotypes will emerge in cdc42/cdc42 mutants. Genetic tests support the importance of suppressing endogenous Cdc42 activities until needed. Thus, bioprobe-assisted analysis uncovers how ubiquitously expressed signaling proteins control cellular events through continual regulation of their activities within animals.
Science 07/2009; 324(5932):1338-40. · 31.20 Impact Factor
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ABSTRACT: 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.
PLoS Genetics 05/2009; 5(4):e1000441. · 8.69 Impact Factor
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ABSTRACT: A spinning disk confocal attachment is added to a full-field real-time frequency-domain fluorescence lifetime-resolved imaging microscope (FLIM). This provides confocal 3-D imaging while retaining all the characteristics of the normal 2-D FLIM. The spinning disk arrangement allows us to retain the speed of the normal 2-D full field FLIM while gaining true 3-D resolution. We also introduce the use of wavelet image transformations into the FLIM analysis. Wavelets prove useful for selecting objects according to their morphology, denoising and background subtraction. The performance of the instrument and the analysis routines are tested with quantitative physical samples and examples are presented with complex biological samples.
Journal of Fluorescence 04/2008; 18(5):929-42. · 2.11 Impact Factor
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ABSTRACT: The molecular mechanisms that generate dendrites in the CNS are poorly understood. The diffusible signal molecule Slit and the neuronally expressed receptor Robo mediate growth cone collapse in vivo. However, in cultured neurons, these molecules promote dendritic development. Here we examine the aCC motoneuron, one of the first CNS neurons to generate dendrites in Drosophila. Slit displays a dynamic concentration topography that prefigures aCC dendrogenesis. Genetic deletion of Slit leads to complete loss of aCC dendrites. Robo is cell-autonomously required in aCC motoneurons to develop dendrites. Our results demonstrate that Slit and Robo control the development of dendrites in the embryonic CNS.
Development 12/2007; 134(21):3795-804. · 6.60 Impact Factor
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ABSTRACT: 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.
Molecular and Cellular Biology 10/2006; 26(17):6598-608. · 5.53 Impact Factor
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ABSTRACT: Midline glia are a source of cues for neuronal navigation and differentiation in the Drosophila CNS. Despite their importance, how glia and neurons communicate during the development is not fully understood. Here, we examined dynamic morphology of midline glia and assessed their direct cellular interactions with neurons within the embryonic CNS. Midline glia extend filopodia-like "gliopodia" from the onset of axogenesis through the near completion of embryonic neural development. The most abundant and stable within the commissures, gliopodia frequently contact neurites extending from the neuropil on either side of the midline. Misexpression of Rac1N17 in midline glia not only reduces the number of gliopodia but also shifts the position of neuropils towards the midline. Midline-secreted signaling protein Slit accumulates along the surface of gliopodia. Mutant analysis supports the idea that gliopodia contribute to its presentation on neuronal surfaces at both the commissures and neuropils. We propose that gliopodia extend the range of direct glia-neuron communication during CNS development.
Molecular and Cellular Neuroscience 02/2006; 31(1):123-30. · 3.66 Impact Factor
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ABSTRACT: Visual information received from the three types of photoreceptor neurons (R1-R6, R7 and R8) in the fly compound eyes converges to the external part of the medulla neuropil (M1-M6 layers) in a layer-specific fashion: R7 and R8 axons terminate at the M6 and M3 layers, respectively, whereas lamina neurons (L1-L5) relay R1-R6 to multiple medulla layers (M1-M5). Here, we show that during development, R7 and R8 neurons establish layer-specific projections in two separate stages: during the first stage, R7 and R8 axons sequentially target to the R7- and R8-temporary layers, respectively; and at the second stage, R7 and R8 growth cones progress synchronously to their destined layers. Using a set of mutations that delete different afferent subsets or alter R7 connectivity, we defined the mechanism of layer selection. We observed that R8, R7 and L1-L5 afferents target to their temporary layers independently, suggesting that afferent-target, but not afferent-afferent, interactions dictate the targeting specificity. N-cadherin is required in the first stage for R7 growth cones to reach and remain in the R7-temporary layer. The Ncad gene contains three pairs of alternatively spliced exons and encodes 12 isoforms. However, expressing a single Ncad isoform in Ncad mutant R7s is sufficient to rescue mistargeting phenotypes. Furthermore, Ncad isoforms mediate promiscuous heterophilic interactions in an in vitro cell-aggregation assay. We propose that Ncad isoforms do not form an adhesion code; rather, they provide permissive adhesion between R7 growth cones and their temporary targets.
Development 04/2005; 132(5):953-63. · 6.60 Impact Factor
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ABSTRACT: Like axons, dendrites need guidance for proper orientation and positioning within the brain. Guidance determines synaptic connectivity as well as the strength of transmission. Recent in vivo studies have demonstrated that several cell-surface receptors, previously known as axon guidance molecules, are also responsible for the directed outgrowth of dendrites. Collectively, these studies reveal that the function of guidance molecules in individual neurons and individual processes is diverse and likely to be specifically regulated. Here, these studies are reviewed and emerging issues and implications are discussed.
Trends in Neurosciences 05/2004; 27(4):194-202. · 14.23 Impact Factor
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ABSTRACT: The Rho GTPases Rac1 and Cdc42 have been implicated in the regulation of axon outgrowth and guidance. However, the downstream effector pathways through which these GTPases exert their effects on axon development are not well characterized. Here, we report that axon outgrowth defects within specific subsets of motoneurons expressing constitutively active Drosophila Rac1 largely persist even with the addition of an effector-loop mutation to Rac1 that disrupts its ability to bind to p21-activated kinase (Pak) and other Cdc42/Rac1 interactive-binding (CRIB)-motif effector proteins. While hyperactivation of Pak itself does not lead to axon outgrowth defects as when Rac1 is constitutively activated, live analysis reveals that it can alter filopodial activity within specific subsets of neurons similar to constitutive activation of Cdc42. Moreover, we show that the axon guidance defects induced by constitutive activation of Cdc42 persist even in the absence of Pak activity. Our results suggest that (1) Rac1 controls axon outgrowth through downstream effector pathways distinct from Pak, (2) Cdc42 controls axon guidance through both Pak and other CRIB effectors, and (3) Pak's primary contribution to in vivo axon development is to regulate filopodial dynamics that influence growth cone guidance.
Developmental Biology 11/2003; 262(2):282-93. · 4.07 Impact Factor
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ABSTRACT: Synaptic partner cells recognize one another by utilizing a variety of molecular cues. Prior to neuromuscular synapse formation, Drosophila embryonic muscles extend dynamic actin-based filopodia called "myopodia." In wild-type animals, myopodia are initially extended randomly from the muscle surface but become gradually restricted to the site of motoneuron innervation, a spatial redistribution we call "clustering." Previous experiments with prospero mutant embryos demonstrated that myopodia clustering does not occur in the absence of motoneuron outgrowth into the muscle field. However, whether myopodia clustering is due to a general signal from passing axons or is a result of the specific interactions between synaptic partners remained to be investigated. Here, we have examined the relationship of myopodia to the specific events of synaptic target recognition, the stable adhesion of synaptic partners. We manipulated the embryonic expression of alphaPS2 integrin and Toll, molecules known to affect synaptic development, to specifically alter synaptic targeting on identified muscles. Then, we used a vital single-cell labeling approach to visualize the behavior of myopodia in these animals. We demonstrate a strong positive correlation between myopodia activity and synaptic target recognition. The frequency of myopodia clustering is lowered in cases where synaptic targeting is disrupted. Myopodia clustering seems to result from the adherence of a subset of myopodia to the innervating growth cone while the rest are eliminated. The data suggest that postsynaptic cells play a dynamic role in the process of synaptic target recognition.
Journal of Neurobiology 05/2003; 55(1):31-40. · 3.05 Impact Factor
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ABSTRACT: Neuronal connectivity is established by the axo-dendritic polarity, correct guidance and targeting of neurons. Unlike for axons, the mechanisms responsible for directed outgrowth of dendrites are not well understood. Using single-cell labeling, we describe specific guidance defects in dendrites of identified neurons in frazzled, robo, netrin and commissureless mutant embryos of Drosophila melanogaster. We found that the cell-surface molecules Frazzled and Robo work as guidance molecules not only for axons but also for dendrites as they navigate within the CNS. Furthermore, we report that each neuron showed a cell-autonomous and independent use of guidance molecules.
Nature Neuroscience 04/2003; 6(3):223-30. · 15.53 Impact Factor
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ABSTRACT: Although evidence exists that activation of the Rho family GTPase Cdc42 affects axonal development, its specific roles within a growth cone are not well delineated. To evaluate the model that Cdc42 activation regulates growth cone navigation by promoting filopodial activity, we adopted a live analysis strategy that uses transgenic Drosophila lines in which neurons coexpressed constitutively active Cdc42 (Cdc42(V12)) and membrane-targeted green fluorescent protein. We found that growth cones that displayed pathfinding defects exhibited little change in their filopodial activity, whereas others without pathfinding defects exhibited an similar50% increase in their filopodial activity. Moreover, effector loop mutations that were added to the constitutively active Cdc42 (Cdc42(V12C40) and Cdc42(V12A37)) exerted little influence over filopodial activity caused by Cdc42 activation but suppressed the pathfinding defects of the growth cones. Together, these data suggest that Cdc42 controls filopodial activity in axonal growth cones independently of its effects on their pathfinding.
Journal of Neuroscience 04/2002; 22(5):1794-806. · 7.11 Impact Factor
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ABSTRACT: Understanding the establishment of functional neuronetworks is one of the frontiers of developmental neurobiology. The use of axons and dendrites from Drosophila nervous system as a model allows the identification of molecular mechanisms which give neurons the ability to guide their processes en route to connect precisely with their partners. By focusing on selected Drosophila model systems, we discuss the recent advances in our understanding of the molecular mechanisms regulating guidance, branching and targeting of axons and dendrites required for the establishment of a functional neuronetwork.
Neurosignals 13(1-2):37-49. · 2.11 Impact Factor
