Iris Salecker

MRC National Institute for Medical Research, London, ENG, United Kingdom

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Publications (24)280.07 Total impact

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    ABSTRACT: Visualization of single neurons within their complex environment is a pivotal step towards uncovering the mechanisms that control neural circuit development and function. This chapter provides detailed technical information on how to use Drosophila variants of the mouse Brainbow-2 system, called Flybow, for stochastic labeling of cells with different fluorescent proteins in one sample. We first describe the genetic strategies and the heat shock regime required for induction of recombination events. This is followed by a detailed protocol as to how to prepare samples for imaging. Finally, we provide specifications to facilitate multichannel image acquisition using confocal microscopy.
    Methods in molecular biology (Clifton, N.J.) 01/2014; 1082:57-69.
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    ABSTRACT: Tightly controlled DNA replication and RNA transcription are essential for differentiation and tissue growth in multicellular organisms. Histone chaperones, including the FACT (facilitates chromatin transcription) complex, are central for these processes and act by mediating DNA access through nucleosome reorganisation. However, their roles in vertebrate organogenesis are poorly understood. Here, we report the identification of zebrafish mutants for the gene encoding Structure specific recognition protein 1a (Ssrp1a), which, together with Spt16, forms the FACT heterodimer. Focussing on the liver and eye, we show that zygotic Ssrp1a is essential for proliferation and differentiation during organogenesis. Specifically, gene expression indicative of progressive organ differentiation is disrupted and RNA transcription is globally reduced. Ssrp1a-deficient embryos exhibit DNA synthesis defects and prolonged S phase, uncovering a role distinct from that of Spt16, which promotes G1 phase progression. Gene deletion/replacement experiments in Drosophila show that Ssrp1b, Ssrp1a and N-terminal Ssrp1a, equivalent to the yeast homologue Pob3, can substitute Drosophila Ssrp function. These data suggest that (1) Ssrp1b does not compensate for Ssrp1a loss in the zebrafish embryo, probably owing to insufficient expression levels, and (2) despite fundamental structural differences, the mechanisms mediating DNA accessibility by FACT are conserved between yeast and metazoans. We propose that the essential functions of Ssrp1a in DNA replication and gene transcription, together with its dynamic spatiotemporal expression, ensure organ-specific differentiation and proportional growth, which are crucial for the forming embryo.
    Development 03/2013; · 6.60 Impact Factor
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    ABSTRACT: During the development of locomotion circuits it is essential that motoneurons with distinct subtype identities select the correct trajectories and target muscles. In vertebrates, the generation of motoneurons and myelinating glia depends on Olig2, one of the five Olig family bHLH transcription factors. We investigated the so far unknown function of the single Drosophila homolog Oli. Combining behavioral and genetic approaches, we demonstrate that oli is not required for gliogenesis, but plays pivotal roles in regulating larval and adult locomotion, and axon pathfinding and targeting of embryonic motoneurons. In the embryonic nervous system, Oli is primarily expressed in postmitotic progeny, and in particular, in distinct ventral motoneuron subtypes. oli mediates axonal trajectory selection of these motoneurons within the ventral nerve cord and targeting to specific muscles. Genetic interaction assays suggest that oli acts as part of a conserved transcription factor ensemble including Lim3, Islet and Hb9. Moreover, oli is expressed in postembryonic leg-innervating motoneuron lineages and required in glutamatergic neurons for walking. Finally, over-expression of vertebrate Olig2 partially rescues the walking defects of oli-deficient flies. Thus, our findings reveal a remarkably conserved role of Drosophila Oli and vertebrate family members in regulating motoneuron development, while the steps that require their function differ in detail.
    Developmental Biology 07/2012; 369(2):261-76. · 3.87 Impact Factor
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    ABSTRACT: A shared feature of many neural circuits is their organization into synaptic layers. However, the mechanisms that direct neurites to distinct layers remain poorly understood. We identified a central role for Netrins and their receptor Frazzled in mediating layer-specific axon targeting in the Drosophila visual system. Frazzled is expressed and cell autonomously required in R8 photoreceptors for directing their axons to the medulla-neuropil layer M3. Netrin-B is specifically localized in this layer owing to axonal release by lamina neurons L3 and capture by target neuron-associated Frazzled. Ligand expression in L3 is sufficient to rescue R8 axon-targeting defects of Netrin mutants. R8 axons target normally despite replacement of diffusible Netrin-B by membrane-tethered ligands. Finally, Netrin localization is instructive because expression in ectopic layers can retarget R8 axons. We propose that provision of localized chemoattractants by intermediate target neurons represents a highly precise strategy to direct axons to a positionally defined layer.
    Neuron 07/2012; 75(1):80-93. · 15.77 Impact Factor
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    ABSTRACT: To facilitate studies of neural network architecture and formation, we generated three Drosophila melanogaster variants of the mouse Brainbow-2 system, called Flybow. Sequences encoding different membrane-tethered fluorescent proteins were arranged in pairs within cassettes flanked by recombination sites. Flybow combines the Gal4-upstream activating sequence binary system to regulate transgene expression and an inducible modified Flp-FRT system to drive inversions and excisions of cassettes. This provides spatial and temporal control over the stochastic expression of one of two or four reporters within one sample. Using the visual system, the embryonic nervous system and the wing imaginal disc, we show that Flybow in conjunction with specific Gal4 drivers can be used to visualize cell morphology with high resolution. Finally, we demonstrate that this labeling approach is compatible with available Flp-FRT-based techniques, such as mosaic analysis with a repressible cell marker; this could further support the genetic analysis of neural circuit assembly and function.
    Nature Methods 02/2011; 8(3):260-6. · 23.57 Impact Factor
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    ABSTRACT: The ability of vertebrates and insects to perceive and process information about the visual world is mediated by neural circuits, which share a strikingly conserved architecture of reiterated columnar and layered synaptic units. Recent genetic approaches conferring single-cell resolution have enabled major advances in our understanding of the cellular and molecular strategies that orchestrate visual circuit assembly in Drosophila. Photoreceptor axon targeting relies on a sequence of interdependent developmental steps to achieve temporal coordination with the formation and maturation of partner neurons. Distinct targeting events depend on anterograde and autocrine signaling, neuron-glia interactions, axon tiling and the timely expression of homophilic cell surface molecules. These mediate local adhesive or repulsive interactions of photoreceptor axons with each other and with target neurons.
    Current opinion in neurobiology 02/2011; 21(1):76-84. · 7.21 Impact Factor
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    ABSTRACT: Anaplastic lymphoma kinase (Alk) has been proposed to regulate neuronal development based on its expression pattern in vertebrates and invertebrates; however, its function in vivo is unknown. We demonstrate that Alk and its ligand Jelly belly (Jeb) play a central role as an anterograde signaling pathway mediating neuronal circuit assembly in the Drosophila visual system. Alk is expressed and required in target neurons in the optic lobe, whereas Jeb is primarily generated by photoreceptor axons and functions in the eye to control target selection of R1-R6 axons in the lamina and R8 axons in the medulla. Impaired Jeb/Alk function affects layer-specific expression of three cell-adhesion molecules, Dumbfounded/Kirre, Roughest/IrreC, and Flamingo, in the medulla. Moreover, loss of flamingo in target neurons causes some R8-axon targeting errors observed in Jeb and Alk mosaic animals. Together, these findings suggest that Jeb/Alk signaling helps R-cell axons to shape their environment for target recognition.
    Cell 04/2007; 128(5):961-75. · 31.96 Impact Factor
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    Carole Chotard, Iris Salecker
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    ABSTRACT: In the developing nervous system, building a functional neuronal network relies on coordinating the formation, specification and survival to diverse neuronal and glial cell subtypes. The establishment of neuronal connections further depends on sequential neuron-neuron and neuron-glia interactions that regulate cell-migration patterns and axon guidance. The visual system of Drosophila has a highly regular, retinotopic organization into reiterated interconnected synaptic circuits. It is therefore an excellent invertebrate model to investigate basic cellular strategies and molecular determinants regulating the different developmental processes that lead to network formation. Studies in the visual system have provided important insights into the mechanisms by which photoreceptor axons connect with their synaptic partners within the optic lobe. In this review, we highlight that this system is also well suited for uncovering general principles that underlie glial cell biology. We describe the glial cell subtypes in the visual system and discuss recent findings about their development and migration. Finally, we outline the pivotal roles of glial cells in mediating neural circuit assembly, boundary formation, neural proliferation and survival, as well as synaptic function.
    Neuron Glia Biology 03/2007; 3(1):17-25. · 1.34 Impact Factor
  • Iris Salecker
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    ABSTRACT: How can RNA processing direct specific aspects of nervous system development? In this issue of Neuron, Edenfeld et al. identified a novel function for two regulators of mRNA splicing in Drosophila: peripheral glial cells require Crooked neck (Crn) and Held out wings (HOW) to mediate migration and ensheathment of peripheral axons.
    Neuron 01/2007; 52(6):933-5. · 15.77 Impact Factor
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    EMBO Reports 07/2006; 7(6):585-9. · 7.19 Impact Factor
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    Carole Chotard, Wendy Leung, Iris Salecker
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    ABSTRACT: The transcription factors Glial cells missing (Gcm) and Gcm2 are known to play a crucial role in promoting glial-cell differentiation during Drosophila embryogenesis. Our findings reveal a central function for gcm genes in regulating neuronal development in the postembryonic visual system. We demonstrate that Gcm and Gcm2 are expressed in both glial and neuronal precursors within the optic lobe. Removal of gcm and gcm2 function shows that the two genes act redundantly and are required for the formation of a subset of glial cells. They also cell-autonomously control the differentiation and proliferation of specific neurons. We show that the transcriptional regulator Dachshund acts downstream of gcm genes and is required to make lamina precursor cells and lamina neurons competent for neuronal differentiation through regulation of epidermal growth factor receptor levels. Our findings further suggest that gcm genes regulate neurogenesis through collaboration with the Hedgehog-signaling pathway.
    Neuron 11/2005; 48(2):237-51. · 15.77 Impact Factor
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    Carole Chotard, Iris Salecker
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    ABSTRACT: In the developing nervous system, growth cones follow specific trajectories to reach their target area and ultimately connect with their correct postsynaptic partners. This review focuses on studies in both Drosophila and vertebrates to highlight that mutual interactions between neurons and glia are essential in forming specific neuronal connections. Glia signal to neurons to direct pathfinding and targeting of axons, as well as to stabilize and refine axonal branches within the target area. Equally, neurons provide crucial information to glia, supporting their migration and correct positioning.
    Trends in Neurosciences 12/2004; 27(11):655-61. · 13.58 Impact Factor
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    ABSTRACT: Drosophila photoreceptor neurons (R cells) project their axons to one of two layers in the optic lobe, the lamina or the medulla. The transcription factor Runt (Run) is normally expressed in the two inner R cells (R7 and R8) that project their axons to the medulla. Here we examine the relationship between Run and the ubiquitously expressed nuclear protein Brakeless (Bks), which has previously been shown to be important for axon termination in the lamina. We report that Bks represses Run in two of the outer R cells: R2 and R5. Expression of Run in R2 and R5 causes axonal mistargeting of all six outer R cells (R1-R6) to the inappropriate layer, without altering expression of cell-specific developmental markers.
    Nature Neuroscience 09/2002; 5(8):746-50. · 15.25 Impact Factor
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    ABSTRACT: In the fly visual system, each class of photoreceptor neurons (R cells) projects to a different synaptic layer in the brain. R1-R6 axons terminate in the lamina, while R7 and R8 axons pass through the lamina and stop in the medulla. As R cell axons enter the lamina, they encounter both glial cells and neurons. The cellular requirement for R1-R6 targeting was determined using loss-of-function mutations affecting different cell types in the lamina. nonstop (encoding a ubiquitin-specific protease) is required for glial cell development and hedgehog for neuronal development. Removal of glial cells but not neurons disrupts R1-R6 targeting. We propose that glial cells provide the initial stop signal promoting growth cone termination in the lamina. These findings uncover a novel function for neuron-glial interactions in regulating target specificity.
    Neuron 02/2001; 29(1):99-113. · 15.77 Impact Factor
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    ABSTRACT: Neurons dissociated from the brain of embryonic cockroaches (Periplaneta americana) can be maintained in culture for several weeks. The survival as well as the progressive organization of the neurons into a complex network was studied during a 5-week period under different culture conditions. About 10% of the dissociated cells adhered to the culture dish. This figure remained constant throughout the culture. The cell diameter ranged from 10 to 20 microns and did not change significantly over time in culture. Whereas only a few cells exhibited neurites at the start of the culture, the number of cells exhibiting neurites increased to reach about 99% after 2 weeks. The different cells were then connected to each other, forming a network, which became more and more complex. The number of cells per cluster as well as the length and the diameter of the "connectives" that linked the different clusters were found to increase with time. The morphology of individual neurons within the network was visualized after intracellular injection of biocytin. Labeling with antibodies raised against serotonin or GABA indicated that neurons were able to differentiate and to acquire specific neurotransmitter fates. The serotonergic phenotype was found to appear progressively throughout the culture, in parallel with the formation of the network. Cell density, addition of fetal calf serum, and ecdysone were shown to influence the development of the network.
    Cell and Tissue Research 02/2000; 299(1):129-43. · 3.68 Impact Factor
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    ABSTRACT: Different Drosophila photoreceptors (R cells) connect to neurons in different optic lobe layers. R1-R6 axons project to the lamina; R7 and R8 axons project to separate layers of the medulla. We show a receptor tyrosine phosphatase, PTP69D, is required for lamina target specificity. In Ptp69D mutants, R1-R6 project through the lamina, terminating in the medulla. Genetic mosaics, transgene rescue, and immunolocalization indicate PTP69D functions in R1-R6 growth cones. PTP69D overexpression in R7 and R8 does not respecify their connections, suggesting PTP69D acts in combination with other factors to determine target specificity. Structure-function analysis indicates the extracellular fibronectin type III domains and intracellular phosphatase activity are required for targeting. We propose PTP69D promotes R1-R6 targeting in response to extracellular signals by dephosphorylating substrate(s) in R1-R6 growth cones.
    Neuron 05/1999; 22(4):707-17. · 15.77 Impact Factor
  • Cell 12/1998; 95(5):587-90. · 31.96 Impact Factor
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    ABSTRACT: Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder. Disease alleles contain a trinucleotide repeat expansion of variable length, which encodes polyglutamine tracts near the amino terminus of the HD protein, huntingtin. Polyglutamine-expanded huntingtin, but not normal huntingtin, forms nuclear inclusions. We describe a Drosophila model for HD. Amino-terminal fragments of human huntingtin containing tracts of 2, 75, and 120 glutamine residues were expressed in photoreceptor neurons in the compound eye. As in human neurons, polyglutamine-expanded huntingtin induced neuronal degeneration. The age of onset and severity of neuronal degeneration correlated with repeat length, and nuclear localization of huntingtin presaged neuronal degeneration. In contrast to other cell death paradigms in Drosophila, coexpression of the viral antiapoptotic protein, P35, did not rescue the cell death phenotype induced by polyglutamine-expanded huntingtin.
    Neuron 10/1998; 21(3):633-42. · 15.77 Impact Factor
  • Cell. 01/1998; 95(5):587-590.
  • I Salecker, J Boeckh
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    ABSTRACT: The embryonic development of the hemimetabolous insect Periplaneta americana requires approximately 31 days. Deafferentation experiments were used to investigate the role of ingrowing receptor axons during embryogenesis, specifically their influence 1) on the subdivision of the antennal lobe neuropil into glomeruli, 2) on the morphology and number of glial cells, and 3) on the arborization pattern of central neurons. The flagellum of one antenna was removed from embryos at different developmental stages starting with day 10. Subsequently, they were raised in culture until a total age of 26 days. At day 10, the deutocerebrum has received only a very small number (ca. 0.4%) of antennal receptor axons; deafferentation at this stage allowed us to deprive the deutocerebrum of approximately 99% of its normal antennal input. Deafferentation has marked effects on the organization of the antennal lobe neuropil. The deafferented lobe is reduced in volume compared to the control side; the characteristic glomeruli are missing. During normal development glomeruli are formed between day 19 and 22, first in dorsal and then in ventral antennal lobe regions. By removing the antenna before day 20, their formation is disturbed in all parts of the antennal lobe. If deafferentation is performed after stage 20, glomeruli persist in dorsal regions, but are missing in ventral regions. On day 24 or later, glomeruli in both dorsal and ventral regions are unaffected by deafferentation. Glial cells continue to extend fine processes into the neuropil in the absence of ingrowing receptor axons. The number of glial cells is reduced compared to control lobes. Multiglomerular local interneurons and other gamma-amino butyric acid-immunoreactive neurons, as well as projection neurons, fail to develop glomerular arborization patterns in antennal lobes deprived of sensory axons.
    The Journal of Comparative Neurology 07/1996; 370(2):262-79. · 3.66 Impact Factor

Publication Stats

1k Citations
3 Downloads
969 Views
280.07 Total Impact Points

Institutions

  • 2004–2014
    • MRC National Institute for Medical Research
      • Division of Molecular Neurobiology
      London, ENG, United Kingdom
  • 2011
    • Medical Research Council (UK)
      Londinium, England, United Kingdom
  • 1990–2001
    • Universität Regensburg
      • Institut für Zoologie
      Regensburg, Bavaria, Germany
  • 1996–1999
    • Howard Hughes Medical Institute
      Maryland, United States
  • 1998
    • University of California, Los Angeles
      • Department of Neurology
      Los Angeles, CA, United States