New Methods for Characterizing the Complex Neural Circuitry of the Adult Drosophila Brain

141 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2007.Recently improved genetic mosaic methods permit unprecedented resolution for study of neural circuitry formation in Drosophila melanogaster. This study utilizes new mosaic methods to investigate conserved mechanisms that underlie the development of adult-specific neurons. In addition to providing several novel insights about the genetics of neural circuitry development, this study also provides a framework for increased resolution in examination of lineage and morphology in the Drosophila brain. First, mosaic analysis with a repressible cell marker (MARCM) reveals stereotyped lineage and morphogenesis in the ellipsoid body (EB), a center of locomotor regulation, resembling development of the mushroom bodies (MB's), sites involved heavily in olfactory learning. In addition, EB neuron birth and axon development are widely separated in time making them ideal model neurons for further studies. Second, comparative genetic mosaic analysis of EB and dorsal cluster (DC) neurons suggests that TGF-beta signaling is widely required for maturation and morphogenesis of post-mitotic adult-specific neurons. Third, combined application of MARCM with a newly developed technique, called "Flip-out" MARCM, reveals that the Drosophila Down syndrome cell adhesion molecule (Dscam) is required for suppressing ectopic bifurcation MB axons and for promoting divergent guidance of growth cones. Knockout of Dscam in EB neurons reveals a general role in formation and guidance of axonal branches. Finally, combining mosaic methods to study both cellular genetic requirements and phenotypes suggest a non-autonomous requirement for the atypical receptor tyrosine kinase (RTK), Linotte/Derailed (Lio/Drl), in patterning MB axon lobe architecture. In the development of MB circuitry, Lio/Drl may act to counter Wnt5 signaling, which appears to be generally required for axon extension across the brain midline.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD

Baboon/dSmad2 TGF-beta signaling is required during late larval stage for development of adult-specific neurons

The intermingling of larval functional neurons with adult-specific neurons during metamorphosis contributes to the development of the adult Drosophila brain. To better understand this process, we characterized the development of a dorsal cluster (DC) of Atonal-positive neurons that are born at early larval stages but do not undergo extensive morphogenesis until pupal formation. We found that Baboon(Babo)/dSmad2-mediated TGF-beta signaling, known to be essential for remodeling of larval functional neurons, is also indispensable for proper morphogenesis of these adult-specific neurons. Mosaic analysis reveals slowed development of mutant DC neurons, as evidenced by delays in both neuronal morphogenesis and atonal expression. We observe similar phenomena in other adult-specific neurons. We further demonstrate that Babo/dSmad2 operates autonomously in individual neurons and specifically during the late larval stage. Our results suggest that Babo/dSmad2 signaling prior to metamorphosis may be widely required to prepare neurons for the dynamic environment present during metamorphosis

Baboon/dSmad2 TGF-β signaling is required during late larval stage for development of adult-specific neurons

The intermingling of larval functional neurons with adult-specific neurons during metamorphosis contributes to the development of the adult Drosophila brain. To better understand this process, we characterized the development of a dorsal cluster (DC) of Atonal-positive neurons that are born at early larval stages but do not undergo extensive morphogenesis until pupal formation. We found that Baboon(Babo)/dSmad2-mediated TGF-β signaling, known to be essential for remodeling of larval functional neurons, is also indispensable for proper morphogenesis of these adult-specific neurons. Mosaic analysis reveals slowed development of mutant DC neurons, as evidenced by delays in both neuronal morphogenesis and atonal expression. We observe similar phenomena in other adult-specific neurons. We further demonstrate that Babo/dSmad2 operates autonomously in individual neurons and specifically during the late larval stage. Our results suggest that Babo/dSmad2 signaling prior to metamorphosis may be widely required to prepare neurons for the dynamic environment present during metamorphosis

A GAL4-driver line resource for Drosophila neurobiology.

We established a collection of 7,000 transgenic lines of Drosophila melanogaster. Expression of GAL4 in each line is controlled by a different, defined fragment of genomic DNA that serves as a transcriptional enhancer. We used confocal microscopy of dissected nervous systems to determine the expression patterns driven by each fragment in the adult brain and ventral nerve cord. We present image data on 6,650 lines. Using both manual and machine-assisted annotation, we describe the expression patterns in the most useful lines. We illustrate the utility of these data for identifying novel neuronal cell types, revealing brain asymmetry, and describing the nature and extent of neuronal shape stereotypy. The GAL4 lines allow expression of exogenous genes in distinct, small subsets of the adult nervous system. The set of DNA fragments, each driving a documented expression pattern, will facilitate the generation of additional constructs for manipulating neuronal function

A GAL4-Driver Line Resource for Drosophila Neurobiology

We established a collection of 7,000 transgenic lines of Drosophila melanogaster. Expression of GAL4 in each line is controlled by a different, defined fragment of genomic DNA that serves as a transcriptional enhancer. We used confocal microscopy of dissected nervous systems to determine the expression patterns driven by each fragment in the adult brain and ventral nerve cord. We present image data on 6,650 lines. Using both manual and machine-assisted annotation, we describe the expression patterns in the most useful lines. We illustrate the utility of these data for identifying novel neuronal cell types, revealing brain asymmetry, and describing the nature and extent of neuronal shape stereotypy. The GAL4 lines allow expression of exogenous genes in distinct, small subsets of the adult nervous system. The set of DNA fragments, each driving a documented expression pattern, will facilitate the generation of additional constructs for manipulating neuronal function

Bioresponsive Small Molecule Polyamines as Noncytotoxic Alternative to Polyethylenimine

Nonviral gene therapy continues to require novel synthetic vectors to deliver therapeutic nucleic acids effectively and safely. The majority of synthetic nonviral vectors employed in clinical trials to date have been cationic liposomes; however, cationic polymers are attracting increasing attention. One of the few cationic polymers to enter clinical trials has been polyethylenimine (PEI); however, doubts remain over its cytotoxicity, and in addition it displays lower levels of transfection than viral systems. Herein, we report on the development of a series of small molecule analogues of PEI that are bioresponsive to the presence of pDNA, forming poly(disulfide)s that are capable of efficacious transfection with no associated toxicity. The most effective small molecule developed, a cyclic disulfide based upon a spermine backbone, is shown to form very well-defined polyplexes (100−200 nm in diameter) that mediate murine lung transfection in vivo to within an order of magnitude of in vivo jetPEI, and at the same time display a much improved cytotoxicity profile

Fly Light Split-GAL4 Driver Collection

<p>The data presented on this site are the work of the <a href="http://janelia.org/team-project/fly-light" target="_blank">Janelia FlyLight Project Team</a> and the laboratories of <a href="http://www.janelia.org/lab/rubin-lab" target="_blank">Gerald M. Rubin</a>. </p><p>The split-GAL4 lines can be requested from the Janelia fly facility by performing a search and adding the desired lines to your cart. You will then be able to use the FlyBank website to tell us where to send them. For additional help ordering lines, please contact us at <a href="mailto:flybank.janelia.org">flybank.janelia.org</a></p><p>In publications, please attribute the data presented on this site to one of the following papers, as follows: <br><br>For the overall strategy and methods used to produce the split-GAL4 lines for the mushroom body neurons: <br>Aso, Y., Hattori, D., Yu, Y., Johnston, R. M., Iyer, N., Ngo, T. B., Dionne, H., Abbott, L. F., Axel, R., Tanimoto, H. & Rubin, G. M. . The neuronal architecture of the mushroom body provides a logic for associative learning. <a href="http://elifesciences.org/content/3/e04577" target="_blank">eLife (2014) 3:e04577</a><br><br>For split-GAL4 lines for the Lobula Columnar (LC) visual projection neurons:<br>Wu, M., Nern, A., Williamson, W. R., Morimoto, M. M., Reiser, M. B., Card, G. M. & Rubin, G. M. Visual projection neurons in the Drosophila lobula link feature detection to distinct behavioral programs. under review<br><br>For refinement of the split-GAL4 vectors and methodology: <br>Pfeiffer, B. D., Ngo, T. T., Hibbard, K. L., Murphy, C., Jenett, A., Truman, J. W. & Rubin, G. M. Refinement of tools for targeted gene expression in Drosophila. <a href="http://www.genetics.org/content/186/2/735.long" target="_blank">Genetics (2010) 186: 735-55</a>. <br><br>For Multicolor Flp-out (MCFO) technique and single cell labeling:<br>Nern, A., Pfeiffer, B.D., and Rubin, G.M. Optimized tools for multicolor stochastic labeling reveal diverse stereotyped cell arrangements in the fly visual system. <a href="http://www.pnas.org/content/112/22/E2967.long" target="_blank">Proc Natl Acad Sci USA (2015) 112: E2967-2976</a>. <br><br>Split-GAL4 lines were designed based on the expression patterns of GAL4 driver lines in the adult nervous system: <br>The Janelia collection of lines is described in Jenett, A., Rubin, G.M., Ngo, T.-T. B., Shepherd, D., Murphy, C., Dionne, H., Pfeiffer, B.D., Cavallaro, A., Hall, D., Jeter, J., Iyer, N., Fetter, D., Hausenfluck, J.H., Peng, H., Trautman, E., Svirskas, R., Myers, G.W., Iwinski, Z.R., Aso, Y., DePasquale, G.M., Enos, A., Hulamm, P., Lam, S.C.B., Li, H-H., Laverty, T., Long, F., Qu, L., Murphy, S.D., Rokicki, K., Safford, T., Shaw, K., Simpson, J.H., Sowell, A., Tae, S., Yu, Y., Zugates, C.T. A GAL4-Driver Line Resource for Drosophila Neurobiology. <a href="http://www.cell.com/cell-reports/fulltext/S2211-1247(12)00292-6" target="_blank">Cell Reports (2012) 2: 991-1001</a> <br><br>The VT collection of lines is described in Kvon, E.Z., Kazmar, T., Stampfel, G., Yanez-Cuna, J.O., Pagani, M., Schernhuber, K., Dickson, B.J., and Stark, A. Genome-scale functional characterization of Drosophila developmental enhancers in vivo. <a href="http://www.nature.com/nature/journal/vaop/ncurrent/full/nature13395.html" target="_blank">Nature (2014) 512: 91-95</a> and Barry J. Dickson, unpublished data. <br><br>For opening and viewing h5j and LSM stacks:<br>Use <a href="http://fiji.sc/" target="_blank">Fiji</a> (<a href="http://fiji.sc/" target="_blank">http://fiji.sc</a>). Fiji has a built-in plugin (H5J_Loader_Plugin-1.0.4) for opening stack in h5j format, a "visually lossless" compression format.</p