Through the collaborative efforts of geneticists, histologists, anatomists, computer scientists, and software engineers, we are creating a database of GAL4 and LexA expression patterns in adults and third instar larvae, single-neuron morphologies, and developmental lineages. The Fly Light Team is assembling models of fly brain anatomy and development by applying unique algorithms to a growing collection of confocal fluorescence imagery.
The Drosophila brain is a perplexing society of tens-of-thousands of interwoven cells in perpetual action, and the understanding of its organization is much in need. The Fly Light team is approaching this goal with systematic experimentation at an ambitious scale.
At the heart of the Fly Light project are large scale data production processes that enable us to highlight and study cells at various points in the life of the fruit fly. We have developed and implemented a number of novel methods for brain dissection, optical clearing of tissue, labeling and automated microscopy. We have also developed computational tools for stitching together multi-stack images, aligning brain images into a common framework, annotating expression patterns, separating individual neurons from multicolor images containing several neurons, and for displaying and manipulating image stacks and other anatomical data.
Ultimately, we strive to turn pictures into interactive virtual brains that will help us discern roles for cell morphology, lineage, and connectivity in the fly’s life. Our goal is to enable a genuine worldwide scientific collaboration for understanding the structure, development and function of the fruit fly nervous system.
- Annotate several thousand GAL4 and LexA driver lines that label different sets of neurons —in the adult Drosophila and third instar larva.
- Establish a highly automated, robust pipeline that produces low-resolution confocal images of fruit fly strains at several points in the life of the fly
- Assemble a complete catalog of single neuron morphologies of the Drosophila central nervous system (larval and adult) by random labeling of neurons via DNA excision
- Assemble complete lineage maps for each stem cell in the Drosophila central nervous system
- Devise algorithms to enable comparisons of transgene expression, neuron morphology, and lineage across tens of thousands of organisms
- Enable collaborative annotations and comparisons of imagery by experts
- Build searchable databases of light-level observations of fruit fly brains that enable integration (to be accomplished in collaboration with our colleagues in the Fly Olympiad and Fly EM Project Teams) with behavioral assay, electron microscopy, and other data from laboratories around the world
In an attempt to define subsets of neurons, as a first step we screened 7,200 GAL4 driver lines, constructed by the Rubin Lab, that each targets a subset of neurons in different brain areas. This has allowed us to triage those lines to a set of about 4,000 lines that each express in roughly 10 and 300 cells in the adult CNS and, in aggregate, appear to have several fold coverage of all neuronal cell types. In addition, we are currently in the process of adding about 4,000 GAL4 and LexA lines generated by the Dickson Lab, for additional exploration of new neural cell types in the adult and larval brain.
In collaboration with the Rubin (adult) and Truman (third instar larva) labs, the Fly Light team is generating a comprehensive set of single neuron morphologies using a simple stochastic labeling approach developed in the Rubin lab. Analysis and interpretation of these data utilizes neuron separation and tracing software developed in the Janelia laboratories of Eugene Myers and Hanchuan Peng and an annotation workbench developed by a team of software engineers led by Sean Murphy.
Unique transgenic techniques allow us also to infer distributions of molecules, from the time each cell was born, and the progenitor it came from. Our goal is to apply these tools at a scale commensurate with the complexity of the brain. In collaboration with the laboratories of Tzumin Lee and James Truman here at Janelia Farm Research Campus, we are trying to sequence lineages and follow neuron morphology through development.
We envision that these activities will be synergistic in gaining an insight into how the brain is constructed and how it functions. Once we have neuron-resolution maps of cell shape and lineage for the entire fly nervous system we will produce, integrate, and disseminate information about connectivity, neurotransmitter distributions, and cell activity.
Although transgenic tools will give us a rich catalogue of images, we have to develop unique software to distill these images into meaningful subsets. We (see Myers and Peng lab pages for details) are designing and applying software tools that can extract the shapes of myriad neurons and reassemble them into digital brains of our design. Thus, we can place neurons from different brains onto a standard map and use computer programs to explore their circuits forming. By assessing juxtapositions of tens of thousands of neurons, we will be able to propose testable cellular networks.
In an effort led by Sean Murphy, the Fly Light Team Project is developing a workstation and annotation software package that enables researchers to review imaged data, run algorithms, display results in 3D, and add their knowledge to our database. With our new software tools, researches will be able to trace and model neuron shapes, overlay data, and add notes to images, parts of neurons, and potential cell-cell connections. We plan to test and develop the tool at Janelia Farm Research Campus before offering it to the community.
The Scientific Project Manager manages the Fly Light’s team of highly skilled research technicians and research specialists. Key strategic decisions are made by the Fly Light Steering Committee that consists of the Scientific Project Manager, Managers, Directors, and Lab Heads whose expertise compliment our core mission. Janelia Shared Resources and Scientific Computing contribute significantly to our project.
- Imaged more than 100,000 confocal stacks of adult and larval nervous systems.
- Built a large scale process for preparing samples and capturing images of individual neurons.
- Prepared more than 100,000 fly brains to support the identification of individual neuronal lineages and the sequencing of five antennal lobe neuronal lineages.
- Developed an automatic microscopy system that reduces complicated setups from hours to minutes.
The overall goal of the Fly Light Project Team is to generate genetic reagents and anatomical data that will be of widespread utility to the Drosophila research community. For this reason, we intend to make the reagents and data we generate available in a timely manner.
Our data release policies are intermediate between what is generally accepted practice for individual labs and that expected of large infrastructure projects like the genome project. More specifically, for aspects of the project where a significant fraction (more than one-third) of the effort is supplied by members of research groups (who are not funded by the project budget) then those individuals will be granted control over the use of the results for a period of time sufficient to generate publications needed for them to get credit for their work. In most cases, we envision this period to be approximately one year.
A GAL4-Driver Line Resource for Drosophila Neurobiology.Cell Reports 2012
A. Jenett, G. M. Rubin, T. B. Ngo, D. Shepherd, C. Murphy, H. Dionne, B. D. Pfeiffer, A. Cavallaro, D. Hall, J. Jeter, N. Iyer, D. Fetter, J. H. Hausenfluck, H. Peng, E. T. Trautman, R. R. Svirskas, E. W. Myers, Z. R. Iwinski, Y. Aso, G. M. Depasquale, A. Enos, P. Hulamm, S. Lam, H. Li, T. R. Laverty, F. Long, L. Qu, S. D. Murphy, K. Rokicki, T. Safford, K. Shaw, J. H. Simpson, A. Sowell, S. Tae, Y. Yu, and C. T. Zugates Cell Reports, 2:991-1001 (2012)
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.
synapse was substantially elevated, at the endocytic zone there was no enhanced polymerization activity. We conclude that actin subserves spatially diverse, independently regulated processes throughout spines. Perisynaptic actin forms a uniquely dynamic structure well suited for direct, active regulation of the synapse.
For the overall strategy and methods used to produce the GAL4 lines:
Pfeiffer, B.D., Jenett, A., Hammonds, A.S., Ngo, T.T., Misra, S., Murphy, C., Scully, A., Carlson, J.W., Wan, K.H., Laverty, T.R., Mungall, C., Svirskas, R., Kadonaga, J.T., Doe, C.Q., Eisen, M.B., Celniker, S.E., Rubin, G.M. (2008). Tools for neuroanatomy and neurogenetics in Drosophila. Proc. Natl. Acad. Sci. USA 105, 9715-9720. http://www.pnas.org/content/105/28/9715.full.pdf+html synapse was substantially elevated, at the endocytic zone there was no enhanced polymerization activity. We conclude that actin subserves spatially diverse, independently regulated processes throughout spines. Perisynaptic actin forms a uniquely dynamic structure well suited for direct, active regulation of the synapse.
For data on expression in the embryo:
Manning, L., Purice, M.D., Roberts, J., Pollard, J.L., Bennett, A.L., Kroll, J.R., Dyukareva, A.V., Doan, P.N., Lupton, J.R., Strader, M.E., Tanner, S., Bauer, D., Wilbur, A., Tran, K.D., Laverty, T.R., Pearson, J.C., Crews, S.T., Rubin, G.M., and Doe, C.Q. (2012) Annotated embryonic CNS expression patterns of 5000 GMR GAL4 lines: a resource for manipulating gene expression and analyzing cis-regulatory motifs. Cell Reports (2012) Doi: 10.1016/j.celrep.2012.09.009 http://www.cell.com/cell-reports/fulltext/S2211-1247(12)00290-2 synapse was substantially elevated, at the endocytic zone there was no enhanced polymerization activity. We conclude that actin subserves spatially diverse, independently regulated processes throughout spines. Perisynaptic actin forms a uniquely dynamic structure well suited for direct, active regulation of the synapse.
For data on expression in imaginal discs:
Jory, A., Estella, C., Giorgianni, M.W., Slattery, M., Laverty, T.R., Rubin, G.M., and Mann, R.S. (2012) A survey of 6300 genomic fragments for cis-regulatory activity in the imaginal discs of Drosophila melanogaster. Cell Reports (2012) Doi: 10.1016/j.celrep.2012.09.010 http://www.cell.com/cell-reports/fulltext/S2211-1247(12)00291-4 synapse was substantially elevated, at the endocytic zone there was no enhanced polymerization activity. We conclude that actin subserves spatially diverse, independently regulated processes throughout spines. Perisynaptic actin forms a uniquely dynamic structure well suited for direct, active regulation of the synapse.
For data on expression in the larval nervous system:
Li, H.-H., Kroll, J.R., Lennox, S., Ogundeyi, O., Jeter, J., Depasquale, G., and Truman, J.W. (2013) A GAL4 driver resource for developmental and behavioral studies on the larval CNS of Drosophila. Cell Reports (submitted).
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