Page 362 - 53rd Annual Drosophila Research Conference

Poster Full Abstracts - Techniques and Functional Genomics
Poster board number is above title. The first author is the presenter
360
Developing a quantitative, cellular resolution morphology and gene expression atlas for Drosophila embryogenesis: A digital 'Campos-Ortega and
Hartenstein’. Soile V E Keränen1 , Jonathan T Barron2 , Pablo Arbelaez2, Jitendra Malik 2, Mark D Biggin1 , David W Knowles1 . 1) Lawrence Berkeley Natl
Lab, Berkeley, CA; 2) Electrical Engineering and Computer Science, UC Berkeley, Berkeley, CA.
Animals comprise of dynamic 3D arrays of cells which differ from each other in histological type, shape, size, location, and other characteristics. We are
creating a quantitative, digital, cellular resolution map of morphology and gene expression for multiple stages during Drosophila embryo development. We
have previously published a VirtualEmbryo (http://bdtnp.lbl.gov/Fly-Net/), a digital morphology and gene expression map of cellularizing blastoderm.
However, the late embryo is structurally much more complex, having estimated 40,000 cells, over 70 cell types and the major larval organs. To accurately
capture this complexity, we have made major improvements in both our image acquisition and image analysis strategies. We are initially focusing on the
morphological richness of stage 16 embryos with the goal of assigning all cells in an embryo image to specific tissue types. To enable this, we have
developed feature detection algorithms that accurately detect a single location or a set of points by learning their appearance from given fiducial coordinates
within multiple embryos stained for nuclear DNA. This will eventually allow us to assign cells in each image to any of multiple tissue types without having
to label each embryo with large numbers of probes of genes expressed in individual cell types. The registered data from multiple embryos can then be used
for quantitative comparisons of gene expression and morphology data. As part of this work, we have developed a visualization tool, FlyAnnotator, which the
user can employ to place defined fiducial points within the 3D data and explore cellular resolution maps of the embryo.
857B
Efficient phenotypic analysis using unfixed, uncoated adult Drosophila for scanning electron microscopy. Nicholas J. Tardi, Kevin A. Edwards.
Biological Sciences, Illinois State Univ, Normal, IL.
Projects featuring repetitive phenotypic analysis of insects, such as mutant screens, quantitative genetics, and taxonomic studies, could be greatly
facilitated by a simpler approach to SEM. Here, we have applied Low Vacuum SEM to wild type and mutant Drosophila, and demonstrate that high quality
ultrastructure data can be obtained easily using minimal preparation. Adult flies, frozen live for storage, were mounted on EM stubs with carbon cement and
directly imaged with no chemical treatment or sputter coating. The key imaging parameters were identified and optimized, including pressure, spot size,
accelerating voltage, working distance, and exposure time. Potential artifacts, including apparent water droplets, variations in chamber pressure, charging,
and sample dehydration were investigated. We conclude that our optimized protocol is well suited to large scale imaging of eyes, wings, bristles, and other
adult structures.
858C
OpenSPIM - an open hardware project to bring Selective Plane Illumination Microscopy to the hands of the Drosophila researchers. Pavel
Tomancak, Peter Pitrone, Johannes Schindelin. MPI-CBG, Dresden, Germany.
Selective Plane Illumination Microscopy (SPIM) is an emerging technique that promises to revolutionize developmental biology by allowing in toto
imaging of large samples with high-resolution. Several spectacular, proof of principle set-ups, capable of recording Drosophila embryogenesis at cellular
resolution have been introduced over the past few years.
We are particularly interested in using SPIM to record patterns of gene expression during embryogenesis using live gene expression reporters based on the
FlyFos system (transgeneome.mpi-cbg.de). SPIM imaging of Drosophila embryogenesis takes by definition as long as the embryo develops and therefore in
order to achieve at least medium throughput the only reasonable way to go is to employ several SPIM set-ups in parallel.
We have recently developed a low-cost, open access SPIM set-up designed specifically for imaging of expression patterns in Drosophila embryogenesis. We
will document all details necessary to build this so called, OpenSPIM, via a publicly accessible wiki. We are also working with the Journal of Visual
Experiments (JoVE) to record the assembly and operation of the OpenSPIM set-up. The microscope is driven using MicroManager from within Fiji (Fiji Is
Just ImageJ; fiji.sc) an Open Source image analysis platform for which we developed advanced algorithms for SPIM image processing. Thus we merge the
concept of Open Source software and hardware into a single integrated imaging solution. We hope that the OpenSPIM will nucleate a community of open
hardware 'makers' that will continue to develop the set-up for the imaging needs of Drosophila research and beyond.
Finally, the current OpenSPIM prototype has been designed to fit into a cabin suitcase (the "SPIM in a briefcase" concept) and so if custom regulations
permit it, we will bring the set-up to the meeting for live demonstration of its capabilities.
859A
Morphogen gradients quantified by sub-single embryo RNA-seq. Peter A. Combs1, Michael B. Eisen 2,3. 1) Biophysics Grad Group, UC Berkeley,
Berkeley, CA; 2) Department of Molecular and Cell Biology, Univ California, Berkeley, CA; 3) Howard Hughes Medical Institute, Univ California,
Berkeley, CA.
Genome-scale techniques have been invaluable at illuminating multi-gene interactions at the expense of spatial information; conversely, any conventional
technique that respects spatial dependence works for only a handful of genes at a time. This is particularly troublesome for studying Drosophila patterning,
which has detailed and precise spatial dependence among a network of genes. Previous work from our lab has shown that single Drosophila embryos
provide ample material for RNA-seq. Here I extend this work to quantify gene expression within a spatially-restricted region of a single embryo. I used a
combination of cryo-sectioning and Illumina sequencing to measure gene expression along the anterior-posterior axis of single D. melanogaster embryos.
The sample preparation protocol was specifically optimized for small volume samples, and yielded enough cDNA to perform high density sequencing of 60
micron thick samples. Our analysis focuses on the expression of the gap gene morphogens along the AP axis.
860B
New Tool in the lab: a Robotic System to process Drosophila samples. Joana Branco 1, António Lopes1 , João Salgado1, Rui Cortesão2 , Jorge Batista2 ,
Nuno André Faustino1 . 1) Gene PreDiT, SA Núcleo 4 - Lote 4-A, Ed. Biocant II 3060-119 Cantanhede Portugal; 2) Institute of Systems and Robotics
Electrical and Computer Engineering Department, University of Coimbra 3030-290 Coimbra Portugal.
To overcome some of the constraints due to Human-handling of Drosophila samples, we have developed a robotic system to process adult flies, and
extract its material for subsequent assays. Currently our robotic system allows preparation of samples for wet weight measurement, protein, lipids and sugar