Alex Kalinka, Karolina Jastrzebowska, Stephan Preibisch, Melek Asli Kayserili

We are interested in quantitatively testing the "hourglass model" of morphological evolution, a model derived from Karl von Baer's third "law" of embryonic development proposed in 1828 which states that divergent animal species pass through a similar period of development known as the "phylotypic stage." Although this long-standing model describes a fundamental pattern of animal development, no evidence exists to prove that the phylotypic stage is actively maintained by natural selection or, alternatively, if it is simply a consequence of mechanistic constraints during development.

We collected micro-array time-courses for embryogenesis of six species of fruitflies using custom designed species-specific Agilent arrays interrogating approximately 3500 genes expressed during embryogenesis. Statistical analysis of the data indicates that gene expression levels vary among species more in the beginning and at the end of embryogenesis compared to mid-embryogenesis, in agreement with the predictions of the hourglass model.

In the future, we plan to expand the microarray analysis to the whole genome using 3’ SAGE deep sequencing and to use our FlyFos system and quantitative imaging approaches to assess the divergence of morphology and spatial genes expression patterns before during and after the phylotypic stage. Our work will establish a new paradigm in post-genomic evolutionary developmental biology and reveal the evolutionary mechanism that underpins one of the most fundamental aspects of animal biology.

collaborators: Uwe Ohler (Duke University) and Casey Bergman (University of Manchester), Julia Jarrels (MPI-CBG Microarray facility)

publications: Kalinka, Alex T.; Varga, Karolina M.; Gerrard, Dave T.; Preibisch, Stephan W.; Corcoran, David L.; Jarrels, Julia; Ohler, Uwe; Bergman, Casey M.; Tomancak, Pavel Gene expression divergence recapitulates the developmental hourglass model Nature, 468, pp. 811-814, (2010)

Radoslaw Kamil Ejsmont, Maria Bogdanzaliewa

In order to visualize gene expression patterns in live embryos, we constructed two complementary genomic fosmid libraries for Drosophila melanogaster and Drosophila pseudoobscura that permit seamless modification of large genomic clones by high-throughput recombineering and direct transgenesis. The fosmid transgenes rescue mutant phenotypes, recapitulate endogenous gene expression patterns and in some cases allow imaging of gene products in living animals. The D.pseudoobscura transgenes rescue RNAi phenotypes when introduced into the D.melanogaster genome, providing a convenient control for the specificity of the knockdown. These libraries will, in combination with recombineering technology, enable systematic analysis and manipulation of gene activity across species.

We are currently testing various tags suitable for live imaging. We are developing a universal tagging system that enables exchange of tags in vivo without the time-consuming transgenesis. Finally, we are optimizing a HT liquid-culture recombineering pipeline to introduce deletions into the already tagged fosmid constructs to dissect cis-regulatory sequences.

We will leverage the astonishing efficiency of our toolkit to establish a genome-wide resource of tagged fosmid transgenes and organize a distributed, community-driven transgenesis effort. We will use the toolkit to test specific hypothesis of sequence determinants of gene expression pattern divergence.

collaborators: Mihail Sarov (MPI-CBG Transgenomics facility), Frank Schnorrer (MPI Martinsried), Silke Winkler (MPI-CBG sequencing facility)

publications: Ejsmont, Radoslaw K; Sarov, Mihail; Winkler, Sylke; Lipinski, Kamil A; Tomancák, Pavel A toolkit for high-throughput, cross-species gene engineering in Drosophila. Nature Methods, 6, no. 6, pp. 435-437, (2009)

Stephan Preibisch, Michael Weber, Melek Asli Kayserili

To realize our goal of studying the interplay between gene expression regulation and morphogenesis, we sought a microscopy technique that would enable tracking of all cells throughout the Drosophila embryo. Selective Plane Illumination Microscopy (SPIM) allows in toto imaging of large specimens, such as Drosophila embryos, by acquiring image stacks from multiple angles. We developed an algorithm for registration of multi-angle microscopy acquisitions using fluorescent beads in rigid mounting medium as fiduciary markers. The beads are matched as invariant, local geometric point descriptors and their displacement is minimized for globally optimal, sample-independent registration. We show that the approach can be used for registration of multi-angle acquisition in many imaging modalities.

We recorded Drosophila embryos expressing His-YFP in all cells throughout embryonic development. We are currently exploring the possibilities of segmenting and tracking cells in these datasets. We plan to extend this analysis to record gene expression reporters in the context of the cellular anatomy. SPIM is also ideal for studying cellular level anatomy of fixed specimens, such as C.elegans and Drosophila embryos. We will collect isotropic scans of embryos from different species of fruitflies at different stages of embryonic development and quantify anatomical differences.

collaborators: Carl-Zeiss Microimaging Gmbh

publications: Preibisch, Stephan W.; Saalfeld, Stephan; Schindelin, Johannes; Tomancák, Pavel Software for bead-based registration of selective plane illumination microscopy data Nature Methods, 7, no. 6, pp. 418-419, (2010)

Stephan Saalfeld

Tiled serial section transmission electron microscopy (ssTEM) is increasingly used to describe high-resolution anatomy of large biological specimens. In particular, it is indispensable for analysis of the neural tissue at the level of synaptic connectivity. Our collaborator, Albert Cardona, collects massive ssTEM dataset covering the ENTIRE first instar larval nervous system consisting of several hundred thousands image tiles.

Registration of serial section TEM image mosaics has to preserve the continuity of the 3d-structure of the specimen while compensating the physical distortions applied to the tissue during sectioning and imaging, staining artifacts, missing sections and the fact that certain structures may appear dissimilar in consecutive sections. We developed a fully automatic, non-rigid but as rigid as possible registration approach for large tiled serial section microscopy stacks. We use the Scale Invariant Feature Transform (SIFT) to identify corresponding landmarks within and across sections and globally optimize the pose of each single tile for minimal overall displacement of these landmark correspondences. We evaluate the precision and robustness of the approach using an artificially generated data set designed to mimic the properties of TEM data. We demonstrate the biological application of our approach by registering large ssTEM of Drosophila first instar larval brain consisting of nearly 7000 images.

Recently, we extended the approach to include lens distortion compensation, handling of image discontinuity at section folds and globally optimal elastic registration. We also developed CATMAID, a web-based tool modeled after GoogleMaps, for collaborative annotation of spatially large image datasets.

collaborators: Albert Cardona (ETH Zurich/Janelia Farm), Volker Hartenstein (UCLA)

publications: Saalfeld, Stephan; Cardona, Albert; Hartenstein, Volker; Tomancák, Pavel As-rigid-as-possible mosaicking and serial section registration of large ssTEM datasets Bioinformatics, 26, no. 12, pp. 57-63, (2010)

Stephan Saalfeld, Stephan Preibisch

In order to extract computationally tractable gene expression information from large 3D and 4D recordings of embryos, we need to employ advanced 3D image analysis and computer vision approaches such as rigid and elastic specimen-to-specimen or specimen-to-atlas registration, segmentation, object recognition and motion tracking.

Thus far we developed state-of-the-art approaches to stitching of confocal microscopy data (2D and 3D), registration of massive electron microscopy datasets and various approaches for registration of multi-view microscopy datasets, in particular from SPIM.

We collect our advances under the umbrella of the Fiji (Fiji Is Just ImageJ) which is an open source project build around popular biological image analysis platform ImageJ. Through Fiji we established a community of researchers interested in combining biology and image analysis. We aim to create usable, thoroughly documented tools for the whole biology community (for details on Fiji see open access tab).  

collaborators:  Johannes Schindelin (MPI-CBG Image Processing facility) and the Fiji open source community

Pavel Mejstrik

I established, in the past, large-scale database on gene expression patterns during Drosophila embryogenesis on the tissue level. In collaboration with Eric Lecuyer and Henry Krause, we extended this analysis to reveal widespread occurrence of sub-cellular RNA localization in the early Drosophila development (I contributed exclusively to the bioinformatics of this project).

In Dresden we further expanded the systematic surveys of gene expression patterns to larval imaginal discs. We developed an extremely robust and efficient protocol for mass isolation of imaginal disc tissue. To date we analyzed, by RNA in situ hybridization, the expression of nearly 6000 genes and found more then 100 novel patterns in the discs. We supplement the spatial image data with quantitative measurement of gene expression using Agilent whole genome micro-array on hand sorted disc material. We are developing image analysis pipeline to categorize and analyze the disc patterns.

In the future, we plan to repeat the analysis in Drosophila ovaries, combining fluorescent and histochemical RNA in situ methods to reveal RNA localization events in the germ-line and patterning events in the somatic follicular epithelium. We plan to dissect RNA localization signals with the FlyFos recombineering toolkit. All the data are or will be presented to the scientific community via publicly accessible web interfaces (see open access tab).

collaborators: Eric Lecuyer (IRCM Montreal), Christian Dahmann (MPI-CBG)