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1.  A role for tuned levels of nucleosome remodeler subunit ACF1 during Drosophila oogenesis 
Developmental biology  2016;411(2):217-230.
The Chromatin Accessibility Complex (CHRAC) consists of the ATPase ISWI, the large ACF1 subunit and a pair of small histone-like proteins, CHRAC-14/16. CHRAC is a prototypical nucleosome sliding factor that mobilizes nucleosomes to improve the regularity and integrity of the chromatin fiber. This may facilitate the formation of repressive chromatin. Expression of the signature subunit ACF1 is restricted during embryonic development, but remains high in primordial germ cells. Therefore, we explored roles for ACF1 during Drosophila oogenesis. ACF1 is expressed in somatic and germline cells, with notable enrichment in germline stem cells and oocytes. The asymmetrical localization of ACF1 to these cells depends on the transport of the Acf1 mRNA by the Bicaudal-D/Egalitarian complex. Loss of ACF1 function in the novel Acf17 allele leads to defective egg chambers and their elimination through apoptosis. In addition, we find a variety of unusual 16-cell cyst packaging phenotypes in the previously known Acf11 allele, with a striking prevalence of egg chambers with two functional oocytes at opposite poles. Surprisingly, we found that the Acf11 deletion – despite disruption of the Acf1 reading frame – expresses low levels of a PHD-bromodomain module from the C-terminus of ACF1 that becomes enriched in oocytes. Expression of this module from the Acf1 genomic locus leads to packaging defects in the absence of functional ACF1, suggesting competitive interactions with unknown target molecules. Remarkably, a two-fold overexpression of CHRAC (ACF1 and CHRAC-16) leads to increased apoptosis and packaging defects. Evidently, finely tuned CHRAC levels are required for proper oogenesis.
doi:10.1016/j.ydbio.2016.01.039
PMCID: PMC5012898  PMID: 26851213
Nucleosome remodeling; ISWI ATPase; Germarium; PHD finger; Bromodomain
2.  Comment on “Cortical folding scales universally with surface area and thickness, not number of neurons” 
Science (New York, N.Y.)  2016;351(6275):825.
Mota and Herculano-Houzel (Reports, 3 July 2015, p.74) assign power functions to neuroanatomical data and present a model to account for evolutionary patterns of cortical folding in the mammalian brain. We detail how the model assumptions are in conflict with experimental and observational work and show that the model itself does not accurately fit the data.
doi:10.1126/science.aad2029
PMCID: PMC4881813  PMID: 26912886
3.  Light-sheet microscopy for everyone? Experience of building an OpenSPIM to study flatworm development 
Background
Selective plane illumination microscopy (SPIM a type of light-sheet microscopy) involves focusing a thin sheet of laser light through a specimen at right angles to the objective lens. As only the thin section of the specimen at the focal plane of the lens is illuminated, out of focus light is naturally absent and toxicity due to light (phototoxicity) is greatly reduced enabling longer term live imaging. OpenSPIM is an open access platform (Pitrone et al. 2013 and OpenSPIM.org) created to give new users step-by-step instructions on building a basic configuration of a SPIM microscope, which can in principle be adapted and upgraded to each laboratory’s own requirements and budget. Here we describe our own experience with the process of designing, building, configuring and using an OpenSPIM for our research into the early development of the polyclad flatworm Maritigrella crozieri – a non-model animal.
Results
Our OpenSPIM builds on the standard design with the addition of two colour laser illumination for simultaneous detection of two probes/molecules and dual sided illumination, which provides more even signal intensity across a specimen. Our OpenSPIM provides high resolution 3d images and time lapse recordings, and we demonstrate the use of two colour lasers and the benefits of two color dual-sided imaging. We used our microscope to study the development of the embryo of the polyclad flatworm M. crozieri. The capabilities of our microscope are demonstrated by our ability to record the stereotypical spiral cleavage pattern of M. crozieri with high-speed multi-view time lapse imaging. 3D and 4D (3D + time) reconstruction of early development from these data is possible using image registration and deconvolution tools provided as part of the open source Fiji platform. We discuss our findings on the pros and cons of a self built microscope.
Conclusions
We conclude that home-built microscopes, such as an OpenSPIM, together with the available open source software, such as MicroManager and Fiji, make SPIM accessible to anyone interested in having continuous access to their own light-sheet microscope. However, building an OpenSPIM is not without challenges and an open access microscope is a worthwhile, if significant, investment of time and money. Multi-view 4D microscopy is more challenging than we had expected. We hope that our experience gained during this project will help future OpenSPIM users with similar ambitions.
Electronic supplementary material
The online version of this article (doi:10.1186/s12861-016-0122-0) contains supplementary material, which is available to authorized users.
doi:10.1186/s12861-016-0122-0
PMCID: PMC4929743  PMID: 27363495
4.  A genome-wide resource for the analysis of protein localisation in Drosophila 
eLife  null;5:e12068.
The Drosophila genome contains >13000 protein-coding genes, the majority of which remain poorly investigated. Important reasons include the lack of antibodies or reporter constructs to visualise these proteins. Here, we present a genome-wide fosmid library of 10000 GFP-tagged clones, comprising tagged genes and most of their regulatory information. For 880 tagged proteins, we created transgenic lines, and for a total of 207 lines, we assessed protein expression and localisation in ovaries, embryos, pupae or adults by stainings and live imaging approaches. Importantly, we visualised many proteins at endogenous expression levels and found a large fraction of them localising to subcellular compartments. By applying genetic complementation tests, we estimate that about two-thirds of the tagged proteins are functional. Moreover, these tagged proteins enable interaction proteomics from developing pupae and adult flies. Taken together, this resource will boost systematic analysis of protein expression and localisation in various cellular and developmental contexts.
DOI: http://dx.doi.org/10.7554/eLife.12068.001
eLife digest
The fruit fly Drosophila melanogaster is a popular model organism in biological research. Studies using Drosophila have led to important insights into human biology, because related proteins often fulfil similar roles in flies and humans. Thus, studying the role of a protein in Drosophila can teach us about what it might do in a human.
To fulfil their biological roles, proteins often occupy particular locations inside cells, such as the cell’s nucleus or surface membrane. Many proteins are also only found in specific types of cell, such as neurons or muscle cells. A protein’s location thus provides clues about what it does, however cells contain many thousands of proteins and identifying the location of each one is a herculean task.
Sarov et al. took on this challenge and developed a new resource to study the localisation of all Drosophila proteins during this animal’s development. First, genetic engineering was used to tag thousands of Drosophila proteins with a green fluorescent protein, so that they could be tracked under a microscope.
Sarov et al. tagged about 10000 Drosophila proteins in bacteria, and then introduced almost 900 of them into flies to create genetically modified flies. Each fly line contains an extra copy of the tagged gene that codes for one tagged protein. About two-thirds of these tagged proteins appeared to work normally after they were introduced into flies. Sarov et al. then looked at over 200 of these fly lines in more detail and observed that many of the proteins were found in particular cell types and localized to specific parts of the cells. Video imaging of the tagged proteins in living fruit fly embryos and pupae revealed the proteins’ movements, while other techniques showed which proteins bind to the tagged proteins, and may therefore work together in protein complexes.
This resource is openly available to the community, and so researchers can use it to study their favourite protein and gain new insights into how proteins work and are regulated during Drosophila development. Following on from this work, the next challenge will be to create more flies carrying tagged proteins, and to swap the green fluorescent tag with other experimentally useful tags.
DOI: http://dx.doi.org/10.7554/eLife.12068.002
doi:10.7554/eLife.12068
PMCID: PMC4805545  PMID: 26896675
drosophila; resource; protein tagging; live imaging; recombineering; genetics; D. melanogaster
5.  An automated workflow for parallel processing of large multiview SPIM recordings 
Bioinformatics  2015;32(7):1112-1114.
Summary: Selective Plane Illumination Microscopy (SPIM) allows to image developing organisms in 3D at unprecedented temporal resolution over long periods of time. The resulting massive amounts of raw image data requires extensive processing interactively via dedicated graphical user interface (GUI) applications. The consecutive processing steps can be easily automated and the individual time points can be processed independently, which lends itself to trivial parallelization on a high performance computing (HPC) cluster. Here, we introduce an automated workflow for processing large multiview, multichannel, multiillumination time-lapse SPIM data on a single workstation or in parallel on a HPC cluster. The pipeline relies on snakemake to resolve dependencies among consecutive processing steps and can be easily adapted to any cluster environment for processing SPIM data in a fraction of the time required to collect it.
Availability and implementation: The code is distributed free and open source under the MIT license http://opensource.org/licenses/MIT. The source code can be downloaded from github: https://github.com/mpicbg-scicomp/snakemake-workflows. Documentation can be found here: http://fiji.sc/Automated_workflow_for_parallel_Multiview_Reconstruction.
Contact: schmied@mpi-cbg.de
Supplementary information: Supplementary data are available at Bioinformatics online.
doi:10.1093/bioinformatics/btv706
PMCID: PMC4896369  PMID: 26628585
6.  Introns and gene expression: Cellular constraints, transcriptional regulation, and evolutionary consequences 
Bioessays  2014;37(2):148-154.
A gene's “expression profile” denotes the number of transcripts present relative to all other transcripts. The overall rate of transcript production is determined by transcription and RNA processing rates. While the speed of elongating RNA polymerase II has been characterized for many different genes and organisms, gene-architectural features – primarily the number and length of exons and introns – have recently emerged as important regulatory players. Several new studies indicate that rapidly cycling cells constrain gene-architecture toward short genes with a few introns, allowing efficient expression during short cell cycles. In contrast, longer genes with long introns exhibit delayed expression, which can serve as timing mechanisms for patterning processes. These findings indicate that cell cycle constraints drive the evolution of gene-architecture and shape the transcriptome of a given cell type. Furthermore, a tendency for short genes to be evolutionarily young hints at links between cellular constraints and the evolution of animal ontogeny.
doi:10.1002/bies.201400138
PMCID: PMC4654234  PMID: 25400101
cell cycle constraints; gene length; macro-evolutionary patterns; splicing
7.  Endogenously Tagged Rab Proteins: A Resource to Study Membrane Trafficking in Drosophila 
Developmental Cell  2015;33(3):351-365.
Summary
Membrane trafficking is key to the cell biological mechanisms underlying development. Rab GTPases control specific membrane compartments, from core secretory and endocytic machinery to less-well-understood compartments. We tagged all 27 Drosophila Rabs with YFPMYC at their endogenous chromosomal loci, determined their expression and subcellular localization in six tissues comprising 23 cell types, and provide this data in an annotated, searchable image database. We demonstrate the utility of these lines for controlled knockdown and show that similar subcellular localization can predict redundant functions. We exploit this comprehensive resource to ask whether a common Rab compartment architecture underlies epithelial polarity. Strikingly, no single arrangement of Rabs characterizes the five epithelia we examine. Rather, epithelia flexibly polarize Rab distribution, producing membrane trafficking architectures that are tissue- and stage-specific. Thus, the core machinery responsible for epithelial polarization is unlikely to rely on polarized positioning of specific Rab compartments.
Graphical Abstract
Highlights
•A collection of YFP-tagged rab alleles allows functional in vivo studies•FLYtRAB database links downloadable image data and annotated Rab localization terms•Rab compartment polarity in epithelia is flexible and dynamic•Similar intracellular Rab protein localization can indicate redundant Rab functions
Membrane trafficking is key to the cell biological mechanisms underlying development. As hypothesis-generating tools, Dunst et al. present a genetic resource for systematic visualization and manipulation of all membrane compartments controlled by Rab GTPases in Drosophila and a database summarizing Rab localization in different cell and tissue types.
doi:10.1016/j.devcel.2015.03.022
PMCID: PMC4431667  PMID: 25942626
8.  Systematic imaging reveals features and changing localization of mRNAs in Drosophila development 
eLife  null;4:e05003.
mRNA localization is critical for eukaryotic cells and affects numerous transcripts, yet how cells regulate distribution of many mRNAs to their subcellular destinations is still unknown. We combined transcriptomics and systematic imaging to determine the tissue-specific expression and subcellular distribution of 5862 mRNAs during Drosophila oogenesis. mRNA localization is widespread in the ovary and detectable in all of its cell types—the somatic epithelial, the nurse cells, and the oocyte. Genes defined by a common RNA localization share distinct gene features and differ in expression level, 3′UTR length and sequence conservation from unlocalized mRNAs. Comparison of mRNA localizations in different contexts revealed that localization of individual mRNAs changes over time in the oocyte and between ovarian and embryonic cell types. This genome scale image-based resource (Dresden Ovary Table, DOT, http://tomancak-srv1.mpi-cbg.de/DOT/main.html) enables the transition from mechanistic dissection of singular mRNA localization events towards global understanding of how mRNAs transcribed in the nucleus distribute in cells.
DOI: http://dx.doi.org/10.7554/eLife.05003.001
eLife digest
To make a protein, the DNA sequence that encodes it must first be ‘transcribed’ to build a molecule of messenger RNA (called mRNA for short). Although many mRNA molecules are found throughout a cell, some are ‘localized’ to certain areas; and recent evidence suggests that this mRNA localization may be more common than previously thought.
Not much is known about how cells identify which mRNAs need to be localized, or how these molecules are then transported to their destination. The localization process has been studied in most detail in the developing egg cell—also known as an oocyte—of the fruit fly species Drosophila melanogaster. These studies have identified few mRNA molecules that, if they are not carefully localized within the cell, cause the different parts of the fly embryo to fail to develop correctly when the oocyte is fertilized.
Jambor et al. created an open-access online resource called the ‘Dresden Ovary Table’ that shows how 5862 mRNA molecules are distributed in several cell types involved in oocyte production in the ovary of female D. melanogaster flies. This resource consists of a combination of three-dimensional fluorescent images and measurements of mRNA amounts recorded at different stages in the development of the oocyte.
Using the resource, Jambor et al. demonstrate that all of the cell types that make up the ovary localize many different mRNA molecules to several distinct destinations within the cells. The localized mRNAs share certain features, with mRNAs localized in the same part of the cell showing the most similarities. For example, localized mRNAs have longer so-called 3′ untranslated regions (3′UTR) that carry regulatory information and these sequences are also more evolutionarily conserved. Further, when the mRNA molecules in the oocyte were examined at different times during its development and compared with the embryo, the majority of these mRNAs were found to change where they are localized as the organism develops.
The resource can be used to gain insight into specific genetic features that control the distribution of mRNAs. This information will be instrumental for cracking the ‘RNA localization code’ and understanding how it affects the activity of proteins in cells.
DOI: http://dx.doi.org/10.7554/eLife.05003.002
doi:10.7554/eLife.05003
PMCID: PMC4384636  PMID: 25838129
RNA localization; imaging; genome wide resource; D. melanogaster
9.  An Adaptive Threshold in Mammalian Neocortical Evolution 
PLoS Biology  2014;12(11):e1002000.
A study of the evolutionary history of cortical folding in mammals, its relationship to physiological and life-history traits and the underlying cortical progenitor behavior during embryogenesis, explains the diversity of folding we see across modern mammals. The diversity of neocortical folding among mammals can be explained by two distinct neurogenic programs, which give rise to mammals with a highly folded neocortex and mammals with slightly folded or unfolded neocortex, each occupying a distinct ecological niche.
Expansion of the neocortex is a hallmark of human evolution. However, determining which adaptive mechanisms facilitated its expansion remains an open question. Here we show, using the gyrencephaly index (GI) and other physiological and life-history data for 102 mammalian species, that gyrencephaly is an ancestral mammalian trait. We find that variation in GI does not evolve linearly across species, but that mammals constitute two principal groups above and below a GI threshold value of 1.5, approximately equal to 109 neurons, which may be characterized by distinct constellations of physiological and life-history traits. By integrating data on neurogenic period, neuroepithelial founder pool size, cell-cycle length, progenitor-type abundances, and cortical neuron number into discrete mathematical models, we identify symmetric proliferative divisions of basal progenitors in the subventricular zone of the developing neocortex as evolutionarily necessary for generating a 14-fold increase in daily prenatal neuron production, traversal of the GI threshold, and thus establishment of two principal groups. We conclude that, despite considerable neuroanatomical differences, changes in the length of the neurogenic period alone, rather than any novel neurogenic progenitor lineage, are sufficient to explain differences in neuron number and neocortical size between species within the same principal group.
Author Summary
What are the key differences in the development and evolution of the cerebral cortex that underlie the differences in its size and degree of folding across mammals? Here, we present phylogenetic evidence that the Jurassic era mammalian ancestor may have been a relatively large-brained species with a folded neocortex. We then show that variation in the degree of cortical folding (gyrencephaly index [GI]) does not evolve linearly across species, as previously assumed, but that mammals fall into two principal groups associated with distinct ecological niches: low-GI mammals (such as mice and tarsiers) and high-GI mammals (such as dolphins and humans), which are found to generate on average 14-fold more brain weight per day of gestation. This greater daily brain weight production in mammals with a highly folded neocortex requires a specific class of progenitor cell-type to adopt a special mode of cell division, which is absent in mammals with slightly folded or unfolded neocortices. Differences among mammals within the same GI group (high or low) are not due to different programming, but rather the result of differences in the length of the neurogenic period. So, the impressively large and folded human neocortex, which is three times the size of the chimpanzee neocortex, can be explained by a modest evolutionary extension of the neurogenic period with respect to its closest primate ancestors.
doi:10.1371/journal.pbio.1002000
PMCID: PMC4236020  PMID: 25405475
10.  Efficient Bayesian-based multiview deconvolution 
Nature methods  2014;11(6):645-648.
Light-sheet fluorescence microscopy is able to image large specimens with high resolution by capturing the samples from multiple angles. Multiview deconvolution can substantially improve the resolution and contrast of the images, but its application has been limited owing to the large size of the data sets. Here we present a Bayesian-based derivation of multiview deconvolution that drastically improves the convergence time, and we provide a fast implementation using graphics hardware.
doi:10.1038/nmeth.2929
PMCID: PMC4153441  PMID: 24747812
11.  Drosophila brain development: Closing the gap between a macroarchitectural and microarchitectural approach 
Neurobiologists address neural structure, development and function at the level of “macrocircuits” (how are different brain compartments interconnected, what overall pattern of activity do they produce), and at the level of “microcircuits” (how does connectivity and physiology of individual neurons and their processes within a compartment determine the functional output of this compartment). Work in our lab aims at reconstructing the developing Drosophila brain at both levels. Macrocircuits can be approached conveniently by reconstructing the pattern of brain lineages, which form groups of neurons whose projections form cohesive fascicles interconnecting the compartments of the larval and adult brain. The reconstruction of microcircuits requires serial section electron microscopy, due to the small size of terminal neuronal processes and their synaptic contacts. Because of the amount of labor that traditionally comes with this approach, very little is known about microcircuitry in brains across the animal kingdom. Many of the problems of serial EM reconstruction is now solvable with digital image recording and specialized software for both image acquisition and post-processing. In this paper we introduce our efforts to reconstruct the small Drosophila larval brain, and discuss our results in light of the published data on neuropile ultrastructure in other animal taxa.
doi:10.1101/sqb.2009.74.037
PMCID: PMC3950651  PMID: 20028843
Drosophila; brain; lineage; connectivity; elctron microscopy
12.  Fiji - an Open Source platform for biological image analysis 
Nature methods  2012;9(7):10.1038/nmeth.2019.
Fiji is a distribution of the popular Open Source software ImageJ focused on biological image analysis. Fiji uses modern software engineering practices to combine powerful software libraries with a broad range of scripting languages to enable rapid prototyping of image processing algorithms. Fiji facilitates the transformation of novel algorithms into ImageJ plugins that can be shared with end users through an integrated update system. We propose Fiji as a platform for productive collaboration between computer science and biology research communities.
doi:10.1038/nmeth.2019
PMCID: PMC3855844  PMID: 22743772
13.  Biological Imaging Software Tools 
Nature methods  2012;9(7):697-710.
Few technologies are more widespread in modern biological laboratories than imaging. Recent advances in optical technologies and instrumentation are providing hitherto unimagined capabilities. Almost all these advances have required the development of software to enable the acquisition, management, analysis, and visualization of the imaging data. We review each computational step that biologists encounter when dealing with digital images, the challenges in that domain, and the overall status of available software for bioimage informatics, focusing on open source options.
doi:10.1038/nmeth.2084
PMCID: PMC3659807  PMID: 22743775
14.  Visualization of image data from cells to organisms 
Nature methods  2010;7(3 0):S26-S41.
Advances in imaging techniques and high-throughput technologies are providing scientists with unprecedented possibilities to visualize internal structures of cells, organs and organisms and to collect systematic image data characterizing genes and proteins on a large scale. To make the best use of these increasingly complex and large image data resources, the scientific community must be provided with methods to query, analyze and crosslink these resources to give an intuitive visual representation of the data. This review gives an overview of existing methods and tools for this purpose and highlights some of their limitations and challenges.
doi:10.1038/nmeth.1431
PMCID: PMC3650473  PMID: 20195255
15.  An Excess of Gene Expression Divergence on the X Chromosome in Drosophila Embryos: Implications for the Faster-X Hypothesis 
PLoS Genetics  2012;8(12):e1003200.
The X chromosome is present as a single copy in the heterogametic sex, and this hemizygosity is expected to drive unusual patterns of evolution on the X relative to the autosomes. For example, the hemizgosity of the X may lead to a lower chromosomal effective population size compared to the autosomes, suggesting that the X might be more strongly affected by genetic drift. However, the X may also experience stronger positive selection than the autosomes, because recessive beneficial mutations will be more visible to selection on the X where they will spend less time being masked by the dominant, less beneficial allele—a proposal known as the faster-X hypothesis. Thus, empirical studies demonstrating increased genetic divergence on the X chromosome could be indicative of either adaptive or non-adaptive evolution. We measured gene expression in Drosophila species and in D. melanogaster inbred strains for both embryos and adults. In the embryos we found that expression divergence is on average more than 20% higher for genes on the X chromosome relative to the autosomes; but in contrast, in the inbred strains, gene expression variation is significantly lower on the X chromosome. Furthermore, expression divergence of genes on Muller's D element is significantly greater along the branch leading to the obscura sub-group, in which this element segregates as a neo-X chromosome. In the adults, divergence is greatest on the X chromosome for males, but not for females, yet in both sexes inbred strains harbour the lowest level of gene expression variation on the X chromosome. We consider different explanations for our results and conclude that they are most consistent within the framework of the faster-X hypothesis.
Author Summary
There is a single copy of the X chromosome in males, yet two copies in females. This unique inheritance pattern has long been predicted to influence how the X chromosome evolves. In particular, the theory suggests that the single copy of the X in males could facilitate faster evolution of the X, although this faster evolution could be either adaptive or non-adaptive. We measured gene expression across the chromosomes in several different Drosophila species and also in several inbred strains of D. melanogaster for both embryos and adults. We found that gene expression is evolving significantly faster between species in the embryos, yet harbours significantly less variation within inbred strains. In adults, evolution between species appears to be much slower than in the embryos, yet they also harbour significantly lower levels of gene expression variation on the X chromosome in inbred strains. Overall, our results are consistent with there being an excess of adaptive evolution on the X chromosome in Drosophila embryos. Finally, we underscore the importance of biological context for understanding how chromosomes evolve in different species.
doi:10.1371/journal.pgen.1003200
PMCID: PMC3531489  PMID: 23300473
16.  TrakEM2 Software for Neural Circuit Reconstruction 
PLoS ONE  2012;7(6):e38011.
A key challenge in neuroscience is the expeditious reconstruction of neuronal circuits. For model systems such as Drosophila and C. elegans, the limiting step is no longer the acquisition of imagery but the extraction of the circuit from images. For this purpose, we designed a software application, TrakEM2, that addresses the systematic reconstruction of neuronal circuits from large electron microscopical and optical image volumes. We address the challenges of image volume composition from individual, deformed images; of the reconstruction of neuronal arbors and annotation of synapses with fast manual and semi-automatic methods; and the management of large collections of both images and annotations. The output is a neural circuit of 3d arbors and synapses, encoded in NeuroML and other formats, ready for analysis.
doi:10.1371/journal.pone.0038011
PMCID: PMC3378562  PMID: 22723842
17.  Abundant Occurrence of Basal Radial Glia in the Subventricular Zone of Embryonic Neocortex of a Lissencephalic Primate, the Common Marmoset Callithrix jacchus 
Cerebral Cortex (New York, NY)  2011;22(2):469-481.
Subventricular zone (SVZ) progenitors are a hallmark of the developing neocortex. Recent studies described a novel type of SVZ progenitor that retains a basal process at mitosis, sustains expression of radial glial markers, and is capable of self-renewal. These progenitors, referred to here as basal radial glia (bRG), occur at high relative abundance in the SVZ of gyrencephalic primates (human) and nonprimates (ferret) but not lissencephalic rodents (mouse). Here, we analyzed the occurrence of bRG cells in the embryonic neocortex of the common marmoset Callithrix jacchus, a near-lissencephalic primate. bRG cells, expressing Pax6, Sox2 (but not Tbr2), glutamate aspartate transporter, and glial fibrillary acidic protein and retaining a basal process at mitosis, occur at similar relative abundance in the marmoset SVZ as in human and ferret. The proportion of progenitors in M-phase was lower in embryonic marmoset than developing ferret neocortex, raising the possibility of a longer cell cycle. Fitting the gyrification indices of 26 anthropoid species to an evolutionary model suggested that the marmoset evolved from a gyrencephalic ancestor. Our results suggest that a high relative abundance of bRG cells may be necessary, but is not sufficient, for gyrencephaly and that the marmoset's lissencephaly evolved secondarily by changing progenitor parameters other than progenitor type.
doi:10.1093/cercor/bhr301
PMCID: PMC3256412  PMID: 22114084
brain evolution; cell cycle; gyrencephaly; marmoset; OSVZ
18.  An alignment-free method to identify candidate orthologous enhancers in multiple Drosophila genomes 
Bioinformatics  2010;26(17):2109-2115.
Motivation: Evolutionarily conserved non-coding genomic sequences represent a potentially rich source for the discovery of gene regulatory region such as transcriptional enhancers. However, detecting orthologous enhancers using alignment-based methods in higher eukaryotic genomes is particularly challenging, as regulatory regions can undergo considerable sequence changes while maintaining their functionality.
Results: We have developed an alignment-free method which identifies conserved enhancers in multiple diverged species. Our method is based on similarity metrics between two sequences based on the co-occurrence of sequence patterns regardless of their order and orientation, thus tolerating sequence changes observed in non-coding evolution. We show that our method is highly successful in detecting orthologous enhancers in distantly related species without requiring additional information such as knowledge about transcription factors involved, or predicted binding sites. By estimating the significance of similarity scores, we are able to discriminate experimentally validated functional enhancers from seemingly equally conserved candidates without function. We demonstrate the effectiveness of this approach on a wide range of enhancers in Drosophila, and also present encouraging results to detect conserved functional regions across large evolutionary distances. Our work provides encouraging steps on the way to ab initio unbiased enhancer prediction to complement ongoing experimental efforts.
Availability: The software, data and the results used in this article are available at http://www.genome.duke.edu/labs/ohler/research/transcription/fly_enhancer/
Contact: tomancak@mpi-cbg.de; uwe.ohler@duke.edu
Supplementary information: Supplementary data are available at Bioinformatics online.
doi:10.1093/bioinformatics/btq358
PMCID: PMC2922894  PMID: 20624780
19.  linkcomm: an R package for the generation, visualization, and analysis of link communities in networks of arbitrary size and type 
Bioinformatics  2011;27(14):2011-2012.
Summary: An essential element when analysing the structure, function, and dynamics of biological networks is the identification of communities of related nodes. An algorithm proposed recently enhances this process by clustering the links between nodes, rather than the nodes themselves, thereby allowing each node to belong to multiple overlapping or nested communities. The R package ‘linkcomm’ implements this algorithm and extends it in several aspects: (i) the clustering algorithm handles networks that are weighted, directed, or both weighted and directed; (ii) several visualization methods are implemented that facilitate the representation of the link communities and their relationships; (iii) a suite of functions are included for the downstream analysis of the link communities including novel community-based measures of node centrality; (iv) the main algorithm is written in C++ and designed to handle networks of any size; and (v) several clustering methods are available for networks that can be handled in memory, and the number of communities can be adjusted by the user.
Availability: The program is freely available from the Comprehensive R Archive Network (http://cran.r-project.org/) under the terms of the GNU General Public License (version 2 or later).
Contact: kalinka@mpi-cbg.de
Supplementary information: Supplementary data are available at Bioinformatics online.
doi:10.1093/bioinformatics/btr311
PMCID: PMC3129527  PMID: 21596792
20.  Mapping the complexity of transcription control in higher eukaryotes 
Genome Biology  2010;11(4):115.
Recent large analyses suggest the importance of combinatorial regulation by broadly expressed transcription factors rather than expression domains characterized by highly specific factors.
Recent genomic analyses suggest the importance of combinatorial regulation by broadly expressed transcription factors rather than expression domains characterized by highly specific factors.
doi:10.1186/gb-2010-11-4-115
PMCID: PMC2884534  PMID: 20441601
21.  An Integrated Micro- and Macroarchitectural Analysis of the Drosophila Brain by Computer-Assisted Serial Section Electron Microscopy 
PLoS Biology  2010;8(10):e1000502.
A new software package allows for dense electron microscopy reconstructions of neuronal networks in the fruit fly brain, and reveals specific differences in microcircuits between insects and vertebrates.
The analysis of microcircuitry (the connectivity at the level of individual neuronal processes and synapses), which is indispensable for our understanding of brain function, is based on serial transmission electron microscopy (TEM) or one of its modern variants. Due to technical limitations, most previous studies that used serial TEM recorded relatively small stacks of individual neurons. As a result, our knowledge of microcircuitry in any nervous system is very limited. We applied the software package TrakEM2 to reconstruct neuronal microcircuitry from TEM sections of a small brain, the early larval brain of Drosophila melanogaster. TrakEM2 enables us to embed the analysis of the TEM image volumes at the microcircuit level into a light microscopically derived neuro-anatomical framework, by registering confocal stacks containing sparsely labeled neural structures with the TEM image volume. We imaged two sets of serial TEM sections of the Drosophila first instar larval brain neuropile and one ventral nerve cord segment, and here report our first results pertaining to Drosophila brain microcircuitry. Terminal neurites fall into a small number of generic classes termed globular, varicose, axiform, and dendritiform. Globular and varicose neurites have large diameter segments that carry almost exclusively presynaptic sites. Dendritiform neurites are thin, highly branched processes that are almost exclusively postsynaptic. Due to the high branching density of dendritiform fibers and the fact that synapses are polyadic, neurites are highly interconnected even within small neuropile volumes. We describe the network motifs most frequently encountered in the Drosophila neuropile. Our study introduces an approach towards a comprehensive anatomical reconstruction of neuronal microcircuitry and delivers microcircuitry comparisons between vertebrate and insect neuropile.
Author Summary
Brains contain a vast number of connections between neurons, termed synapses. The precise patterns of these synaptic contacts form the structural underpinning of electrical microcircuits responsible for animal behavior. Due to their small size, synaptic contacts can be conclusively shown using only high-resolution electron microscopy (EM). Therefore, complete series of ultrathin sections are required to reconstruct neuronal microcircuitry. The acquisition and analysis of EM sections (with 15,000 sections per millimeter of tissue) is practical only by computer-assisted means. In this article, we demonstrate the utility of the software package TrakEM2 to model interconnections of nerve fibers from consecutive EM sections and to efficiently reconstruct the neural networks encountered in different parts of a small brain, the early larval brain of the fruit fly Drosophila melanogaster. Neuronal networks are composed of patterns of axons and dendrites (neuronal extensions that transmit and receive signals, respectively), and using TrakEM2, we describe the most common motifs they form. Our study introduces an approach towards a comprehensive anatomical reconstruction of neuronal microcircuitry and delivers microcircuitry comparisons between vertebrate and insect brains.
doi:10.1371/journal.pbio.1000502
PMCID: PMC2950124  PMID: 20957184
22.  In Vivo RNAi Rescue in Drosophila melanogaster with Genomic Transgenes from Drosophila pseudoobscura 
PLoS ONE  2010;5(1):e8928.
Background
Systematic, large-scale RNA interference (RNAi) approaches are very valuable to systematically investigate biological processes in cell culture or in tissues of organisms such as Drosophila. A notorious pitfall of all RNAi technologies are potential false positives caused by unspecific knock-down of genes other than the intended target gene. The ultimate proof for RNAi specificity is a rescue by a construct immune to RNAi, typically originating from a related species.
Methodology/Principal Findings
We show that primary sequence divergence in areas targeted by Drosophila melanogaster RNAi hairpins in five non-melanogaster species is sufficient to identify orthologs for 81% of the genes that are predicted to be RNAi refractory. We use clones from a genomic fosmid library of Drosophila pseudoobscura to demonstrate the rescue of RNAi phenotypes in Drosophila melanogaster muscles. Four out of five fosmid clones we tested harbour cross-species functionality for the gene assayed, and three out of the four rescue a RNAi phenotype in Drosophila melanogaster.
Conclusions/Significance
The Drosophila pseudoobscura fosmid library is designed for seamless cross-species transgenesis and can be readily used to demonstrate specificity of RNAi phenotypes in a systematic manner.
doi:10.1371/journal.pone.0008928
PMCID: PMC2812509  PMID: 20126626
23.  Motif composition, conservation and condition-specificity of single and alternative transcription start sites in the Drosophila genome 
Genome Biology  2009;10(7):R73.
A map of transcription start sites across the Drosophila genome, providing insights into initiation patterns and spatiotemporal conditions.
Background
Transcription initiation is a key component in the regulation of gene expression. mRNA 5' full-length sequencing techniques have enhanced our understanding of mammalian transcription start sites (TSSs), revealing different initiation patterns on a genomic scale.
Results
To identify TSSs in Drosophila melanogaster, we applied a hierarchical clustering strategy on available 5' expressed sequence tags (ESTs) and identified a high quality set of 5,665 TSSs for approximately 4,000 genes. We distinguished two initiation patterns: 'peaked' TSSs, and 'broad' TSS cluster groups. Peaked promoters were found to contain location-specific sequence elements; conversely, broad promoters were associated with non-location-specific elements. In alignments across other Drosophila genomes, conservation levels of sequence elements exceeded 90% within the melanogaster subgroup, but dropped considerably for distal species. Elements in broad promoters had lower levels of conservation than those in peaked promoters. When characterizing the distributions of ESTs, 64% of TSSs showed distinct associations to one out of eight different spatiotemporal conditions. Available whole-genome tiling array time series data revealed different temporal patterns of embryonic activity across the majority of genes with distinct alternative promoters. Many genes with maternally inherited transcripts were found to have alternative promoters utilized later in development. Core promoters of maternally inherited transcripts showed differences in motif composition compared to zygotically active promoters.
Conclusions
Our study provides a comprehensive map of Drosophila TSSs and the conditions under which they are utilized. Distinct differences in motif associations with initiation pattern and spatiotemporal utilization illustrate the complex regulatory code of transcription initiation.
doi:10.1186/gb-2009-10-7-r73
PMCID: PMC2728527  PMID: 19589141
24.  Globally optimal stitching of tiled 3D microscopic image acquisitions 
Bioinformatics  2009;25(11):1463-1465.
Motivation: Modern anatomical and developmental studies often require high-resolution imaging of large specimens in three dimensions (3D). Confocal microscopy produces high-resolution 3D images, but is limited by a relatively small field of view compared with the size of large biological specimens. Therefore, motorized stages that move the sample are used to create a tiled scan of the whole specimen. The physical coordinates provided by the microscope stage are not precise enough to allow direct reconstruction (Stitching) of the whole image from individual image stacks.
Results: To optimally stitch a large collection of 3D confocal images, we developed a method that, based on the Fourier Shift Theorem, computes all possible translations between pairs of 3D images, yielding the best overlap in terms of the cross-correlation measure and subsequently finds the globally optimal configuration of the whole group of 3D images. This method avoids the propagation of errors by consecutive registration steps. Additionally, to compensate the brightness differences between tiles, we apply a smooth, non-linear intensity transition between the overlapping images. Our stitching approach is fast, works on 2D and 3D images, and for small image sets does not require prior knowledge about the tile configuration.
Availability: The implementation of this method is available as an ImageJ plugin distributed as a part of the Fiji project (Fiji is just ImageJ: http://pacific.mpi-cbg.de/).
Contact: tomancak@mpi-cbg.de
doi:10.1093/bioinformatics/btp184
PMCID: PMC2682522  PMID: 19346324
25.  Selective maintenance of Drosophila tandemly arranged duplicated genes during evolution 
Genome Biology  2008;9(12):R176.
Genes occurring in conserved, tandemly-arrayed clusters in Drosophila melanogaster are co-expressed to a much higher extent than other duplicated genes.
Background
The physical organization and chromosomal localization of genes within genomes is known to play an important role in their function. Most genes arise by duplication and move along the genome by random shuffling of DNA segments. Higher order structuring of the genome occurs in eukaryotes, where groups of physically linked genes are co-expressed. However, the contribution of gene duplication to gene order has not been analyzed in detail, as it is believed that co-expression due to recent duplicates would obscure other domains of co-expression.
Results
We have catalogued ordered duplicated genes in Drosophila melanogaster, and found that one in five of all genes is organized as tandem arrays. Furthermore, among arrays that have been spatially conserved over longer periods than would be expected on the basis of random shuffling, a disproportionate number contain genes encoding developmental regulators. Using in situ gene expression data for more than half of the Drosophila genome, we find that genes in these conserved clusters are co-expressed to a much higher extent than other duplicated genes.
Conclusions
These results reveal the existence of functional constraints in insects that retain copies of genes encoding developmental and regulatory proteins as neighbors, allowing their co-expression. This co-expression may be the result of shared cis-regulatory elements or a shared need for a specific chromatin structure. Our results highlight the association between genome architecture and the gene regulatory networks involved in the construction of the body plan.
doi:10.1186/gb-2008-9-12-r176
PMCID: PMC2646280  PMID: 19087263

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