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1.  Maternal Co-ordinate Gene Regulation and Axis Polarity in the Scuttle Fly Megaselia abdita 
PLoS Genetics  2015;11(3):e1005042.
Axis specification and segment determination in dipteran insects are an excellent model system for comparative analyses of gene network evolution. Antero-posterior polarity of the embryo is established through systems of maternal morphogen gradients. In Drosophila melanogaster, the anterior system acts through opposing gradients of Bicoid (Bcd) and Caudal (Cad), while the posterior system involves Nanos (Nos) and Hunchback (Hb) protein. These systems act redundantly. Both Bcd and Hb need to be eliminated to cause a complete loss of polarity resulting in mirror-duplicated abdomens, so-called bicaudal phenotypes. In contrast, knock-down of bcd alone is sufficient to induce double abdomens in non-drosophilid cyclorrhaphan dipterans such as the hoverfly Episyrphus balteatus or the scuttle fly Megaselia abdita. We investigate conserved and divergent aspects of axis specification in the cyclorrhaphan lineage through a detailed study of the establishment and regulatory effect of maternal gradients in M. abdita. Our results show that the function of the anterior maternal system is highly conserved in this species, despite the loss of maternal cad expression. In contrast, hb does not activate gap genes in this species. The absence of this activatory role provides a precise genetic explanation for the loss of polarity upon bcd knock-down in M. abdita, and suggests a general scenario in which the posterior maternal system is increasingly replaced by the anterior one during the evolution of the cyclorrhaphan dipteran lineage.
Author Summary
The basic head-to-tail polarity of an animal is established very early in development. In dipteran insects (flies, midges, and mosquitoes), polarity is established with the help of so-called morphogen gradients. Morphogens are regulatory proteins that are distributed as a concentration gradient, often involving diffusion from a localised source. This graded distribution then leads to the concentration-dependent activation of different target genes along the embryo’s axis. We examine this process, which differs to a surprising extent between dipteran species, in the scuttle fly Megaselia abdita, and compare our results to the model organism Drosophila melanogaster. In this way, we not only gain insights into how the mechanisms that establish polarity function differently in different species, but also how the system has evolved since these two flies shared a common ancestor. Specifically, we pin down the main difference between Drosophila and Megaselia in the altered function of the maternal Hunchback morphogen gradient, which activates target genes in the former, but not the latter species, where it has been completely replaced by the Bicoid morphogen during evolution.
PMCID: PMC4355411  PMID: 25757102
2.  High-resolution gene expression data from blastoderm embryos of the scuttle fly Megaselia abdita  
Scientific Data  2015;2:150005.
Gap genes are involved in segment determination during early development in dipteran insects (flies, midges, and mosquitoes). We carried out a systematic quantitative comparative analysis of the gap gene network across different dipteran species. Our work provides mechanistic insights into the evolution of this pattern-forming network. As a central component of our project, we created a high-resolution quantitative spatio-temporal data set of gap and maternal co-ordinate gene expression in the blastoderm embryo of the non-drosophilid scuttle fly, Megaselia abdita. Our data include expression patterns in both wild-type and RNAi-treated embryos. The data—covering 10 genes, 10 time points, and over 1,000 individual embryos—consist of original embryo images, quantified expression profiles, extracted positions of expression boundaries, and integrated expression patterns, plus metadata and intermediate processing steps. These data provide a valuable resource for researchers interested in the comparative study of gene regulatory networks and pattern formation, an essential step towards a more quantitative and mechanistic understanding of developmental evolution.
PMCID: PMC4423355  PMID: 25977812
3.  BioPreDyn-bench: a suite of benchmark problems for dynamic modelling in systems biology 
Dynamic modelling is one of the cornerstones of systems biology. Many research efforts are currently being invested in the development and exploitation of large-scale kinetic models. The associated problems of parameter estimation (model calibration) and optimal experimental design are particularly challenging. The community has already developed many methods and software packages which aim to facilitate these tasks. However, there is a lack of suitable benchmark problems which allow a fair and systematic evaluation and comparison of these contributions.
Here we present BioPreDyn-bench, a set of challenging parameter estimation problems which aspire to serve as reference test cases in this area. This set comprises six problems including medium and large-scale kinetic models of the bacterium E. coli, baker’s yeast S. cerevisiae, the vinegar fly D. melanogaster, Chinese Hamster Ovary cells, and a generic signal transduction network. The level of description includes metabolism, transcription, signal transduction, and development. For each problem we provide (i) a basic description and formulation, (ii) implementations ready-to-run in several formats, (iii) computational results obtained with specific solvers, (iv) a basic analysis and interpretation.
This suite of benchmark problems can be readily used to evaluate and compare parameter estimation methods. Further, it can also be used to build test problems for sensitivity and identifiability analysis, model reduction and optimal experimental design methods. The suite, including codes and documentation, can be freely downloaded from the BioPreDyn-bench website,
Electronic supplementary material
The online version of this article (doi:10.1186/s12918-015-0144-4) contains supplementary material, which is available to authorized users.
PMCID: PMC4342829  PMID: 25880925
Dynamic modelling; Model calibration; Parameter estimation; Optimization; Benchmarks; Large-scale; Metabolism; Transcription; Signal transduction; development
4.  The Comet Cometh: Evolving Developmental Systems 
Biological Theory  2015;10(1):36-49.
In a recent opinion piece, Denis Duboule has claimed that the increasing shift towards systems biology is driving evolutionary and developmental biology apart, and that a true reunification of these two disciplines within the framework of evolutionary developmental biology (EvoDevo) may easily take another 100 years. He identifies methodological, epistemological, and social differences as causes for this supposed separation. Our article provides a contrasting view. We argue that Duboule’s prediction is based on a one-sided understanding of systems biology as a science that is only interested in functional, not evolutionary, aspects of biological processes. Instead, we propose a research program for an evolutionary systems biology, which is based on local exploration of the configuration space in evolving developmental systems. We call this approach—which is based on reverse engineering, simulation, and mathematical analysis—the natural history of configuration space. We discuss a number of illustrative examples that demonstrate the past success of local exploration, as opposed to global mapping, in different biological contexts. We argue that this pragmatic mode of inquiry can be extended and applied to the mathematical analysis of the developmental repertoire and evolutionary potential of evolving developmental mechanisms and that evolutionary systems biology so conceived provides a pragmatic epistemological framework for the EvoDevo synthesis.
PMCID: PMC4357653  PMID: 25798078
Dynamical systems theory; Epistemology; Evolutionary developmental biology (EvoDevo); Evolutionary systems biology; Natural history of configuration space; Scientific perspectivism
5.  Quantitative system drift compensates for altered maternal inputs to the gap gene network of the scuttle fly Megaselia abdita 
eLife  null;4:e04785.
The segmentation gene network in insects can produce equivalent phenotypic outputs despite differences in upstream regulatory inputs between species. We investigate the mechanistic basis of this phenomenon through a systems-level analysis of the gap gene network in the scuttle fly Megaselia abdita (Phoridae). It combines quantification of gene expression at high spatio-temporal resolution with systematic knock-downs by RNA interference (RNAi). Initiation and dynamics of gap gene expression differ markedly between M. abdita and Drosophila melanogaster, while the output of the system converges to equivalent patterns at the end of the blastoderm stage. Although the qualitative structure of the gap gene network is conserved, there are differences in the strength of regulatory interactions between species. We term such network rewiring ‘quantitative system drift’. It provides a mechanistic explanation for the developmental hourglass model in the dipteran lineage. Quantitative system drift is likely to be a widespread mechanism for developmental evolution.
eLife digest
Similar biological phenomena can result from different processes occurring in different organisms. For example, the early stages of how an insect develops from an egg can vary substantially between different species. Nonetheless, all insects have a body plan that develops in segments. The same outcome occurring as a result of different developmental steps is known as ‘system drift’, but the mechanisms underlying this phenomenon are largely unknown.
How the body segments of the fruit fly Drosophila develop has been extensively studied. First, a female fruit fly adds messenger RNA (or mRNA) molecules copied from a number of genes into her egg cells. These mRNA molecules are then used to produce proteins whose concentration varies along the length of each egg. These proteins in turn switch on so-called ‘gap genes’ in differing amounts in different locations throughout the fruit fly embryo. The activity of these genes goes on to define the position and extent of specific segments along the fruit fly's body.
Like the fruit fly, the scuttle fly Megaselia abdita has a segmented body. However, mothers of this species deposit somewhat different protein gradients into their eggs. How the regulation of development differs in the scuttle fly to compensate for this change is unknown. Now, Wotton et al. have studied, in detail, how gap genes are regulated in this less well-understood fly species to understand the mechanisms responsible for a specific example of system drift.
In the fruit fly, gap genes normally switch-off (or reduce the expression of) other gap genes within the same developing body segment, and Wotton et al. found that the same kind of interactions tended to occur in the scuttle fly. As such, the overall structure of the gap gene network was fairly similar between scuttle and fruit flies. There were, however, differences in the strength of these interactions in the two fly species. These quantitative differences result in a different way of making the same segmental pattern in the embryo. In this way, Wotton et al. show how tinkering with the strength of specific gene interactions can provide an explanation for system drift.
PMCID: PMC4337606  PMID: 25560971
evolutionary developmental biology (evo-devo); developmental system drift; gap gene network; RNA interference; data quantification; Megaselia abdita; D. melanogaster; other
6.  SuperFly: a comparative database for quantified spatio-temporal gene expression patterns in early dipteran embryos 
Nucleic Acids Research  2014;43(Database issue):D751-D755.
We present SuperFly (, a relational database for quantified spatio-temporal expression data of segmentation genes during early development in different species of dipteran insects (flies, midges and mosquitoes). SuperFly has a special focus on emerging non-drosophilid model systems. The database currently includes data of high spatio-temporal resolution for three species: the vinegar fly Drosophila melanogaster, the scuttle fly Megaselia abdita and the moth midge Clogmia albipunctata. At this point, SuperFly covers up to 9 genes and 16 time points per species, with a total of 1823 individual embryos. It provides an intuitive web interface, enabling the user to query and access original embryo images, quantified expression profiles, extracted positions of expression boundaries and integrated datasets, plus metadata and intermediate processing steps. SuperFly is a valuable new resource for the quantitative comparative study of gene expression patterns across dipteran species. Moreover, it provides an interesting test set for systems biologists interested in fitting mathematical gene network models to data. Both of these aspects are essential ingredients for progress toward a more quantitative and mechanistic understanding of developmental evolution.
PMCID: PMC4383950  PMID: 25404137
7.  Classification of transient behaviours in a time-dependent toggle switch model 
BMC Systems Biology  2014;8:43.
Waddington’s epigenetic landscape is an intuitive metaphor for the developmental and evolutionary potential of biological regulatory processes. It emphasises time-dependence and transient behaviour. Nowadays, we can derive this landscape by modelling a specific regulatory network as a dynamical system and calculating its so-called potential surface. In this sense, potential surfaces are the mathematical equivalent of the Waddingtonian landscape metaphor. In order to fully capture the time-dependent (non-autonomous) transient behaviour of biological processes, we must be able to characterise potential landscapes and how they change over time. However, currently available mathematical tools focus on the asymptotic (steady-state) behaviour of autonomous dynamical systems, which restricts how biological systems are studied.
We present a pragmatic first step towards a methodology for dealing with transient behaviours in non-autonomous systems. We propose a classification scheme for different kinds of such dynamics based on the simulation of a simple genetic toggle-switch model with time-variable parameters. For this low-dimensional system, we can calculate and explicitly visualise numerical approximations to the potential landscape. Focussing on transient dynamics in non-autonomous systems reveals a range of interesting and biologically relevant behaviours that would be missed in steady-state analyses of autonomous systems. Our simulation-based approach allows us to identify four qualitatively different kinds of dynamics: transitions, pursuits, and two kinds of captures. We describe these in detail, and illustrate the usefulness of our classification scheme by providing a number of examples that demonstrate how it can be employed to gain specific mechanistic insights into the dynamics of gene regulation.
The practical aim of our proposed classification scheme is to make the analysis of explicitly time-dependent transient behaviour tractable, and to encourage the wider use of non-autonomous models in systems biology. Our method is applicable to a large class of biological processes.
PMCID: PMC4109741  PMID: 24708864
8.  A Staging Scheme for the Development of the Scuttle Fly Megaselia abdita 
PLoS ONE  2014;9(1):e84421.
Model organisms, such as Drosophila melanogaster, provide powerful experimental tools for the study of development. However, approaches using model systems need to be complemented by comparative studies for us to gain a deeper understanding of the functional properties and evolution of developmental processes. New model organisms need to be established to enable such comparative work. The establishment of new model system requires a detailed description of its life cycle and development. The resulting staging scheme is essential for providing morphological context for molecular studies, and allows us to homologise developmental processes between species. In this paper, we provide a staging scheme and morphological characterisation of the life cycle for an emerging non-drosophilid dipteran model system: the scuttle fly Megaselia abdita. We pay particular attention to early embryogenesis (cleavage and blastoderm stages up to gastrulation), the formation and retraction of extraembryonic tissues, and the determination and formation of germ (pole) cells. Despite the large evolutionary distance between the two species (approximately 150 million years), we find that M. abdita development is remarkably similar to D. melanogaster in terms of developmental landmarks and their relative timing.
PMCID: PMC3883658  PMID: 24409295
9.  A Staging Scheme for the Development of the Moth Midge Clogmia albipunctata 
PLoS ONE  2014;9(1):e84422.
Model organisms, such as Drosophila melanogaster, allow us to address a wide range of biological questions with experimental rigour. However, studies in model species need to be complemented by comparative studies if we are to fully understand the functional properties and evolutionary history of developmental processes. The establishment of new model organisms is crucial for this purpose. One of the first essential steps to establish a species as an experimental model is to carefully describe its life cycle and development. The resulting staging scheme serves as a framework for molecular studies, and allows us to homologise developmental processes between species. In this paper, we have characterised the life cycle and development of an emerging non-drosophilid dipteran model system: the moth midge Clogmia albipunctata. In particular, we focus on early embryogenesis (cleavage and blastoderm cycles before gastrulation), on formation and retraction of extraembryonic tissues, and on formation of the germ line. Considering the large evolutionary distance between the two species (approximately 250 million years), we find that the development of C. albipunctata is remarkably conserved compared to D. melanogaster. On the other hand, we detect significant differences in morphology and timing affecting the development of extraembryonic tissues and the germ line. Moreover, C. albipunctata shows several heterochronic shifts, and lacks head involution and associated processes during late stages of development.
PMCID: PMC3883683  PMID: 24409296
10.  A quantitative atlas of Even-skipped and Hunchback expression in Clogmia albipunctata (Diptera: Psychodidae) blastoderm embryos 
EvoDevo  2014;5:1.
Comparative studies of developmental processes are one of the main approaches to evolutionary developmental biology (evo-devo). Over recent years, there has been a shift of focus from the comparative study of particular regulatory genes to the level of whole gene networks. Reverse-engineering methods can be used to computationally reconstitute and analyze the function and dynamics of such networks. These methods require quantitative spatio-temporal expression data for model fitting. Obtaining such data in non-model organisms remains a major technical challenge, impeding the wider application of data-driven mathematical modeling to evo-devo.
We have raised antibodies against four segmentation gene products in the moth midge Clogmia albipunctata, a non-drosophilid dipteran species. We have used these antibodies to create a quantitative atlas of protein expression patterns for the gap gene hunchback (hb), and the pair-rule gene even-skipped (eve). Our data reveal differences in the dynamics of Hb boundary positioning and Eve stripe formation between C. albipunctata and Drosophila melanogaster. Despite these differences, the overall relative spatial arrangement of Hb and Eve domains is remarkably conserved between these two distantly related dipteran species.
We provide a proof of principle that it is possible to acquire quantitative gene expression data at high accuracy and spatio-temporal resolution in non-model organisms. Our quantitative data extend earlier qualitative studies of segmentation gene expression in C. albipunctata, and provide a starting point for comparative reverse-engineering studies of the evolutionary and developmental dynamics of the segmentation gene system.
PMCID: PMC3897886  PMID: 24393251
Clogmia albipunctata; Non-drosophilid diptera; Non-model organism; Pattern formation; Comparative network analysis; Segmentation gene network; Hunchback; Even-skipped; Image bioinformatics; Quantitative expression data
11.  Reverse-Engineering Post-Transcriptional Regulation of Gap Genes in Drosophila melanogaster 
PLoS Computational Biology  2013;9(10):e1003281.
Systems biology proceeds through repeated cycles of experiment and modeling. One way to implement this is reverse engineering, where models are fit to data to infer and analyse regulatory mechanisms. This requires rigorous methods to determine whether model parameters can be properly identified. Applying such methods in a complex biological context remains challenging. We use reverse engineering to study post-transcriptional regulation in pattern formation. As a case study, we analyse expression of the gap genes Krüppel, knirps, and giant in Drosophila melanogaster. We use detailed, quantitative datasets of gap gene mRNA and protein expression to solve and fit a model of post-transcriptional regulation, and establish its structural and practical identifiability. Our results demonstrate that post-transcriptional regulation is not required for patterning in this system, but is necessary for proper control of protein levels. Our work demonstrates that the uniqueness and specificity of a fitted model can be rigorously determined in the context of spatio-temporal pattern formation. This greatly increases the potential of reverse engineering for the study of development and other, similarly complex, biological processes.
Author Summary
The analysis of pattern-forming gene networks is largely focussed on transcriptional regulation. However, post-transcriptional events, such as translation and regulation of protein stability also play important roles in the establishment of protein expression patterns and levels. In this study, we use a reverse-engineering approach—fitting mathematical models to quantitative expression data—to analyse post-transcriptional regulation of the Drosophila gap genes Krüppel, knirps and giant, involved in segment determination during early embryogenesis. Rigorous fitting requires us to establish whether our models provide a robust and unique solution. We demonstrate, for the first time, that this can be done in the context of a complex spatio-temporal regulatory system. This is an important methodological advance for reverse-engineering developmental processes. Our results indicate that post-transcriptional regulation is not required for pattern formation, but is necessary for proper regulation of gap protein levels. Specifically, we predict that translation rates must be tuned for rapid early accumulation, and protein stability must be increased for persistence of high protein levels at late stages of gap gene expression.
PMCID: PMC3814631  PMID: 24204230
12.  Lack of tailless leads to an increase in expression variability in Drosophila embryos☆ 
Developmental Biology  2013;377(1):305-317.
Developmental processes are robust, or canalised: dynamic patterns of gene expression across space and time are regulated reliably and precisely in the presence of genetic and environmental perturbations. It remains unclear whether canalisation relies on specific regulatory factors (such as heat-shock proteins), or whether it is based on more general redundancy and distributed robustness at the network level. The latter explanation implies that mutations in many regulatory factors should exhibit loss of canalisation. Here, we present a quantitative characterisation of segmentation gene expression patterns in mutants of the terminal gap gene tailless (tll) in Drosophila melanogaster. Our analysis provides new insights into the dynamic mechanisms underlying gap gene regulation, and reveals significantly increased variability of gene expression in the mutant compared to the wild-type background. We show that both position and timing of posterior segmentation gene expression domains vary strongly from embryo-to-embryo in tll mutants. This variability must be caused by a vulnerability in the regulatory system which is hidden or buffered in the wild-type, but becomes uncovered by the deletion of tll. Our analysis provides evidence that loss of canalisation in mutants could be more widespread than previously thought.
► We present a quantitative analysis of spatial gene expression in Drosophila mutants. ► Dynamic gap domain shifts do not depend on expression of terminal gap genes or hb. ► Expression variability is greatly increased in a tll mutant background. ► This indicates de-canalisation (loss of developmental robustness) in the mutant. ► Such de-canalisation is a common phenomenon in mutants of developmental regulators.
PMCID: PMC3635121  PMID: 23333944
Drosophila embryogenesis; Segmentation gene network; Quantitative expression analysis; Pattern formation; Robustness/canalisation; Genetic capacitance
13.  Evolution and expression of BMP genes in flies 
Development Genes and Evolution  2013;223(5):335-340.
Bone morphogenetic proteins (BMPs) play key roles in development. In Drosophila melanogaster, there are three BMP-encoding genes: decapentaplegic (dpp), glass bottom boat (gbb) and screw (scw). dpp and gbb are found in all groups of insects. In contrast, the origin of scw via duplication of an ancestral gbb homologue is more recent, with new evidence placing it within the Diptera. Recent studies show that scw appeared basal to the Schizophora, since scw orthologues exist in aschizan cyclorrhaphan flies. In order to further localise the origin of scw, we have utilised new genomic resources for the nematoceran moth midge Clogmia albipunctata (Psychodidae). We identified the BMP subclass members dpp and gbb from an early embryonic transcriptome and show that their expression patterns in the blastoderm differ considerably from those seen in cyclorrhaphan flies. Further searches of the genome of C. albipunctata were unable to identify a scw-like gbb duplicate, but confirm the presence of dpp and gbb. Our phylogenetic analysis shows these to be clear orthologues of dpp and gbb from other non-cyclorrhaphan insects, with C. albipunctata gbb branching ancestrally to the cyclorrhaphan gbb/scw split. Furthermore, our analysis suggests that scw is absent from all Nematocera, including the Bibionomorpha. We conclude that the gbb/scw duplication occurred between the separation of the lineage leading to Brachycera and the origin of cyclorrhaphan flies 200–150 Ma ago.
Electronic supplementary material
The online version of this article (doi:10.1007/s00427-013-0445-9) contains supplementary material, which is available to authorized users.
PMCID: PMC3744649  PMID: 23595982
Bone morphogenetic proteins (BMPs); Phylogenetic analysis; Gene duplication; Diptera; Clogmia albipunctata
14.  Comparative transcriptomics of early dipteran development 
BMC Genomics  2013;14:123.
Modern sequencing technologies have massively increased the amount of data available for comparative genomics. Whole-transcriptome shotgun sequencing (RNA-seq) provides a powerful basis for comparative studies. In particular, this approach holds great promise for emerging model species in fields such as evolutionary developmental biology (evo-devo).
We have sequenced early embryonic transcriptomes of two non-drosophilid dipteran species: the moth midge Clogmia albipunctata, and the scuttle fly Megaselia abdita. Our analysis includes a third, published, transcriptome for the hoverfly Episyrphus balteatus. These emerging models for comparative developmental studies close an important phylogenetic gap between Drosophila melanogaster and other insect model systems. In this paper, we provide a comparative analysis of early embryonic transcriptomes across species, and use our data for a phylogenomic re-evaluation of dipteran phylogenetic relationships.
We show how comparative transcriptomics can be used to create useful resources for evo-devo, and to investigate phylogenetic relationships. Our results demonstrate that de novo assembly of short (Illumina) reads yields high-quality, high-coverage transcriptomic data sets. We use these data to investigate deep dipteran phylogenetic relationships. Our results, based on a concatenation of 160 orthologous genes, provide support for the traditional view of Clogmia being the sister group of Brachycera (Megaselia, Episyrphus, Drosophila), rather than that of Culicomorpha (which includes mosquitoes and blackflies).
PMCID: PMC3616871  PMID: 23432914
Non-drosophilid diptera; Clogmia albipunctata; Megaselia abdita; Episyrphus balteatus; Comparative transcriptomics; RNA-seq; De novo assembly; Automated annotation; Evolutionary developmental biology; Phylogenomics
15.  Medium-Throughput Processing of Whole Mount In Situ Hybridisation Experiments into Gene Expression Domains 
PLoS ONE  2012;7(9):e46658.
Understanding the function and evolution of developmental regulatory networks requires the characterisation and quantification of spatio-temporal gene expression patterns across a range of systems and species. However, most high-throughput methods to measure the dynamics of gene expression do not preserve the detailed spatial information needed in this context. For this reason, quantification methods based on image bioinformatics have become increasingly important over the past few years. Most available approaches in this field either focus on the detailed and accurate quantification of a small set of gene expression patterns, or attempt high-throughput analysis of spatial expression through binary pattern extraction and large-scale analysis of the resulting datasets. Here we present a robust, “medium-throughput” pipeline to process in situ hybridisation patterns from embryos of different species of flies. It bridges the gap between high-resolution, and high-throughput image processing methods, enabling us to quantify graded expression patterns along the antero-posterior axis of the embryo in an efficient and straightforward manner. Our method is based on a robust enzymatic (colorimetric) in situ hybridisation protocol and rapid data acquisition through wide-field microscopy. Data processing consists of image segmentation, profile extraction, and determination of expression domain boundary positions using a spline approximation. It results in sets of measured boundaries sorted by gene and developmental time point, which are analysed in terms of expression variability or spatio-temporal dynamics. Our method yields integrated time series of spatial gene expression, which can be used to reverse-engineer developmental gene regulatory networks across species. It is easily adaptable to other processes and species, enabling the in silico reconstitution of gene regulatory networks in a wide range of developmental contexts.
PMCID: PMC3460907  PMID: 23029561
16.  Efficient Reverse-Engineering of a Developmental Gene Regulatory Network 
PLoS Computational Biology  2012;8(7):e1002589.
Understanding the complex regulatory networks underlying development and evolution of multi-cellular organisms is a major problem in biology. Computational models can be used as tools to extract the regulatory structure and dynamics of such networks from gene expression data. This approach is called reverse engineering. It has been successfully applied to many gene networks in various biological systems. However, to reconstitute the structure and non-linear dynamics of a developmental gene network in its spatial context remains a considerable challenge. Here, we address this challenge using a case study: the gap gene network involved in segment determination during early development of Drosophila melanogaster. A major problem for reverse-engineering pattern-forming networks is the significant amount of time and effort required to acquire and quantify spatial gene expression data. We have developed a simplified data processing pipeline that considerably increases the throughput of the method, but results in data of reduced accuracy compared to those previously used for gap gene network inference. We demonstrate that we can infer the correct network structure using our reduced data set, and investigate minimal data requirements for successful reverse engineering. Our results show that timing and position of expression domain boundaries are the crucial features for determining regulatory network structure from data, while it is less important to precisely measure expression levels. Based on this, we define minimal data requirements for gap gene network inference. Our results demonstrate the feasibility of reverse-engineering with much reduced experimental effort. This enables more widespread use of the method in different developmental contexts and organisms. Such systematic application of data-driven models to real-world networks has enormous potential. Only the quantitative investigation of a large number of developmental gene regulatory networks will allow us to discover whether there are rules or regularities governing development and evolution of complex multi-cellular organisms.
Author Summary
To better understand multi-cellular organisms we need a better and more systematic understanding of the complex regulatory networks that govern their development and evolution. However, this problem is far from trivial. Regulatory networks involve many factors interacting in a non-linear manner, which makes it difficult to study them without the help of computers. Here, we investigate a computational method, reverse engineering, which allows us to reconstitute real-world regulatory networks in silico. As a case study, we investigate the gap gene network involved in determining the position of body segments during early development of Drosophila. We visualise spatial gap gene expression patterns using in situ hybridisation and microscopy. The resulting embryo images are quantified to measure the position of expression domain boundaries. We then use computational models as tools to extract regulatory information from the data. We investigate what kind, and how much data are required for successful network inference. Our results reveal that much less effort is required for reverse-engineering networks than previously thought. This opens the possibility of investigating a large number of developmental networks using this approach, which in turn will lead to a more general understanding of the rules and principles underlying development in animals and plants.
PMCID: PMC3395622  PMID: 22807664
17.  A matter of timing and precision 
PMCID: PMC3010115  PMID: 21081921
18.  The gap gene network 
Gap genes are involved in segment determination during the early development of the fruit fly Drosophila melanogaster as well as in other insects. This review attempts to synthesize the current knowledge of the gap gene network through a comprehensive survey of the experimental literature. I focus on genetic and molecular evidence, which provides us with an almost-complete picture of the regulatory interactions responsible for trunk gap gene expression. I discuss the regulatory mechanisms involved, and highlight the remaining ambiguities and gaps in the evidence. This is followed by a brief discussion of molecular regulatory mechanisms for transcriptional regulation, as well as precision and size-regulation provided by the system. Finally, I discuss evidence on the evolution of gap gene expression from species other than Drosophila. My survey concludes that studies of the gap gene system continue to reveal interesting and important new insights into the role of gene regulatory networks in development and evolution.
PMCID: PMC3016493  PMID: 20927566
Developmental biology; Evolution; Segment determination; Pattern formation; Gene regulatory network; Transcriptional regulation; Patterning precision; Size regulation
19.  Reverse engineering a gene network using an asynchronous parallel evolution strategy 
BMC Systems Biology  2010;4:17.
The use of reverse engineering methods to infer gene regulatory networks by fitting mathematical models to gene expression data is becoming increasingly popular and successful. However, increasing model complexity means that more powerful global optimisation techniques are required for model fitting. The parallel Lam Simulated Annealing (pLSA) algorithm has been used in such approaches, but recent research has shown that island Evolutionary Strategies can produce faster, more reliable results. However, no parallel island Evolutionary Strategy (piES) has yet been demonstrated to be effective for this task.
Here, we present synchronous and asynchronous versions of the piES algorithm, and apply them to a real reverse engineering problem: inferring parameters in the gap gene network. We find that the asynchronous piES exhibits very little communication overhead, and shows significant speed-up for up to 50 nodes: the piES running on 50 nodes is nearly 10 times faster than the best serial algorithm. We compare the asynchronous piES to pLSA on the same test problem, measuring the time required to reach particular levels of residual error, and show that it shows much faster convergence than pLSA across all optimisation conditions tested.
Our results demonstrate that the piES is consistently faster and more reliable than the pLSA algorithm on this problem, and scales better with increasing numbers of nodes. In addition, the piES is especially well suited to further improvements and adaptations: Firstly, the algorithm's fast initial descent speed and high reliability make it a good candidate for being used as part of a global/local search hybrid algorithm. Secondly, it has the potential to be used as part of a hierarchical evolutionary algorithm, which takes advantage of modern multi-core computing architectures.
PMCID: PMC2850326  PMID: 20196855
20.  Gene Circuit Analysis of the Terminal Gap Gene huckebein 
PLoS Computational Biology  2009;5(10):e1000548.
The early embryo of Drosophila melanogaster provides a powerful model system to study the role of genes in pattern formation. The gap gene network constitutes the first zygotic regulatory tier in the hierarchy of the segmentation genes involved in specifying the position of body segments. Here, we use an integrative, systems-level approach to investigate the regulatory effect of the terminal gap gene huckebein (hkb) on gap gene expression. We present quantitative expression data for the Hkb protein, which enable us to include hkb in gap gene circuit models. Gap gene circuits are mathematical models of gene networks used as computational tools to extract regulatory information from spatial expression data. This is achieved by fitting the model to gap gene expression patterns, in order to obtain estimates for regulatory parameters which predict a specific network topology. We show how considering variability in the data combined with analysis of parameter determinability significantly improves the biological relevance and consistency of the approach. Our models are in agreement with earlier results, which they extend in two important respects: First, we show that Hkb is involved in the regulation of the posterior hunchback (hb) domain, but does not have any other essential function. Specifically, Hkb is required for the anterior shift in the posterior border of this domain, which is now reproduced correctly in our models. Second, gap gene circuits presented here are able to reproduce mutants of terminal gap genes, while previously published models were unable to reproduce any null mutants correctly. As a consequence, our models now capture the expression dynamics of all posterior gap genes and some variational properties of the system correctly. This is an important step towards a better, quantitative understanding of the developmental and evolutionary dynamics of the gap gene network.
Author Summary
Currently, there are two very different approaches to the study of pattern formation: Traditional developmental genetics investigates the role of particular factors in great mechanistic detail, while newly developed systems-biology methods study many factors in parallel but usually remain rather general in their conclusions. Here, we attempt to bridge the gap between the two by studying the expression pattern and function of a particular developmental gene—the terminal gap gene huckebein (hkb) in the fruit fly Drosophila melanogaster—in great quantitative detail using a systems-level approach called the gene circuit method. Gene circuits are mathematical models which allow us to reconstitute a developmental process in the computer. This allows us to study the function of the hkb gene in its wild-type regulatory context with unprecedented accuracy and resolution. Our results confirm earlier, qualitative evidence, and show that hkb plays a small, but crucial role in gap gene regulation. Understanding hkb's regulatory contributions is essential for our wider understanding of dynamic shifts in the position of gap gene expression domains which play important roles during both development and evolution.
PMCID: PMC2760955  PMID: 19876378
21.  Getting the Measure of Positional Information 
PLoS Biology  2009;7(3):e1000081.
Quantitative measurements and mathematical modeling finally allow us to probe the limits of precision in developmental systems and reveal the importance of feedback regulation for developmental robustness.
PMCID: PMC2661971  PMID: 19338391
22.  Parameter estimation and determinability analysis applied to Drosophila gap gene circuits 
BMC Systems Biology  2008;2:83.
Mathematical modeling of real-life processes often requires the estimation of unknown parameters. Once the parameters are found by means of optimization, it is important to assess the quality of the parameter estimates, especially if parameter values are used to draw biological conclusions from the model.
In this paper we describe how the quality of parameter estimates can be analyzed. We apply our methodology to assess parameter determinability for gene circuit models of the gap gene network in early Drosophila embryos.
Our analysis shows that none of the parameters of the considered model can be determined individually with reasonable accuracy due to correlations between parameters. Therefore, the model cannot be used as a tool to infer quantitative regulatory weights. On the other hand, our results show that it is still possible to draw reliable qualitative conclusions on the regulatory topology of the gene network. Moreover, it improves previous analyses of the same model by allowing us to identify those interactions for which qualitative conclusions are reliable, and those for which they are ambiguous.
PMCID: PMC2586632  PMID: 18817540
23.  Reverse Engineering the Gap Gene Network of Drosophila melanogaster  
PLoS Computational Biology  2006;2(5):e51.
A fundamental problem in functional genomics is to determine the structure and dynamics of genetic networks based on expression data. We describe a new strategy for solving this problem and apply it to recently published data on early Drosophila melanogaster development. Our method is orders of magnitude faster than current fitting methods and allows us to fit different types of rules for expressing regulatory relationships. Specifically, we use our approach to fit models using a smooth nonlinear formalism for modeling gene regulation (gene circuits) as well as models using logical rules based on activation and repression thresholds for transcription factors. Our technique also allows us to infer regulatory relationships de novo or to test network structures suggested by the literature. We fit a series of models to test several outstanding questions about gap gene regulation, including regulation of and by hunchback and the role of autoactivation. Based on our modeling results and validation against the experimental literature, we propose a revised network structure for the gap gene system. Interestingly, some relationships in standard textbook models of gap gene regulation appear to be unnecessary for or even inconsistent with the details of gap gene expression during wild-type development.
Modeling dynamical systems involves determining which elements of the system interact with which, and what is the nature of the interaction. In the context of modeling gene expression dynamics, this question equates to determining regulatory relationships between genes. Perkins and colleagues present a new computational method for fitting differential equation models of time series data, and apply it to expression data from the well-known segmentation network of Drosophila melanogaster. The method is orders of magnitude faster than other approaches that produce fits of comparable quality, such as Simulated Annealing. The authors show that it is possible to detect interactions de novo as well as to test existing regulatory hypotheses, and they propose a revised network structure for the gap gene system, based on their modeling efforts and on other experimental literature.
PMCID: PMC1463021  PMID: 16710449

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