A bistable autoregulatory module in the developing embryo commits cells to binary expression fates.

Bistable autoactivation has been proposed as a mechanism for cells to adopt binary fates during embryonic development. However, it is unclear whether the autoactivating modules found within developmental gene regulatory networks are bistable, unless their parameters are quantitatively determined. Here, we combine in vivo live imaging with mathematical modeling to dissect the binary cell fate dynamics of the fruit fly pair-rule gene fushi tarazu (ftz), which is regulated by two known enhancers: the early (non-autoregulating) element and the autoregulatory element. Live imaging of transcription and protein concentration in the blastoderm revealed that binary Ftz fates are achieved as Ftz expression rapidly transitions from being dictated by the early element to the autoregulatory element. Moreover, we discovered that Ftz concentration alone is insufficient to activate the autoregulatory element, and that this element only becomes responsive to Ftz at a prescribed developmental time. Based on these observations, we developed a dynamical systems model and quantitated its kinetic parameters directly from experimental measurements. Our model demonstrated that the ftz autoregulatory module is indeed bistable and that the early element transiently establishes the content of the binary cell fate decision to which the autoregulatory module then commits. Further in silico analysis revealed that the autoregulatory element locks the Ftz fate quickly, within 35 min of exposure to the transient signal of the early element. Overall, our work confirms the widely held hypothesis that autoregulation can establish developmental fates through bistability and, most importantly, provides a framework for the quantitative dissection of cellular decision-making

How complex an intron may be? The example of the first intron of the CTP synthase gene of Drosophila melanogaster

In eukaryotes, maturation of primary transcripts into mature messenger RNAs involves the elimination of parts of the gene called ‘introns’. The biological significance of introns is not yet completely understood. It has been demonstrated that introns may contain other genes, or regulatory sequences that may be involved in transcriptional control, or also being involved in alternative splicing mechanisms. However, these functions explain the role of only a small number of them, and it is very difficult to formulate any generalization. The CTP synthase gene of Drosophila melanogaster is characterized by the presence of a long first intron (approximately 7.2 kilobases) whose role is currently unknown. In the present report we analyzed in silico the content of this intron, and found that it contains at least three interesting sub-sequences. Two of them are homologous to the CTP synthase itself and to a putative nucleotide pyrophosphatase, respectively. The third is a short stretch of DNA able to fold into a thermodynamically stable hairpin and showing homology with other 19 sequences from 21 genes inside the D. melanogaster genome. These findings suggest a complex yet very accurate way of controlling gene expression inside the fruit fly

Neurofly 2008 abstracts : the 12th European Drosophila neurobiology conference 6-10 September 2008 Wuerzburg, Germany

This volume consists of a collection of conference abstracts

Su(H)-Mediated Repression Positions Gene Boundaries along the Dorsal-Ventral Axis of Drosophila Embryos

In Drosophila embryos, a nuclear gradient of the Dorsal (Dl) transcription factor directs differential gene expression along the dorsoventral (DV) axis, translating it into distinct domains that specify future mesodermal, neural, and ectodermal territories. However, the mechanisms used to differentially position gene expression boundaries along this axis are not fully understood. Here, using a combination of approaches, including mutant phenotype analyses and chromatin immunoprecipitation, we show that the transcription factor Suppressor of Hairless, Su(H), helps define dorsal boundaries for many genes expressed along the DV axis. Synthetic reporter constructs also provide molecular evidence that Su(H) binding sites support repression and act to counterbalance activation through Dl and the ubiquitous activator Zelda. Our study highlights a role for broadly expressed repressors, like Su(H), and organization of transcription factor binding sites within cis-regulatory modules as important elements controlling spatial domains of gene expression to facilitate flexible positioning of boundaries across the entire DV axis

Topology and Robustness in the Drosophila Segment Polarity Network

A complex hierarchy of genetic interactions converts a single-celled Drosophila melanogaster egg into a multicellular embryo with 14 segments. Previously, von Dassow et al. reported that a mathematical model of the genetic interactions that defined the polarity of segments (the segment polarity network) was robust (von Dassow et al. 2000). As quantitative information about the system was unavailable, parameters were sampled randomly. A surprisingly large fraction of these parameter sets allowed the model to maintain and elaborate on the segment polarity pattern. This robustness is due to the positive feedback of gene products on their own expression, which induces individual cells in a model segment to adopt different stable expression states (bistability) corresponding to different cell types in the segment polarity pattern. A positive feedback loop will only yield multiple stable states when the parameters that describe it satisfy a particular inequality. By testing which random parameter sets satisfy these inequalities, I show that bistability is necessary to form the segment polarity pattern and serves as a strong predictor of which parameter sets will succeed in forming the pattern. Although the original model was robust to parameter variation, it could not reproduce the observed effects of cell division on the pattern of gene expression. I present a modified version that incorporates recent experimental evidence and does successfully mimic the consequences of cell division. The behavior of this modified model can also be understood in terms of bistability in positive feedback of gene expression. I discuss how this topological property of networks provides robust pattern formation and how large changes in parameters can change the specific pattern produced by a network

Evolution of the extraembryonic tissue in flies: from Megaselia abdita to Drosophila melanogaster

Embryonic development establishes the body plan, organs, and the shape of the adult animal organism. This process involves cells and tissues that eventually will not be part of the growing embryo, so-called extraembryonic tissues. In insects, extraembryonic tissues contribute to embryonic development by fulfilling important roles during specific morphogenetic movements, such as blastokinesis, germband retraction and dorsal closure, but also in the protection of the embryo against egg desiccation and pathogens. Within insects, extraembryonic tissues differ in number and topology, they may display diverse morphologies even between closely related species, and it is currently not yet clear which specific function each extraembryonic tissue fulfills and how its development is genetically regulated. Most of our current understanding of extraembryonic development and function in insects stems from studies in Tribolium castaneum and Drosophila melanogaster. The two species show several morphological differences, not only at the extraembryonic level but also in the morphology of the embryo. Specifically, T. castaneum has two extraembryonic tissues called amnion and serosa: the serosa separates from the embryo, grows over it, and eventually encloses the embryo, the amnion stays attached to the embryo and covers its ventral side. D. melanogaster, by contrast, develops only one single extraembryonic tissue called amnioserosa that remains in constant contact with the embryo and stays on its dorsal side. The diversity in form, development, and function of extraembryonic tissues in insect species provides an outstanding model to address how form and function of specific epithelia evolved, and how these changes were genetically encoded. In my thesis, I have taken advantage of intermediate characters in extraembryonic development of Megaselia abdita (Diptera, Phoridae), which features a similar embryonic development as D. melanogaster but maintained two extraembryonic tissues and thus part of the ancestral extraembryonic development described in T. castaneum. I have focused my attention on a detailed in vivo analysis of extraembryonic development at a morphogenetic and cellular level by establishing and using light-sheet microscopy. I acquired evidence that links extraembryonic tissues behavior in M. abdita to orthologues of the T-box transcription factor Dorsocross and the tumor necrosis factor Eiger, which in D. melanogaster are key genes that contribute to specification and morphogenesis of the amnioserosa. In vivo observations and functional studies suggest an important interaction of the extraembryonic tissues of M. abdita with the extracellular matrix that seems to be finely regulated. In conclusion, the results of this study increase our knowledge on morphology and development of extraembryonic tissues in M. abdita and provided an in vivo technique for non-model organisms to study in toto dynamics of early development

Inferring Drosophila gap gene regulatory network: a parameter sensitivity and perturbation analysis

<p>Abstract</p> <p>Background</p> <p>Inverse modelling of gene regulatory networks (GRNs) capable of simulating continuous spatio-temporal biological processes requires accurate data and a good description of the system. If quantitative relations between genes cannot be extracted from direct measurements, an efficient method to estimate the unknown parameters is mandatory. A model that has been proposed to simulate spatio-temporal gene expression patterns is the connectionist model. This method describes the quantitative dynamics of a regulatory network in space. The model parameters are estimated by means of model-fitting algorithms. The gene interactions are identified without making any prior assumptions concerning the network connectivity. As a result, the inverse modelling might lead to multiple circuits showing the same quantitative behaviour and it is not possible to identify one optimal circuit. Consequently, it is important to address the quality of the circuits in terms of model robustness.</p> <p>Results</p> <p>Here we investigate the sensitivity and robustness of circuits obtained from reverse engineering a model capable of simulating measured gene expression patterns. As a case study we use the early gap gene segmentation mechanism in <it>Drosophila melanogaster</it>. We consider the limitations of the connectionist model used to describe GRN Inferred from spatio-temporal gene expression. We address the problem of circuit discrimination, where the selection criterion within the optimization technique is based of the least square minimization on the error between data and simulated results.</p> <p>Conclusion</p> <p>Parameter sensitivity analysis allows one to discriminate between circuits having significant parameter and qualitative differences but exhibiting the same quantitative pattern. Furthermore, we show that using a stochastic model derived from a deterministic solution, one can introduce fluctuations within the model to analyze the circuits' robustness. Ultimately, we show that there is a close relation between circuit sensitivity and robustness to fluctuation, and that circuit robustness is rather modular than global. The current study shows that reverse engineering of GRNs should not only focus on estimating parameters by minimizing the difference between observation and simulation but also on other model properties. Our study suggests that multi-objective optimization based on robustness and sensitivity analysis has to be considered.</p