John Reinitz

Dynamic control of positional information in the early Drosophila embryo

Morphogen gradients contribute to pattern formation by determining positional information in morphogenetic fields. Interpretation of positional information is thought to rely on direct, concentration-threshold-dependent mechanisms for establishing multiple differential domains of target gene expression. In Drosophila, maternal gradients establish the initial position of boundaries for zygotic gap gene expression, which in turn convey positional information to pair-rule and segment-polarity genes, the latter forming a segmental pre-pattern by the onset of gastrulation. Here we report, on the basis of quantitative gene expression data, substantial anterior shifts in the position of gap domains after their initial establishment. Using a data-driven mathematical modelling approach, we show that these shifts are based on a regulatory mechanism that relies on asymmetric gap–gap cross-repression and does not require the diffusion of gap proteins. Our analysis implies that the threshold-dependent interpretation of maternal morphogen concentration is not sufficient to determine shifting gap domain boundary positions, and suggests that establishing and interpreting positional information are not independent processes in the Drosophila blastoderm.

Canalization of gene expression in the Drosophila blastoderm by gap gene cross regulation.

Developing embryos exhibit a robust capability to reduce phenotypic variations that occur naturally or as a result of experimental manipulation. This reduction in variation occurs by an epigenetic mechanism called canalization, a phenomenon which has resisted understanding because of a lack of necessary molecular data and of appropriate gene regulation models. In recent years, quantitative gene expression data have become available for the segment determination process in the Drosophila blastoderm, revealing a specific instance of canalization. These data show that the variation of the zygotic segmentation gene expression patterns is markedly reduced compared to earlier levels by the time gastrulation begins, and this variation is significantly lower than the variation of the maternal protein gradient Bicoid. We used a predictive dynamical model of gene regulation to study the effect of Bicoid variation on the downstream gap genes. The model correctly predicts the reduced variation of the gap gene expression patterns and allows the characterization of the canalizing mechanism. We show that the canalization is the result of specific regulatory interactions among the zygotic gap genes. We demonstrate the validity of this explanation by showing that variation is increased in embryos mutant for two gap genes, Krüppel and knirps, disproving competing proposals that canalization is due to an undiscovered morphogen, or that it does not take place at all. In an accompanying article in PLoS Computational Biology (doi:10.1371/journal.pcbi.1000303), we show that cross regulation between the gap genes causes their expression to approach dynamical attractors, reducing initial variation and providing a robust output. These results demonstrate that the Bicoid gradient is not sufficient to produce gap gene borders having the low variance observed, and instead this low variance is generated by gap gene cross regulation. More generally, we show that the complex multigenic phenomenon of canalization can be understood at a quantitative and predictive level by the application of a precise dynamical model.

Quantitative and predictive model of transcriptional control of the Drosophila melanogaster even skipped gene

Here we present a quantitative and predictive model of the transcriptional readout of the proximal 1.7 kb of the control region of the Drosophila melanogaster gene even skipped (eve). The model is based on the positions and sequence of individual binding sites on the DNA and quantitative, time-resolved expression data at cellular resolution. These data demonstrated new expression features, first reported here. The model correctly predicts the expression patterns of mutations in trans, as well as point mutations, insertions and deletions in cis. It also shows that the nonclassical expression of stripe 7 driven by this fragment is activated by the protein Caudal (Cad), and repressed by the proteins Tailless (Tll) and Giant (Gt).

Canalization of gene expression and domain shifts in the Drosophila blastoderm by dynamical attractors.

The variation in the expression patterns of the gap genes in the blastoderm of the fruit fly Drosophila melanogaster reduces over time as a result of cross regulation between these genes, a fact that we have demonstrated in an accompanying article in PLoS Biology (see Manu et al., doi:10.1371/journal.pbio.1000049). This biologically essential process is an example of the phenomenon known as canalization. It has been suggested that the developmental trajectory of a wild-type organism is inherently stable, and that canalization is a manifestation of this property. Although the role of gap genes in the canalization process was established by correctly predicting the response of the system to particular perturbations, the stability of the developmental trajectory remains to be investigated. For many years, it has been speculated that stability against perturbations during development can be described by dynamical systems having attracting sets that drive reductions of volume in phase space. In this paper, we show that both the reduction in variability of gap gene expression as well as shifts in the position of posterior gap gene domains are the result of the actions of attractors in the gap gene dynamical system. Two biologically distinct dynamical regions exist in the early embryo, separated by a bifurcation at 53% egg length. In the anterior region, reduction in variation occurs because of stability induced by point attractors, while in the posterior, the stability of the developmental trajectory arises from a one-dimensional attracting manifold. This manifold also controls a previously characterized anterior shift of posterior region gap domains. Our analysis shows that the complex phenomena of canalization and pattern formation in the Drosophila blastoderm can be understood in terms of the qualitative features of the dynamical system. The result confirms the idea that attractors are important for developmental stability and shows a richer variety of dynamical attractors in developmental systems than has been previously recognized.

A database for management of gene expression data in situ

MOTIVATION To create a spatiotemporal atlas of Drosophila segmentation gene expression at cellular resolution. RESULTS The expression of segmentation genes plays a crucial role in the establishment of the Drosophila body plan. Using the IBM DB2 Relational Database Management System we have designed and implemented the FlyEx database. FlyEx contains 2832 images of 14 segmentation gene expression patterns obtained from 954 embryos and 2,073,662 quantitative data records. The averaged data is available for most of segmentation genes at eight time points. FlyEx supports operations on images of gene expression patterns. The database can be used to examine the quality of data, analyze the dynamics of formation of segmentation gene expression domains, as well as estimate the variability of gene expression patterns. We also provide the capability to download data of interest. AVAILABILITY http://urchin.spbcas.ru/flyex, http://flyex.ams.sunysb.edu/flyex

FlyEx, the quantitative atlas on segmentation gene expression at cellular resolution.

The datasets on gene expression are the valuable source of information about the functional state of an organism. Recently, we have acquired the large dataset on expression of segmentation genes in the Drosophila blastoderm. To provide efficient access to the data, we have developed the FlyEx database (http://urchin.spbcas.ru/flyex). FlyEx contains 4716 images of 14 segmentation gene expression patterns obtained from 1579 embryos and 9 500 000 quantitative data records. Reference data are available for all segmentation genes in cycles 11–13 and all temporal classes of cycle 14A. FlyEx supports operations on images of gene expression patterns. The database can be used to examine the quality of data, analyze the dynamics of formation of segmentation gene expression domains, as well as to estimate the variability of gene expression patterns. Currently, a user is able to monitor and analyze the dynamics of formation of segmentation gene expression domains over the whole period of segment determination, that amounts to 1.5 h of development. FlyEx supports the data downloads and construction of personal reference datasets, that makes it possible to more effectively use and analyze data.

Known Maternal Gradients are not Sufficient for the Establishment of Gap Domains in Drosophila melanogaster

Gap genes are among the first transcriptional targets of maternal morphogen gradients in the early Drosophila embryo. However, it remains unclear whether these gradients are indeed sufficient to establish the boundaries of localized gap gene expression patterns. In this study, we address this question using gap gene circuits, which are data-driven mathematical tools for extracting regulatory information from quantitative wild-type gene expression data. We present new, quantitative data on the mRNA expression patterns for the gap genes Krüppel (Kr), knirps (kni) and giant (gt) during the early blastoderm stage of Drosophila development. This data set shows significant differences in timing of gap gene expression compared to previous observations, and reveals that early gap gene expression is highly variable in both space and time. Gene circuit models fit to this data set were used for a detailed regulatory analysis of early gap gene expression. Our analysis shows that the proper balance of maternal repression and activation is essential for the correct positioning of gap domains, and that such balance can only be achieved for early expression of kni. In contrast, our results suggest that early expression of gt requires local neutralization of repressive input in the anterior region of the embryo, and that known maternal gradients are completely insufficient to position the boundaries of the early central Kr domain, or in fact any Kr-like domain in the central region of the blastoderm embryo. Based on this, we propose that unknown additional regulators must be involved in early gap gene regulation.

A high-throughput method for quantifying gene expression data from early Drosophila embryos

We describe an automated high-throughput method to measure protein levels in single nuclei in blastoderm embryos of Drosophila melanogaster by means of immunofluorescence. The method consists of a chain of specific algorithms assembled into an image processing pipeline. This pipeline transforms a confocal scan of an embryo stained with fluorescently tagged antibodies into a text file. This text file contains a numerical identifier for each nucleus, the coordinates of its centroid, and the average concentrations of three proteins in that nucleus. The central algorithmic component of the method is the automatic identification of nuclei by edge detection with the use of watersheds as an error-correction step. This method provides high-throughput quantification at cellular resolution.

Removal of background signal from in situ data on the expression of segmentation genes in Drosophila

Here we present a method for the removal of nonspecific background signal from fluorescently localized expression patterns of Drosophila segmentation genes. Our algorithm for removal of background signal brings the data to a common standard form with zero background and removes systematic error in gene expression levels caused by the presence of background. The method is based on the discovery, reported here, that background is well fit by a very broad two-dimensional paraboloid. The paraboloid is determined from the area of the embryo in which a given gene is not expressed and the whole pattern is then normalized by this paraboloid to remove background from the entire embryo. The software implementing this algorithm is available from the authors.

Transcriptional Control in Drosophila

We present a new model of transcriptional control. A central goal of this model is to show how modular enhancers arise from groups of binding sites. The model has a three-layer organization. The first layer describes the binding of activators and repressors to the regulatory region of a gene and incorporates the effects of repression by competition and quenching. The second layer describes adapter molecules binding to DNA-bound activators, and incorporates the effect of direct repression. Finally, the activation of transcription is modeled by an Arrhenius mechanism in which activating adapters lower the activation energy barrier. We show that this model is testable against transcription data derived from early Drosophila embryos. We believe this model is sufficiently refined to give a realistic account of the physiological consequences of complex interactions of regulatory molecules. The present approach supplements and supports, in an essential way, the insights into multigenic regulation derived from work aimed at formulating logical design principles for regulatory networks.