Hilde Janssens

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 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.

A quantitative atlas of Even-skipped and Hunchback expression in Clogmia albipunctata (Diptera: Psychodidae) blastoderm embryos

BackgroundComparative 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.ResultsWe 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.ConclusionsWe 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.

Gene Circuit Analysis of the Terminal Gap Gene huckebein

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.

Regulation of phototransduction responsiveness and retinal degeneration by a phospholipase D–generated signaling lipid

Drosophila melanogaster phototransduction proceeds via a phospholipase C (PLC)–triggered cascade of phosphatidylinositol (PI) lipid modifications, many steps of which remain undefined. We describe the involvement of the lipid phosphatidic acid and the enzyme that generates it, phospholipase D (Pld), in this process. Pld null flies exhibit decreased light sensitivity as well as a heightened susceptibility to retinal degeneration. Pld overexpression rescues flies lacking PLC from light-induced, metarhodopsin-mediated degeneration and restores visual signaling in flies lacking the PI transfer protein, which is a key player in the replenishment of the PI 4,5-bisphosphate (PIP2) substrate used by PLC to transduce light stimuli into neurological signals. Altogether, these findings suggest that Pld facilitates phototransduction by maintaining adequate levels of PIP2 and by protecting the visual system from metarhodopsin-induced, low light degeneration.

Pipeline for acquisition of quantitative data on segmentation gene expression from confocal images.

We describe a data pipeline developed to extract the quantitative data on segmentation gene expression from confocal images of gene expression patterns in Drosophila. The pipeline consists of 5 steps: image segmentation, background removal, temporal characterization of an embryo, data registration and data averaging. This pipeline was successfully applied to obtain quantitative gene expression data at cellular resolution in space and at the 6.5 minute resolution in time, as well as to construct a spatiotemporal atlas of segmentation gene expression. Each data pipeline step can be easily adapted to process a wide range of images of gene expression patterns.

Reverse-engineering post-transcriptional regulation of gap genes in Drosophila melanogaster.

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.