Towards a dynamic quantitative understanding of axis formation in Drosophila – AXOMORPH

The project is composed of five Tasks which are largely independent except for Task 1 (coordination). Each of the other Tasks (Task 2 to 5) corresponds to one of the four specific objectives of the proposal. Namely, Task 2 will be devoted to the dynamics of the Bicoid molecules, Task 3 to the mechanism concerning the thresholds required to set up boundaries, Task 4 to the memorization process and Task 5 to the mechanism of spatial averaging. The four research Tasks will performed in parallel by the various members of the consortium.
Importantly, given the interdisciplinary approaches proposed, all Tasks involve at least two Partners of the proposal. They are all organized to provide a favorable context to collaborations between the three Partners and the synergism required for efficient and rapid progress beyond the limit of knowledge regarding the specific question asked.

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In parallel to its power in helping to solve fundamental questions of developmental biology, the Bicoid morphogen in the Drosophila embryo provide a unique simple system to examine protein diffusion and the onset of de novo transcription within a developmental field. The originality and the novelty of this proposal rely on the development of challenging experimental approaches of leading-edge imaging to tackle these questions. They include i) fluorescence correlation spectroscopy, ii) multi-scale collection of fluorescent in situ hybridization data at the resolution of each nucleus on the whole organism and at different nuclear cycle and iii) the development of a strategy to follow the process of transcription in living organisms with a similar multi-scale resolution than with FISH. The accumulation of a huge amount of data will require their processing in order to understand at best their meaning and will provide strong support for modeling. The proposed models will quantitatively challenge our understanding of the molecular processes governing morphogen formation and activity, and in turns propose new quantitative experiments to test their theoretical predictions. The interplay of these two approaches will allow for a full understanding of the precision of morphogen gradients. Finally, these approaches should provide key landmark studies to adapt these new experimental and theoretical developments to more complex systems. In the longer term, this research should facilitate the development of reliable technologies enabling the quantitative monitoring of biological processes including disease detection and progression, or, therapeutic efficacy.

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Although much is known about the molecular components involved in signal transduction and gene expression, a challenging issue in developmental biology is to understand how the correct genes are turned on at the correct location in space and at the correct time during development to produce and coordinate the numerous cell types present in the adult organism. To understand the coordinated spatio-temporal expression of genes during development, it is now essential to understand the interplay between the molecular components involved in a quantitative manner. A plethora of transcription factors play critical role in development, from the acquisition of cell identity (patterning) to terminal differentiation. It is also clear that sufficient amounts of a given transcription factor are required for its activity and that threshold concentration limits are critical for appropriate responses. In extreme situations, some of these factors are distributed as concentration gradients and able to induce the expression of different sets of target genes as a function of their concentration. These factors, called morphogens, control molecular cascades transforming the information contained in their concentration gradient into spatial information allowing for the formation of discrete domains, each associated with a specific cell fate. Although the critical role of morphogens in axial patterning is now well established, it remains unclear how precisely is the morphogen concentration detected by the cell and how precise each step in the gene expression process acting downstream is. Understanding how these concentration gradients are established, how threshold activities emerge from these smooth gradients and how precise are these processes, in front of, for instance, varying size and environmental conditions constitutes a novel challenge in developmental biology, and is the general objective of this proposal.

To answer these questions, we propose to focus on one of the simplest examples of a morphogenetic gradient, the Bicoid gradient in the Drosophila fruit fly embryo. Our goal is to decipher the function of this gradient in a precise manner using quantitative approaches and their theoretical evaluation. This would be achieved through better understandings of both the mobility of the Bicoid protein in syncytial embryos and its transcriptional activity during the nuclear cycles 9 to 14. Towards these goals, we will use leading edge cell imaging approaches, providing quantitative and dynamic analyses of this gradient and its activity in the context of the developing embryo. The data collected at different scales (whole embryo, nucleus and given genomic locus) and as a function of time will be analyzed for correlations and statistical significance aiming to extract essential information for the understanding of these processes. In parallel, these data will be confronted to theoretical models and the possibility to use genetics will allow manipulating the system to create perturbation and test the accuracy of these models. Altogether, this project should allow determining how a precise Bicoid concentration gradient is established in about one hour in the embryo and how the transcriptional machinery is able to measure precisely these concentrations and provide in less than two hours precise transcriptional responses.

The strength of this proposal relies on an already existing fruitful collaboration between the team of Nathalie Dostatni Lantieri (Partner 1, Institut Curie, Paris, France) in Drosophila genetics and the team of Cécile Fradin (Partner 2, Mc Master University, Hamilton, Canada) in experimental biophysics. In addition, the accumulation of quantitative data on this system will highly benefit from the expertise of Mathieu Coppey (Partner 3, Laboratoire Kasstler Brossel, ENS, Paris) for data mining and Aleksandra Walczak (Partner 3, Laboratoire de physique théorique, ENS, Paris) for the elaboration of theoretical models.