Date : 19/7/2010

Internship proposal for : Master 1 or Master 2

Laboratory

Nuclear dynamics and genome plasticity
UMR 218 CNRS / Institut Curie
Pavillon Pasteur 26 rue d'Ulm 75248 PARIS
Director : Geneviève Almouzni
Website : http://www.curie.fr/recherche/themes/detail_equipe.cfm/lang/_fr/id_equipe/62.htm
Main discipline : Molecular biology/ Molecular genetics

Supervisor

Nathalie DOSTATNI
email : Cet e-mail est protégé contre les robots collecteurs de mails, votre navigateur doit accepter le Javascript pour le voir
phone : +33 156246690

Subjects / Tools-Methodologies

1 : Quantitative approche of transcription/Genetics in drosophila

2 : Morphogen concentration gradients/Morphogen gradients are used by various organisms to establish polarity along embryonic axes or within organ systems. In these systems, positional information is provided by the concentration of the morphogen which is detected by each cell in the target tissue and which give rise to differential expression of the various target genes responsible for the determination of cell identities. Although the critical role of morphogens in axial patterning is now well established, important mechanistic questions concerning their mode of action remain unanswered: how do morphogens become gradually distributed in a field of cells; how are they able to differentially control the expression of different target genes at different threshold levels, and finally to what extent do they contribute, directly or indirectly, to the precision of patterning. The Drosophila Bicoid transcription factor was the first morphogen identified. It is acting in a syncytial embryo, which has recently become approachable to multi-scale quantitative 4D analysis, owing to the development of in situ cytochemistry and live cell imaging and provides thus an ideal system to address these questions in a quantitative manner [1]. Recent studies indicate that the Bicoid gradient is precisely established in Drosophila embryos after eight nuclear divisions (cycle-9) [1] and that target protein expression is specified, five divisions later (cycle-14), with a precision corresponding to a relative difference of Bicoid concentration of 10% [2]. To understand how such precision is achieved, we used recently fluorescent in situ hybridization (FISH) to detect nascent transcripts of the hunchback target gene at their site of synthesis. Using this approach, we show that, as early as the onset of zygotic transcription (i.e. much earlier than previously proposed [3]), the expression of hunchback is precise and highly synchronous among most anterior hunchback loci [4]. To understand how these precision and synchrony were achieved so promptly after the gradient establishment, we revisited the mobility of Bicoid using fluorescent correlation spectroscopy (FCS) and found that a large fraction of nuclear Bcd-EGFP was much more mobile than observed by FRAP in the cytoplasm [5]. This faster mobility of Bicoid was also captured in the nucleus [4]. It explains how the transcriptional response of Bicoid can reach precision and synchrony so rapidly and resets the system within its physical limits [4, 5]. However, given the rapidity of nuclear divisions at these early stages and given the interruption of transcription during mitosis, this fast mobility of Bicoid remains too slow to allow a novel reading of its concentration at each interphase and suggests the existence of a memorization process that recalls this information from earlier cycles. Interestingly, maternal Hunchback is required for the synchrony of the response and is therefore likely involved in this memorization process [4].

3 : fluorescent in situ hybridization/The goal of the M2 project is to begin the quantitative exploration of the mechanism underlying the precision of the Bicoid transcriptional response. This will be achieved by determining to what extent Bicoid provides positional information along the Antero-posterior axis according to a threshold mechanism. Previous analysis of hunchback expression using RNA-FISH indicates that its transcription border is positioned at quantitatively different Bicoid concentrations thresholds in embryos expressing one versus two doses of Bicoid [4]. This suggests that another source of quantitative positional information, unevenly distributed in the embryo, contributes to the positioning of this border. The goal of the project will be to identify the other sources of maternal positional information involved in the spatial precision of the response. Toward this goal, the expression of hunchback will be analyzed by RNA-FISH in embryos expressing one or two doses of Bicoid in various mutants. Candidates are part of the classical maternal effects mutants of the anterior, posterior or terminal systems [1] and will be tested individually or in combination. This analysis will likely uncover new functions for these maternal determinants. This project highlights the power of the Drosophila embryo to develop cutting-edge quantitative approaches and to provide a unique system to examine, at the fundamental level, the variability in the onset of de novo transcription within a developmental field. Given the general importance of the concept of morphogenetic gradients in development and the multidisciplinary approach taken here, this project should be of interest to students in Biology or Physics concerned by fundamental questions in biological systems that can be quantitatively approached in order to be modeled at the level of an organism.

References:
1.Porcher A. and Dostatni N. The Bicoid morphogen system. Current Biology, 2010. 20, R249-R254.
2.Gregor T, Wieschaus EF, McGregor AP, Bialek W, and Tank DW. 2007. Cell 130:141. Stability and Nuclear Dynamics of the Bicoid Morphogen Gradient.
3.Gregor T, Tank DW, Wieschaus EF, and Bialek W. 2007. Cell 130:153. Probing the Limits to Positional Information.
4.Porcher A., Abu-Arish A., Huart S., Roelens B., Fradin C. and N Dostatni. The time to measure positional information: maternal Hunchback is required for the synchrony of the Bicoid transcriptional response at the onset of zygotic transcription. 2010. Development. 137:2795-2804.
5.Abu-Arish A., Porcher A., Czerwonka A., Dostatni N and C. Fradin. Fast mobility of Bicoid captured by fluorence correlation spectroascopy : Implication for the rapid establishment of its gradient. Biophysical Journal. 2010. 99 : .