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http://hdl.handle.net/11375/24604
Title: | Noise and Robustness downstream of a morphogen gradient: Quantitative approach by imaging transcription dynamics in living embryos |
Authors: | Perez Romero, Carmina Angelica |
Advisor: | Fradin, Cecile Dostatni, Nathalie |
Department: | Biochemistry |
Keywords: | embryonic development;bicoid;drosphila melanogaster;hunchback;gene networks;quantitative live imaging |
Publication Date: | 2019 |
Abstract: | During development, cell differentiation frequently occurs upon signaling from concentration or activity gradients of molecules called morphogens. These molecules control in a dose-dependent manner the expression of sets of target genes that determine cell identity. A simple paradigm to study morphogens is the Bicoid gradient, which determines antero-posterior patterning in fruit fly embryos. The Bicoid transcription factor allows the rapid step-like expression of its major target gene hunchback, expressed only in the anterior half of the embryo. The general goal of my thesis was to understand how the information contained in the Bicoid morphogen gradient is rapidly interpreted to provide the precise expression pattern of its target. Using the MS2 system to fluorescently tag specific RNA in living embryos, we were able to show that the ongoing transcription process at the hunchback promoter is bursty and likely functions according to a two-state model. At each nuclear interphase, transcription is first observed in the anterior and it rapidly spreads towards the posterior, as expected for a Bicoid dose-dependent activation process. Surprisingly, it takes only 3 minutes from the first hints of transcription at the anterior to reach steady state with the setting of a sharp expression border in the middle of the embryo. Using modeling taking into account this very fast dynamics, we show that the presence of only 6 Bicoid binding sites (known number of sites in the hunchback promoter) in the promoter, is not sufficient to explain the establishment of a sharp expression border in such a short time. Thus, either more Bicoid binding sites or inputs from other transcription factors could help reconcile the model to the data. To better understand the role of transcription factors other than Bicoid in this process, I used a two-pronged strategy involving synthetic MS2 reporters combined with the analysis of the hunchback MS2 reporter in various mutant backgrounds. I show that the pioneer factor Zelda and the Hunchback protein itself are also critical for hunchback expression, maternal Hunchback acting at nuclear cycle 11-12, while zygotic Hunchback is acting later at nuclear cycle 13-14. The synthetic reporter approach indicate that in contrast to Hunchback and Caudal, Bicoid is able to activate transcription on its own when bound to the promoter. However, the presence of 6 Bicoid binding sites only leads to stochastic activation of the target loci. Interestingly, the binding of Hunchback to the Bicoid-dependent promoter reduces this stochasticity while Caudal might act as a posterior repressor gradient. Confronting these experimental data to theoretical models is ongoing and should allow to better understand the role of transcription factors, other than Bicoid, in hunchback expression at the mechanistic level. |
Description: | This thesis was done in collaboration with Sorbonne University as part of a double degree Cotutelle. |
URI: | http://hdl.handle.net/11375/24604 |
Appears in Collections: | Open Access Dissertations and Theses |
Files in This Item:
File | Description | Size | Format | |
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PerezRomero_Carmina_A_201904_PhDBiochemistry.pdf | 17.21 MB | Adobe PDF | View/Open |
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