Quantitative analysis of gene regulation in Drosophila
In this project, characterization of the MAPK signaling cascade was done using the terminal patterning of Drosophila embryo as an experimental system. MAPK acts downstream of the Torso receptor tyrosine kinase (RTK) whose activation is limited to the poles of the embryo. This results in a localized activation of MAPK which is essential for the specification of the embryonic termini. Active MAPK controls the expression of the tailless (tll) and huckebein (hkb) gap genes by downregulating a transcriptional repressor, Capicua (Cic). In this work we are interested in understanding the mechanisms that allow differential specification and precise positioning of gene expression boundaries. We quantitatively analyzed information flow from MAPK to Cic and the two target genes, tll and hkb, by constructing input-output maps in wild type embryos. Our results show that the sharp MAPK activation is translated into a graded gradient of Cic concentration and switch-like responses of tll and hkb genes. This indicates that the ultrasensitive response occurs at the level of the Cic repression which led us to test the hypothesis that Cic alone could be sufficient for tll and hkb boundary specification. However, data from mutants containing lower levels of Cic showed that boundaries of two genes become indistinguishable in terms of Cic concentration while still clearly different in terms of position. This suggests that MAPK downregulation of Cic alone is not sufficient to explain the observed localization and that, other factors are required for the robust patterning of the termini of the Drosophila embryo. To find the missing components of this regulatory system we have started an in vitro Yeast One Hybrid screen. This technique, allowing detection of protein-DNA interaction, was optimized for high-throughput screening. Together with a library Drosophila transcription factors we hope to identify the missing regulators of tll and hkb genes.
Department of Chemical Engineering and Lewis-Sigler Institute for Integrative Genomics, Princeton University; Laboratory of Systems Biology and Genetics; EPFL
Record created on 2010-11-05, modified on 2016-08-08