Abstract
Fluorescent transcriptional reporters are widely used as signaling reporters and biomarkers to monitor pathway activities and determine cell type identities. However, a large amount of dynamic information is lost due to the long half-life of the fluorescent proteins. To better detect dynamics, fluorescent transcriptional reporters can be destabilized to shorten their half-lives. However, applications of this approach in vivo are limited due to significant reduction of signal intensities. To overcome this limitation, we enhanced translation of a destabilized fluorescent protein and demonstrate the advantages of this approach by characterizing spatio-temporal changes of transcriptional activities in Drosophila. In addition, by combining a fast-folding destabilized fluorescent protein and a slow-folding long-lived fluorescent protein, we generated a dual-color transcriptional timer that provides spatio-temporal information about signaling pathway activities. Finally, we demonstrate the use of this transcriptional timer to identify new genes with dynamic expression patterns.
Highlights
The “on” kinetic of fluorescent proteins (FPs) have been improved by engineering fast folding FPs, which shorten the maturation time of FPs from more than 1 hr to less than 10 min[11,12]
Concluding Remarks In this study, we described a general and straightforward strategy to use destabilized transcriptional reporters in vivo and demonstrated its power in revealing the spatio-temporal dynamics of gene expression, which is missed by conventional transcriptional reporters
We generated a dualcolor TransTimer that encodes the transcriptional dynamics into a green-to-red color ratio which can be analyzed in fixed tissues
Summary
The copyright holder for this preprint It is made available under. Changes in gene expression are one of the key mechanisms that organisms use during both development and homeostasis. Gene expression is a highly dynamic process, which bears critical information about regulatory mechanisms and controls the fate of many biological processes[1,2]. Oscillatory or constant expression of the Notch effector Hes[1] dictates the choice of neuron stem cells between proliferation and differentiation[3]. During the development of fly compound eyes, simultaneous activation of EGF and Notch signals determines a cone cell fate[5], while cells that experience sequential expression of EGF and the Notch-ligand Delta differentiate into photoreceptor cells[6]
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