In vivostudy of gene expression with an enhanced dual-color fluorescent transcriptional timer

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.

A dynamic cell recruitment process drives growth of the Drosophila wing by overscaling the Vestigial expression pattern

“…So far, the evidence of a cell recruitment mechanism that patterns the Vg gradient has been indirect. In order to investigate more directly the dynamics of the cell recruitment process during normal development, we used a recently-developed tool of dual-color fluorescent reporters, known as TransTimer (He et al 2019), to examine the dynamics of the vg QE . The TransTimer expresses a rapid yet unstable GFP and a more stable, but slower RFP under vg QE Gal4 control.…”

“…For the evaluation of the vg QE dynamics (Fig. 4), UAS-TransTimer reporter, a destabilized GFP (dGFP) protein combined with a stable RFP (He et al 2019), was placed under vg QE Gal4 driver to quantify cells that recently activated this enhancer. Taking advantage of the different maturation rates of both proteins (dGFP about 0.1 h, and RFP about 1.5 h), we selected all the GFP pixels with intensity values above a threshold in the wing pouch for each disc; this threshold was set by ranking all the pixels within by their GFP intensity in three different groups using the machine learning K-Means function from scikit-learn python package (https://scikit-learn.org/stable/index.html) and all the pixels in the lower group were taken away.…”

“…If this ratio was lower than 0.4 at a specific pixel (this value corresponds to the half maximum for GFP taken from Fig. 3b in He et al 2019), we marked this pixel and considered it as new activation of the vg QE (Fig. 4A).…”