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

Thank you for submitting your article "In vivo study of gene expression with an enhanced dual-color fluorescent transcriptional timer" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by K VijayRaghavan as the Senior Editor and Hugo Bellen as the Reviewing Editor. The following individual involved in the review of your submission has agreed to reveal their identity: Julie Simpson (Reviewer #3).

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

Summary:

In this paper, He and colleagues generated a new in vivo reporter to detect spatial and temporal activity of gene expression by combining a fast-folding destabilized fluorescent protein (FP) and a slow-folding long-lived FP. While existing transcriptional reporters combined with stable FPs are successful in detecting signaling activities, this is at the expense of detecting dynamic changes due to the long-lived fluorescent signals. The authors improved spatial and temporal resolution of these reporters by generating short-lived GFP through the inclusion of PEST peptides (GFP-PEST). Since the shorter half-life of GST-PEST caused significant loss of signals, the authors combined super-folder GFP and translational enhancing elements. The utility of these features were demonstrated by generating STAT and Notch reporters. The authors next combined short-lived GFP/long-lived RFP reporters, and generated STAT reporters that can detect dynamic STAT activity in embryos by live imaging (or in fixed sample by ratiometric imaging of the FPs). To expand this tool for broader contexts (and requiring less genetic crosses), the authors generated a clever construct they called TransTimer that includes the self-cleaving 2A peptide between edGFP and RFP under the control of a single promoter. Using TransTimer with enhancer-trapped GAL4 lines or through insertion near an enhancer region allowed the discovery of new genes showing dynamic expression changes. Indeed, the authors found that a new gene, CG30159, is required for the maintenance of intestinal stem cells. Finally, the authors generated TransTimerLex, which achieved both detection and manipulation of gene expression at the same time.

This is a nicely designed study with strong proof of principle. Together, these different transcriptional reporters enhance the current tools/methods available to study dynamic transcriptional changes. They will be very useful to the wider Drosophila research committee, and as pointed out by the authors. The design elements should be transferrable to other research organisms as well. In summary, we believe this is a very clever tool that will be useful for the community and eLife readership.

Essential revisions:

1) When Transtimer is used as a direct reporter construct under the control of a gene's promoter and enhancer, it directly reflects the expression dynamics of that gene. It is more complex when Transtimer is used as a UAS construct. This use employs GAL4 which is a stable intermediary. The authors should model how using a stable intermediary such as GAL4 affects detection of signaling dynamics. This would strengthen the conclusion that can be reached using the Transtimer as a UAS construct. If there are already GAL4 reagents (e.g. generated as part of Flylight project) for any of the genes for which the authors have a direct reporter, a head to head comparison would be beneficial.