Transcriptional Reporters: Watching gene expression in color

A combination of two fluorescent proteins with different half-lives allows gene expression to be followed with improved time resolution.
  1. Julie H Simpson  Is a corresponding author
  1. University of California, Santa Barbara, United States

Cells are dynamic places and the levels of gene products – RNA molecules and proteins – inside a given cell change over time. Moreover, different types of cells contain different constellations of RNA molecules and proteins. These aspects of cell identity are controlled by gene expression – the process by which genes are transcribed to form messenger RNA (mRNA) molecules, some of which are then translated to produce proteins.

Many techniques are available to study gene expression in cells. Single-cell RNA sequencing provides a global view of the transcriptional profiles of cells (Bates et al., 2019). In fixed tissue samples, in situ hybridization can be used to detect mRNA molecules, while immunohistochemistry techniques involving antibodies can detect proteins. 'Enhancer bashing' methods have been used to identify the regulatory elements that govern when and where particular genes are expressed (Borok et al., 2010). 'Enhancer traps' and 'protein traps' rely on reporters – this is, genes that produce an easily detectable protein – to provide information on the expression of neighboring genes of interest (St Johnston, 2002). 

Fluorescent proteins are widely used as reporters for gene expression. When illuminated with certain wavelengths of light, these proteins emit fluorescent light of a characteristic color that can then be detected. There are also fluorescent proteins that change color over time or when exposed to light of a specific wavelength (Lin and Tsien, 2010). Most cells do not produce their own fluorescent proteins, so DNA constructs containing the sequence for the fluorescent protein have to be introduced. The insertion can happen either at the normal locus of the gene or at a defined landing site. Fluorescent proteins placed under the regulatory sequences of a gene of interest can then be used to report on the expression of that gene.

Previously, relationships between cells could be detected by inducing dividing cells to express one of several fluorescent proteins at specific time-points during development (Lee and Luo, 1999; Cachero and Jefferis, 2011). Now, in eLife, Li He, Norbert Perrimon and colleagues at Harvard Medical School, Chongqing University and Tufts University report how they have combined two fluorescent proteins with different half-lives to make a reporter (which they call a transcriptional timer or TransTimer) that can be used to explore the dynamics of gene expression in fruit flies (He et al., 2019).

A bright, fast-folding version of green fluorescent protein was selected and modified to speed up its translation. First, He et al. optimized protein synthesis by ensuring that the most common triplet codons available in the fly were used to make the protein. Next, sequences were added to make more of the fluorescent protein by increasing translation initiation, increasing transport of mRNA to the cytoplasm, and efficiently poly-adenylating the mRNA so it could be found by the translation machinery (Pfeiffer et al., 2012). Last, they added a sequence to target the protein for degradation in order to ensure a short half-life. This kind of careful protein engineering – with emphasis on temporal precision of the off switch – is similar to that which led to dramatic improvements in the calcium sensors that can report action potentials in neurons (Dana et al., 2019).

In vitro and in vivo tests showed that these modifications resulted in a rapid increase and decrease of the fluorescent signal, with green fluorescence being detected within 10 minutes and disappearing inside of two hours. A slow-folding, stable red fluorescent protein which can be detected after 1.5 hours, and which lasts for more than 20 hours, was added to generate the TransTimer reporter.

Due to the different folding and degradation times of the two proteins in the TransTimer, the onset of the green fluorescence is fast, followed by a more gradual rise in red fluorescence. If a gene is stably expressed, both green and red fluorescence will be detected. On the other hand, if a gene is dynamically expressed, after an initial period during which both colors can be detected, only red fluorescence will be observed. These fluorescent proteins are bright enough that the changes in color can be watched in living tissue, and potentially tracked in experiments involving long-term imaging of developing embryos (Royer et al., 2016).

He et al. demonstrated some of the applications of the TransTimer in the fruit fly. First, they showed that it can be used to detect short bursts of gene expression in fixed tissues (by measuring the proportions of green to red fluorescent proteins), and that it can help identify new genes with dynamic regulation through a TransTimer enhancer trap. Next it was shown that the TransTimer can be used to follow the gene expression history of different cell types. For example, neurons are sequentially produced by neuroblast stem cells, and key genes in this differentiation are transiently expressed. With the TransTimer, cells that currently express a gene will fluoresce both green and red, while cells that have already switched it off will only emit red fluorescence. In tissues like the eye or wing disc, this resulted in a leading edge of green/red cells followed by a wave of red ones.

The TransTimer could also have other applications in flies. First, it could allow the study of gene expression in dynamic processes such as circadian rhythms and metamorphosis. Second, it could provide real-time information about gene expression during cell fate assignments, complementing mRNA sequencing data, which only provides a snapshot of this process (Bates et al., 2019). Finally, the TransTimer's ability to record gene expression history could be used to answer mechanistic questions about the gene expression cascades that establish neuron identity in developing Drosophila brains (Doe, 2017). It should also be possible to adapt the TransTimer for use in other organisms.

The regulation of gene expression is complex and many questions remain. By showing when, where and how much a gene is expressed, reporters such as the TransTimer will be valuable tools in our efforts to better understand this process.

References

Article and author information

Author details

  1. Julie H Simpson

    Julie H Simpson is in the Department of Molecular, Cellular, and Developmental Biology and the Neuroscience Research Institute at the University of California, Santa Barbara, United States

    For correspondence
    jhsimpson@lifesci.ucsb.edu
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6793-7100

Publication history

  1. Version of Record published: July 26, 2019 (version 1)

Copyright

© 2019, Simpson

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 5,297
    views
  • 267
    downloads
  • 0
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Julie H Simpson
(2019)
Transcriptional Reporters: Watching gene expression in color
eLife 8:e49414.
https://doi.org/10.7554/eLife.49414

Further reading

    1. Developmental Biology
    Siyuan Cheng, Ivan Fan Xia ... Stefania Nicoli
    Research Article

    Vascular smooth muscle cells (VSMCs) envelop vertebrate brain arteries and play a crucial role in regulating cerebral blood flow and neurovascular coupling. The dedifferentiation of VSMCs is implicated in cerebrovascular disease and neurodegeneration. Despite its importance, the process of VSMC differentiation on brain arteries during development remains inadequately characterized. Understanding this process could aid in reprogramming and regenerating dedifferentiated VSMCs in cerebrovascular diseases. In this study, we investigated VSMC differentiation on zebrafish circle of Willis (CoW), comprising major arteries that supply blood to the vertebrate brain. We observed that arterial specification of CoW endothelial cells (ECs) occurs after their migration from cranial venous plexus to form CoW arteries. Subsequently, acta2+ VSMCs differentiate from pdgfrb+ mural cell progenitors after they were recruited to CoW arteries. The progression of VSMC differentiation exhibits a spatiotemporal pattern, advancing from anterior to posterior CoW arteries. Analysis of blood flow suggests that earlier VSMC differentiation in anterior CoW arteries correlates with higher red blood cell velocity and wall shear stress. Furthermore, pulsatile flow induces differentiation of human brain PDGFRB+ mural cells into VSMCs, and blood flow is required for VSMC differentiation on zebrafish CoW arteries. Consistently, flow-responsive transcription factor klf2a is activated in ECs of CoW arteries prior to VSMC differentiation, and klf2a knockdown delays VSMC differentiation on anterior CoW arteries. In summary, our findings highlight blood flow activation of endothelial klf2a as a mechanism regulating initial VSMC differentiation on vertebrate brain arteries.

    1. Developmental Biology
    Zhimin Xu, Zhao Wang ... Yingchuan B Qi
    Research Article

    Precise developmental timing control is essential for organism formation and function, but its mechanisms are unclear. In C. elegans, the microRNA lin-4 critically regulates developmental timing by post-transcriptionally downregulating the larval-stage-fate controller LIN-14. However, the mechanisms triggering the activation of lin-4 expression toward the end of the first larval stage remain unknown. We demonstrate that the transmembrane transcription factor MYRF-1 is necessary for lin-4 activation. MYRF-1 is initially localized on the cell membrane, and its increased cleavage and nuclear accumulation coincide with lin-4 expression timing. MYRF-1 regulates lin-4 expression cell-autonomously and hyperactive MYRF-1 can prematurely drive lin-4 expression in embryos and young first-stage larvae. The tandem lin-4 promoter DNA recruits MYRF-1GFP to form visible loci in the nucleus, suggesting that MYRF-1 directly binds to the lin-4 promoter. Our findings identify a crucial link in understanding developmental timing regulation and establish MYRF-1 as a key regulator of lin-4 expression.