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We present the LiveFly toolbox for quantitative analysis of transcription dynamics in live Drosophila embryos. The toolbox allows users to process two-color 3D confocal movies acquired using nuclei-labeling and the fluorescent RNA-tagging system described in the previous chapter and export the nuclei's position as a function of time, their lineages and the intensity traces of the active loci. The toolbox, which is tailored for the context of Drosophila early development, is semiautomatic, and requires minimal user intervention. It also includes a tool to combine data from multiple movies and visualize several features of the intensity traces and the expression pattern.

Live imaging has been used in recent years for the understanding of dynamic processes in biology, such as embryo development. This was made possible by a combination of advancements in microscopy, leading to improved signal-to-noise ratios and better spatial and temporal resolutions, and by the development of new fluorescence markers, allowing for the quantification of protein expression and transcriptional dynamics in vivo. Here we describe a general protocol, which can be used in standard confocal microscopes to image early Drosophila melanogaster embryos, in order to learn about the transcriptional dynamics of a fluorescently labeled RNA.

Morphogen gradients provide concentration-dependent positional information along polarity axes. Although the dynamics of the establishment of these gradients is well described, precision and noise in the downstream activation processes remain elusive. A simple paradigm to address these questions is the Bicoid morphogen gradient that elicits a rapid step-like transcriptional response in young fruit fly embryos. Focusing on the expression of the major Bicoid target, hunchback (hb), at the onset of zygotic transcription, we used the MS2-MCP approach which combines fluorescent labeling of nascent mRNA with live imaging at high spatial and temporal resolution. Removing 36 putative Zelda binding sites unexpectedly present in the original MS2 reporter, we show that the 750 bp of the hb promoter are sufficient to recapitulate endogenous expression at the onset of zygotic transcription. After each mitosis, in the anterior, expression is turned on to rapidly reach a plateau with all nuclei expressing the reporter. Consistent with a Bicoid dose-dependent activation process, the time period required to reach the plateau increases with the distance to the anterior pole. Despite the challenge imposed by frequent mitoses and high nuclei-to-nuclei variability in transcription kinetics, it only takes 3 minutes at each interphase for the MS2 reporter loci to distinguish subtle differences in Bicoid concentration and establish a steadily positioned and steep (Hill coefficient ~ 7) expression boundary. Modeling based on the cooperativity between the 6 known Bicoid binding sites in the hb promoter region, assuming rate limiting concentrations of the Bicoid transcription factor at the boundary, is able to capture the observed dynamics of pattern establishment but not the steepness of the boundary. This suggests that a simple model based only on the cooperative binding of Bicoid is not sufficient to describe the spatiotemporal dynamics of early hb expression., Author summary During development, the first thing that an embryo needs to know is the orientation of its body. We study how the head-to-tail axis forms in the fruit fly embryo. To position the axis, the embryo relies on proteins called morphogens broadcasting instructions to other genes, so that cells know, depending on where they are along the axis, what they should become. The concentration of the Bicoid morphogen is much higher in the head of the embryo and lower towards the tail. Bicoid activates hunchback, which divides the embryo in two parts. By visualizing this process in real time in living embryos, we see the hunchback gene activated very efficiently in the head part, where each nucleus expresses the gene, whereas it is not expressed at all in the tail part. Intriguingly, the boundary separating the two domains is precisely positioned and becomes very steep in no more than three minutes. We use a modeling approach to understand how this is achieved so rapidly. Given the parameters of the system, we find that although our model is able to reproduce the fast dynamics of the process, it fails to reproduce the steepness of the boundary suggesting that a more complex approach is needed to capture the additional mechanisms involved.

Elongation of a short telomere depends on the action of multiple telomerase molecules, which are visible as telomerase RNA foci or clusters associated with telomeres in yeast and mammalian cells. How several telomerase molecules act on a single short telomere is unknown. Herein, we report that the telomeric noncoding RNA TERRA is involved in the nucleation of telomerase molecules into clusters prior to their recruitment at a short telomere. We find that telomere shortening induces TERRA expression, leading to the accumulation of TERRA molecules into a nuclear focus. Simultaneous time-lapse imaging of telomerase RNA and TERRA reveals spontaneous events of telomerase nucleation on TERRA foci in early S phase, generating TERRA-telomerase clusters. This cluster is subsequently recruited to the short telomere from which TERRA transcripts originate during S phase. We propose that telomere shortening induces noncoding RNA expression to coordinate the recruitment and activity of telomerase molecules at short telomeres.

The simultaneous expression of the hunchback gene in the numerous nuclei of the developing fly embryo gives us a unique opportunity to study how transcription is regulated in living organisms. A recently developed MS2-MCP technique for imaging nascent messenger RNA in living Drosophila embryos allows us to quantify the dynamics of the developmental transcription process. The initial measurement of the morphogens by the hunchback promoter takes place during very short cell cycles, not only giving each nucleus little time for a precise readout, but also resulting in short time traces of transcription. Additionally, the relationship between the measured signal and the promoter state depends on the molecular design of the reporting probe. We develop an analysis approach based on tailor made autocorrelation functions that overcomes the short trace problems and quantifies the dynamics of transcription initiation. Based on live imaging data, we identify signatures of bursty transcription initiation from the hunchback promoter. We show that the precision of the expression of the hunchback gene to measure its position along the anterior-posterior axis is low both at the boundary and in the anterior even at cycle 13, suggesting additional post-transcriptional averaging mechanisms to provide the precision observed in fixed embryos., Author Summary The fly embryo provides a natural laboratory to study the dynamics of transcription and its implications for the developing organism. Using live imaging experiments we investigate the nature of transcription regulation of the hunchback gene—the first to read out the maternal Bicoid gradient. While traditional time trace analysis methods based on OFF time distributions or autocorrelation functions fail for short signals, our tailored autocorrelation function overcomes these limitations revealing bursty dynamics that is reproducible between cell cycles and embryos. The inferred rates result in a lot of variability in the readout of nuclei sensing similar Bicoid concentrations, suggesting additional readout mechanisms than a one-to-one mapping of the input onto the output.