A sensitive mNeonGreen reporter system to measure transcriptional dynamics in Drosophila development

Abstract

The gene regulatory network governing anterior-posterior axis formation in Drosophila is a well-established paradigm to study transcription in developmental biology. The rapid temporal dynamics of gene expression during early stages of development, however, are difficult to track with standard techniques. We optimized the bright and fast-maturing fluorescent protein mNeonGreen as a real-time, quantitative reporter of enhancer expression. We derive enhancer activity from the reporter fluorescence dynamics with high spatial and temporal resolution, using a robust reconstruction algorithm. By comparing our results with data obtained with the established MS2-MCP system, we demonstrate the higher detection sensitivity of our reporter. We used the reporter to quantify the activity of variants of a simple synthetic enhancer, and observe increased activity upon reduction of enhancer-promoter distance or addition of binding sites for the pioneer transcription factor Zelda. Our reporter system constitutes a powerful tool to study spatio-temporal gene expression dynamics in live embryos.

Fig. 1. A mNeonGreen reporter system to measure enhancer expression in living Drosophila melanogaster embryos.

a Composition of the mNeonGreen reporter construct including: Enhancer, basal DSCP promoter, the translation enhancer sequence IVS21 at the 5′UTR, the mNeonGreen fused to three nuclear localization signals and the terminator sequence p10. b Illustration of the use of a fluorescent protein as a transcriptional reporter. The time course of protein fluorescence (in green) is different in different portions of the embryo and carries information on the underlying dynamics of the total mRNA production (in orange), which can be computed using the reconstruction algorithm described in Supplementary Fig. 1. c–e Representative confocal slices of embryos carrying hb_ant-mNeonRep at three different time points during embryo development, showing mNeonGreen fluorescence in false colors. f Fluorescence time course traces corresponding to the average signal in 2% bins along the AP axis of the embryo, color coded by their position along the axis. g Fluorescence expression patterns along the AP axis. Each track corresponds to a different time of embryo development. h Time course of cumulative mRNA production in 2% bins along the AP axis of the embryo, color coded by their position along the axis. i Total mRNA production patterns at selected times of embryo development.

Fig. 2. The mNeonGreen reporter detects weaker expression patterns than the MS2 system.

a Confocal slice of the anterior region of an hb_ant-mNeonRep embryo, showing mNeonGreen fluorescence in false colors b Cumulative mRNA production patterns at different times of embryo development measured with the mNeonGreen reporter. c Confocal slice of the anterior region of a hb_ant-DSCP-MS2-yellow embryo, MCP-GFP fluorescence represented in false colors. d Cumulative mRNA production patterns for the hb_ant enhancer as a function of AP position, measured with the MS2-MCP system. Since the field of view is limited to about 30% of the embryo, data were pooled from two independent experiments, focusing on the regions 0–30 and 30–60% AP, respectively. The shaded area represents a region of the embryo for which no data was collected. e Comparison of the cumulative mRNA production patterns measured with the mNeonRep and MS2-MCP reporter systems, at all times and positions. r = Pearson’s correlation coefficient. f Confocal slice of the anterior region of a Bcd3-mNeonRep embryo, showing mNeonGreen fluorescence in false colors (g) Cumulative mRNA production patterns (maximum production level indicated by an arrow) at different times of embryo development measured with the mNeonGreen reporter. h Confocal slice of the anterior tip of a Bcd3-DSCP-MS2-yellow embryo, MCP-GFP fluorescence represented in false colors. The arrows indicate exemplary fluorescent spots. i Cumulative mRNA production patterns for the Bcd3 enhancer as function of AP position, measured with the MS2 system.

Fig. 3. Activity and dynamics of embryos carrying native Drosophila enhancers using the mNeon reporter system.

a and b. Confocal fluorescence sections of hb_ant and Kr_CD2 embryos just before gastrulation. c and d. total mRNA levels along the AP axis over time. Averages from three replicates of each enhancer. The inset in (d) zooms in on the evolution of the central stripe of the Kr_CD2 enhancer. The red line highlights the dynamic shift of the position of the expression peak. e and f. Total mRNA levels as a function of time, with AP position color-coded as indicated. Averages from three replicates of each enhancer. g Temporal dynamics for the anterior domain (dots) and the posterior/central stripe (crosses) of the two enhancers.

Fig. 4. Applying the mNeonGreen reporter to measure transcriptional dynamics of synthetic enhancers.

a–c Dynamics of the activity of the Bcd3-proximal, Bcd3 and Zld3Bcd3 enhancers, respectively. From top to bottom: sketch of enhancer architectures, illustrating arrangement of binding sites and distance from TSS. Cumulative mRNA production patterns along the AP axis, color coded by the time of embryo development (average of 3 biological replicates). Time course of cumulative mRNA production, color coded by position along the AP axis (average of 3 biological replicates). d Ratio of the cumulative mRNA production for Bcd3-proximal and Bcd3 enhancers as a function of time, color coded by the position along the AP axis. e Ratio of the cumulative mRNA production for Zld3Bcd3 and Bcd3 enhancers as a function of time, color coded by the position along the AP axis.