Dynamics of hunchback translation in real-time and at single-mRNA resolution in the Drosophila embryo

Abstract

The Hunchback (Hb) transcription factor is critical for anterior-posterior patterning of the Drosophila embryo. Despite the maternal hb mRNA acting as a paradigm for translational regulation, due to its repression in the posterior of the embryo, little is known about the translatability of zygotically transcribed hb mRNAs. Here we adapt the SunTag system, developed for imaging translation at single mRNA resolution in tissue culture cells, to the Drosophila embryo to study the translation dynamics of zygotic hb mRNAs. Using single-molecule imaging in fixed and live embryos, we provide evidence for translational repression of zygotic SunTag-hb mRNAs. While the proportion of SunTag-hb mRNAs translated is initially uniform, translation declines from the anterior over time until it becomes restricted to a posterior band in the expression domain. We discuss how regulated hb mRNA translation may help establish the sharp Hb expression boundary, which is a model for precision and noise during developmental patterning. Overall, our data show how use of the SunTag method on fixed and live embryos is a powerful combination for elucidating spatiotemporal regulation of mRNA translation in Drosophila.

Modulating the bicoid gradient in space and time

“…Recently, it was demonstrated that the translation efficiency of ribosomes of the early Drosophila egg is not linear along the A-P axis, exemplified by an analysis on the gap gene hunchback ( hb ) [ 67 ]. A completely new hallmark of this report was that hb expression levels would not only be dependent on the binding of the necessary transcription factors driving the gene, e.g.…”

Live and fixed imaging of translation sites at single mRNA resolution in the Drosophila embryo

“…Introduction of the SunTag array into the protein of interest (POI), and subsequent binding of a single chain antibody-fluorescent protein fusion (scFv-FP) to the nascent SunTag peptides ( Figure 1 A), allows translation to be visualized in the Drosophila embryo ( Dufourt et al., 2021 ; Vinter et al., 2021 ). Insertion of SunTag at the start of the coding sequence enables rapid visualization of translation sites and maximizes their signal, facilitating early detection and quantitation.…”

“…Single molecule fluorescent in situ hybridization (smFISH) is used with SunTag labeling ( Figure 1 B) to visualize translation of individual mRNAs in fixed embryos, whereas the MS2/MCP system for mRNA detection is exploited for live imaging of translation. Insertion of 128 MS2 copies gives the required sensitivity for single mRNA detection ( Vinter et al. 2021 ; Dufourt et al.…”

“…Simultaneous detection of individual mRNAs using the MS2/MCP system or by smFISH allows translation sites to be identified and quantified. For complete information on the generation and use of this protocol, please refer to Vinter et al. (2021) .…”

“…For complete information on the generation and use of this protocol, please refer to Vinter et al. (2021) .…”