Verbrugghe, M. | bioRxiv Science

bioRxiv2024

Imaging Translation in Early Embryo Development

The ultimate output of gene expression is to ensure that proteins are synthesized at the right levels, locations, and timings. Recently different imaging-based methods have been developed to visualize the translation of single mRNA molecules. These methods rely on signal amplification with the introduction of an array of a short peptide sequence (a tag such as SunTag), recognized by a genetically encodable single-chain antibody (a detector such as scFv). In this chapter, we discuss such methods to image and quantify translation dynamics in the early Drosophila embryo and provide examples based on a twist-32XSunTag reporter. We outline a step-by-step protocol to light-up translation in living embryos. We also detail a combinatorial strategy in fixed samples (smFISH-IF), allowing to distinguish single mRNA molecules engaged in translation.

developmental biology↗

bioRxiv2024

Regulation of bicoid mRNA throughout oogenesis and early embryogenesis impacts protein gradient formation

The transcription factor Bicoid (Bcd) protein guides early Drosophila patterning and is the best-characterised morphogen. The source of the morphogen, bcd mRNA, is maternally deposited during oogenesis and localised to the anterior pole of the mature oocyte. While the spatiotemporal interpretation of the Bcd morphogen gradient has been intensely studied, when and where Bcd protein is produced and how this protein gradient is dynamically shaped remains contentious. Here, we use the SunTag reporter system to quantitatively examine the spatiotemporal profile of bcd mRNA translation in vivo. We show that association with Processing bodies (P bodies) in mature oocytes prevent premature bcd mRNA translation. Following egg activation, bcd mRNA dissociates from P bodies and translation is observed at the anterior pole. Translation remains restricted to the anterior domain throughout early development, even after nuclear migration in the syncytial blastoderm. At cellularisation, translation ceases and the remaining bcd mRNA associates with reformed P bodies, which appear to block any further translation. We use these observations to create a new modified source-diffusion-degradation model of Bcd gradient formation that has spatiotemporally varying production. Overall, our study reveals that bcd mRNA translation is tightly controlled in space and time during oogenesis and early embryogenesis.

developmental biology↗