Live imaging and quantitation of nascent transcription using the MS2/MCP system in the Drosophila embryo

Abstract

Summary Visualizing transcription live in Drosophila is providing important new insights into the spatiotemporal regulation of transcription. Here, we describe a protocol to visualize and quantitate transcription from gene loci that are tagged with MS2 stem-loop sequences in the Drosophila embryo. MS2 stem-loop sequences are recognized by a coat protein fused to a fluorescent protein and visualized with microscopy. We also describe an analysis pipeline to extract and subsequently quantify transcription dynamics. For complete details on the use and execution of this protocol, please refer to Hoppe et al. (2020) .

An improved Erk biosensor reveals oscillatory Erk dynamics driven by mitotic erasure during early development

“…Live imaging of Drosophila embryos was carried out as described 62 . Embryos were dechorionated in bleach and positioned laterally on top of a coverslip (No.…”

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

“… Use nail polish to secure the embryo coverslip to the other coverslips. Note: A movie of this mounting technique can be found in ( Hoppe and Ashe, 2021b ). …”

“…Depending on the movie length this can be time consuming. For a semi-automated way to remove background fluorescence see ( Hoppe and Ashe, 2021b ).…”

Far from home: the role of glial mRNA localization in synaptic plasticity

“…Translation of the glial protein Sparc, a negative regulator of excitatory synaptogenesis, was also detected locally in PAPs, but no studies were done to prove that excitatory synaptogenesis is impaired or impossible without Sparc mRNA in astrocytic projections (Sakers et al, 2017) Neuronal mRNAs have previously been successfully tagged with the MS2 loops which results in mRNA decorated with fluorescent proteins fused to the MS2 coat protein (MCP) (Park et al, 2014;Bertrand et al, 1998;Weil, Parton and Davis, 2010). MS2-MCP enables live tracking of mRNAs transport in neurons, as well as observing the activity-dependent behavior of transcripts of interest (Hoppe and Ashe, 2021), and has been successfully used in radial glia (Pilaz et al, 2016). However, as mentioned above, radial glia are a stem cell type and some radial glia do differentiate into neurons.…”

“…Neuronal mRNAs have previously been successfully tagged with the MS2 loops which results in mRNA decorated with fluorescent proteins fused to the MS2 coat protein (MCP) ( Bertrand et al 1998 ; Weil et al 2010 ; Park et al 2014 ). MS2–MCP enables live tracking of mRNAs transport in neurons, as well as observing the activity-dependent behavior of transcripts of interest ( Hoppe and Ashe 2021 ), and has been successfully used in radial glia ( Pilaz et al 2016 ). However, as mentioned above, radial glia are a stem cell type and some radial glia do differentiate into neurons.…”