Visualization of Individual Scr mRNAs during Drosophila Embryogenesis Yields Evidence for Transcriptional Bursting

Abstract

SUMMARY The detection and counting of transcripts within single cells using Fluorescent in situ Hybridization (FISH) [1–6] has allowed researchers to ask quantitative questions about gene expression at the level of individual cells. This method is often preferable to quantitative RT-PCR [7–9], as it does not necessitate destruction of the cells being probed, and maintains spatial information that may be of interest. Until now studies using FISH at single molecule resolution have only been rigorously carried out in isolated cells (eg. yeast cells or mammalian cell culture). Here we describe the detection and counting of transcripts within single cells of fixed, whole-mount Drosophila embryos using a combination of FISH, immunohistochemistry, and image segmentation. Our method takes advantage of inexpensive, long RNA probes detected with antibodies [10, 11], and we present novel evidence to show that we can robustly detect single mRNA molecules. We use this method to characterize transcription at the endogenous locus of the Hox gene Sex combs reduced (Scr), by comparing a stably expressing group of cells to a group that only transiently expresses the gene. Our data provide evidence for transcriptional bursting [2, 5, 12–16], as well for divergent "accumulation" and “maintenance” phases of gene activity at the Scr locus.

Dynamic regulation of eve stripe 2 expression reveals transcriptional bursts in living Drosophila embryos

“…These fluctuations are evocative of transcriptional bursts reported in a number of other systems subject to live-image analysis, including bacteria, yeast, Dictyostelium, and cultured mammalian cells (18). Moreover, previous analysis based on fixed Drosophila embryos found evidence of transcriptional bursting at the Hox gene Scr (27) and in gap gene expression (17).…”

Live imaging of nascent RNA dynamics reveals distinct types of transcriptional pulse regulation

“…Within individual cells, transcription occurs as a series of irregular pulses, interspersed by long, irregular periods of inactivity. Pulsing (or bursting) is a fundamental feature of transcription, conserved from prokaryotes to mammalian cells (1)(2)(3)(4)(5). These phenomena have strong implications for our understanding of transcriptional mechanism and may provide a major source of stochasticity in gene expression (6), a driver of cell diversity in differentiation and disease (7,8).…”

Experimental and Modeling Approaches for Understanding the Effect of Gene Expression Noise in Biological Development

“…Intronic probes for nascent transcripts were used to demonstrate transcriptional bursting in segmentation genes [27]. Similar to a unicellular population, early Bcd-activated hb expression can be heterogeneous [28], but becomes more synchronized as spatial pattern matures [29]; this may be aided by a persistence of the transcriptional state through cell divisions [30].…”