Imaging nascent transcription in wholemount vertebrate embryos to characterize zygotic genome activation

Abstract

A major event in early embryo development is the awakening of the embryonic genome, a process of large-scale transcriptional induction termed zygotic genome activation (ZGA). To understand how ZGA is controlled temporally and spatially, tools are required to image and quantify nascent transcription in wholemount embryos. In this chapter, we describe a metabolic labeling approach that leverages 5-ethynyl uridine (5-EU) incorporation into newly transcribed RNAs. Subsequently, click chemistry is used to conjugate these nascent transcripts to fluorophores for wholemount confocal imaging or biotin for RNA sequencing. Such an approach facilitates direct visualization of the global transcriptional state of each cell during early embryogenesis and provides a spatial map of gene expression activity. We describe this procedure for imaging nascent transcription in a vertebrate embryo Xenopus laevis, and use it as our model the onset of large-scale ZGA. Unlike cell culture systems in which 5-EU can be added to the media, metabolic labeling in Xenopus embryos requires microinjection in one-cell or two-cell stage embryos. This method is a powerful tool to quantify the nascent transcriptome at a single-cell level and to dissect mechanisms that control ZGA. We propose that this methodology can be applied broadly in other embryonic systems, and demonstrate the feasibility using zebrafish cleavage stage embryos. Finally, we demonstrate how to sequence the nascent transcriptome via 5-EU incorporation and separation of zygotic vs maternal RNAs. Altogether, our generalizable methodology will facilitate new insights into gene regulation and spatial patterning of ZGA during early embryogenesis.

Keywords: 5-Ethynyl uridine; Early embryogenesis; Nascent transcription; Single-cell transcriptome; Wholemount imaging; Zygotic genome activation.