Abstract
Early embryo development requires maternal-to-zygotic transition, during which transcriptionally silent nuclei begin widespread gene expression during zygotic genome activation (ZGA).1-3 ZGA is vital for early cell fating and germ-layer specification,3,4 and ZGA timing is regulated by multiple mechanisms.1-5 However, controversies remain about whether these mechanisms are interrelated and vary among species6-10 and whether the timing of germ-layer-specific gene activation is temporally ordered.11,12 In some embryonic models, widespread ZGA onset is spatiotemporally graded,13,14 yet it is unclear whether the transcriptome follows this pattern. A major challenge in addressing these questions is to accurately measure the timing of each gene activation. Here, we metabolically label and identify the nascent transcriptome using 5-ethynyl uridine (5-EU) in Xenopus blastula embryos. We find that EU-RNA-seq outperforms total RNA-seq in detecting the ZGA transcriptome, which is dominated by transcription from maternal-zygotic genes, enabling improved ZGA timing determination. We uncover discrete spatiotemporal patterns for individual gene activation, a majority following a spatial pattern of ZGA that is correlated with a cell size gradient.14 We further reveal that transcription necessitates a period of developmental progression and that ZGA can be precociously induced by cycloheximide, potentially through elongation of interphase. Finally, most ectodermal genes are activated earlier than endodermal genes, suggesting a temporal orchestration of germ-layer-specific genes, potentially linked to the spatially graded pattern of ZGA. Together, our study provides fundamental new insights into the composition and dynamics of the ZGA transcriptome, mechanisms regulating ZGA timing, and its role in the onset of early cell fating.