Summary. During early development, specification of the three primordial germ layers, ectoderm, mesoderm, and endoderm, is the first major step in defining embryonic tissues, and dysregulation of this process leads to developmental defects. Germ layer formation is a fundamental focus of developmental biology; however, little is known about an emerging concept that the initiation of their expression follows a temporal hierarchy. The ordering is debated in different embryonic models and importantly it is not known whether proper coordination of timing is essential for development. The cell differentiation that accompanies germ layer formation largely occurs in gastrulation. Yet, many tissue-specific genes that specify individual germ layers initiate their expression in blastula stages following genome activation, requiring methods to track the onset of their nascent transcription. We developed a new method for quantifying the blastula nascent transcriptome using 5-ethynyl uridine (EU) incorporation and isolation, termed EU-RNA-seq. This method has increased sensitivity relative to conventional RNA-seq and enables delicate measurement of time-of-onset for maternal-zygotic transcripts. By analyzing germ layer expression in the model vertebrate Xenopus laevis, we discovered that the ectoderm genes are expressed earlier than endoderm genes, suggesting a hierarchical onset of germ layer expression that is consistent with recent observations made in mouse. However, the mechanisms underlying the hierarchical onset of germ layer expression remain largely unknown. Interestingly, the phased onset is consistent with our recent finding that large-scale zygotic transcription initiates in a spatially graded manner, first in cells at the animal pole (AP, the presumptive ectoderm) and delayed in cells at the vegetal pole (VP, the presumptive endoderm) in blastula embryos. Epigenetic regulation, including chromatin accessibility and histone modifications, have been shown to play a central role in gene regulation in early development. We hypothesize that differential epigenetic regulation may underlie these patterns of genome activation and differential timing of germ layer induction. We found a strong positive correlation between nascent transcription and transcription factor binding sites (TFBS) enrichment for pioneer factors FOXA4 and POU3F1 and a negative correlation with the histone demethylase KDM2B. To gain deeper mechanistic insights into the origin of hierarchical germ layer initiation, we aim to probe chromatin accessibility in distinct regions for the presumptive germ layers in the blastula embryos and determine binding of FOXA4 and POU3F1. Moreover, to understand the developmental consequences of dysregulated germ layer timing, we implemented an approach to spatially alter cell division timing and alter the pattern of genome activation in blastula embryos. We will characterize the timing of germ layer gene expression programs in the desynchronized embryo...