The Spatio-Temporal Control of Zygotic Genome Activation

Abstract

7One of the earliest and most significant events in embryonic development is zygotic 8 genome activation (ZGA). In several species, bulk transcription begins at the mid-9 blastula transition (MBT) when, after a certain number of cleavages, the embryo attains 10 a particular nuclear-to-cytoplasmic (N/C) ratio, maternal repressors become sufficiently 11 diluted, and the cell cycle slows down. Here we resolve the frog ZGA in time and space 12 by profiling RNA polymerase II (RNAPII) engagement and its transcriptional readout. We 13 detect a gradual increase in both the quantity and the length of RNAPII elongation before 14 the MBT, revealing that >1,000 zygotic genes disregard the N/C timer for their activation, 15 and that the sizes of newly transcribed genes are not necessarily constrained by cell 16 cycle duration. We also find that Wnt, Nodal and BMP signaling together generate most 17 of the spatio-temporal dynamics of regional ZGA, directing the formation of orthogonal 18 body axes and proportionate germ layers. 102respectively. Gene activation reached its peak at the MBT (~4.5 hpf), with 1,854 newly-103 activated genes, before dropping to 724 genes at the early-to-mid gastrula stage (~7.5 hpf) and 104 increasing again to 1,214 genes towards the end of gastrulation (~10 hpf) (Figures 1B,C and 105 S1E and Table S2). The dramatic increase in transcriptional activity that occurs in the 1.5 hours 106 between the 128-cell stage and the MBT can be illustrated by Hilbert curves ( Figure 1C), which 107 Page 4 of 43 provide a genome-wide overview of RNAPII enrichment by folding chromosomes into two-108 dimensional space-filling and position-preserving plots (Gu et al., 2016). While most zygotic 109 genes remain active beyond the mid-gastrula stage, 197 (including siamois2 [sia2], nodal5 and 110 znf470) of the 4,836 zygotic genes (~4%) are deactivated within ~6 h of development (Figures 1111C and S1F,G). Slightly less than one third of the activated genes were differentially expressed 112 along either or both of the animal-vegetal and dorso-ventral axes ( Figures 1D and S1F). 113The temporal order of enriched biological processes supported by ZGA matched the regulatory 114 flow of gene expression, starting with nucleosome assembly, nucleic acid synthesis, mRNA 115 metabolism and production, post-translational modification and degradation of proteins (Figure 116 1E). The earliest transcriptional engagement, beginning at the 32 to 128-cell stages, was 117 detected in gene clusters of tens to hundreds of kilobases (Figures 1A,C and S1H-J). These 118 clusters featured close relatives of the same genes, some of which are critical to Nodal signaling 119 (Nodal ligands), the formation of the Spemann organiser (Siamois homeobox transcription 120 factors), nucleosome assembly (histones), mRNA decay (MIR-427), and ongoing gene 121 regulation (zinc finger [ZF] transcription factors with on average 10 Cys2-His2 [C2H2] domains; 122 Figure S1J). These earliest activated genes were shorter and encoded smaller proteins than 123 those wi...