Highlights d Embryos can be made with 33 DNA differences but identical X. tropicalis ZGA templates d Decreased genomic DNA per cell leads to delayed zygotic gene expression at the MBT d The N/C ratio can regulate transcription independent from its role in the cell cycle d The DNA-to-cytoplasm ratio regulates the timing of most zygotic genes at the MBT

In multicellular animals, the first major event after fertilization is the switch from maternal to zygotic control of development. During this transition, zygotic gene transcription is broadly activated in an otherwise quiescent genome in a process known as zygotic genome activation (ZGA). In fast developing embryos, ZGA often overlaps with the slowing of initially synchronous cell divisions at the mid-blastula transition (MBT). Initial studies of the MBT led to the nuclear-to-cytoplasmic ratio model where MBT timing is regulated by the exponentially increasing amounts of some nuclear component N titrated against a fixed cytoplasmic component C. However, more recent experiments have been interpreted to suggest that ZGA is independent of the N/C ratio. To determine the role of the N/C ratio in ZGA, we generated Xenopus frog embryos with ~3-fold differences in genomic DNA (i.e., N) by using X. tropicalis sperm to fertilize X. laevis eggs with or without their maternal genome. Resulting embryos have otherwise identical X. tropicalis genome template amounts, embryo sizes, and X. laevis maternal environments. We used the X. tropicalis paternally derived mRNA to identify a high confidence set of exclusively zygotic transcripts. Both ZGA and the increase in cell cycle duration are delayed in embryos with ~3-fold less DNA per cell. Thus, DNA is an important component of the N/C ratio, which is indeed a critical regulator of zygotic genome activation in Xenopus embryos.