The nuclear to cytoplasmic ratio directly regulates zygotic transcription in Drosophila through multiple modalities.

Early embryos must rapidly generate large numbers of cells to form an organism. Many species accomplish this through a series of rapid, reductive, and transcriptionally silent cleavage divisions. Previous work has demonstrated that the number of divisions before both cell cycle elongation and zygotic genome activation (ZGA) is regulated by the ratio of nuclear content to cytoplasm (N/C). To understand how the N/C ratio affects the timing of ZGA, we directly assayed the behavior of several previously identified N/C ratio-dependent genes using the MS2-MCP reporter system in living Drosophila embryos with altered ploidy and cell cycle durations. For every gene that we examined, we found that nascent RNA output per cycle is delayed in haploid embryos. Moreover, we found that the N/C ratio influences transcription through three overlapping modes of action. For some genes (knirps, fushi tarazu, and snail), the effect of ploidy can be primarily attributed to changes in cell cycle duration. However, additional N/C ratio-mediated mechanisms contribute significantly to transcription delays for other genes. For giant and bottleneck, the kinetics of transcription activation are significantly disrupted in haploids, while for frühstart and Krüppel, the N/C ratio controls the probability of transcription initiation. Our data demonstrate that the regulatory elements of N/C ratio-dependent genes respond directly to the N/C ratio through multiple modes of regulation.