Coupling of zygotic transcription to mitotic control at the Drosophila mid-blastula transition

Xuemin Lu 2 Jennifer M. Li 1 Olivier Elemento 0 Saeed Tavazoie 0 Eric F. Wieschaus 2 3 0 Lewis-Sigler Institute for Integrative Genomics 1 Department of Molecular and Cellular Biology, Harvard University , Cambridge, MA 02138 , USA 2 Department of Molecular Biology, Princeton University , Princeton, NJ 08544 , USA 3 Howard Hughes Medical Institute, Princeton University , Princeton, NJ 08544 , USA One of the most prominent features at the mid-blastula transition (MBT) observed in most embryos is a pause in cell cycle regulated by the nucleocytoplasmic (N/C) ratio. By using chromosome rearrangements to manipulate the DNA content of embryos, we determined that the threshold for this cell cycle pause in Drosophila is about 70% of the DNA content normally present at cycle 14. Embryos with DNA contents around this value show intermediate cell cycle behaviors. Some pause at cycle 14, some at cycle 15, and some form patches arrested in different mitotic cycles. A second feature at MBT is a massive increase in zygotic transcription and a parallel degradation of maternally supplied RNAs. To determine whether these changes in gene expression are governed by the same N/C ratio that controls cell cycle pause, we compared gene expression in haploid and diploid Drosophila embryos. We find that most maternal RNA degradation and most new transcription correlate with absolute time or developmental stage, and are timed independently of the N/C ratio. We identify a class of zygotically active genes whose expression depends on the N/C ratio and which are only expressed at cycle 15 in haploids. In embryos with patchy cell cycle behavior due to threshold DNA contents, the expression of these genes correlates tightly with the boundaries of the mitotic patches, suggesting either that the mechanism that pauses the mitotic cycle is the same as the one that measures the N/C ratio, or that it is tightly coupled to the mechanism controlling zygotic transcription of N/C ratio genes at the MBT. INTRODUCTION The development of most metazoan embryos is characterized by a series of rapid, synchronous cell divisions, followed by a dramatic slowing of the cell cycle, the onset of asynchronous cleavages and the initiation of morphogenetic movements (OFarrell et al., 2004). The alteration in cell cycle behavior occurs coordinately with both the onset of bulk transcription from the zygotic genome and the degradation of maternal RNAs required for early development prior to the activation of the zygotic genome (McKnight and Miller, 1976; Zalokar, 1976). Despite the near universality of this developmental switch, known as the mid-blastula transition (MBT), the molecular mechanisms that regulate the MBT and coordinate alterations in the cell cycle timing with the activation of zygotic transcription and the degradation of maternal messages remain largely unknown. In most organisms, the MBT occurs after a defined number of cell cycles and thus at a fixed time after fertilization. However, previous work has shown that the transition is not controlled by the absolute time after fertilization or by a cell cycle counting mechanism. Instead, the ratio of nuclear content to cytoplasmic volume (the N/C ratio) controls the timing of the MBT and the onset of zygotic transcription in organisms as diverse as Drosophila and Xenopus. In Xenopus embryos, where wholesale zygotic transcription normally begins after 12 cycles of DNA replication and mitosis, manipulations that artificially increase the DNA content cause premature activation of the zygotic genome (Newport and Kirschner, 1982a; Newport and Kirschner, 1982b). Similarly, in Drosophila embryos, which normally undergo 13 rapid cleavage divisions, reduction of the DNA content by half in haploid embryos results in an extra cell cycle, with a corresponding delay of the MBT (Edgar et al., 1986). These observations have led to a model in which the exponential increase in DNA content during cleavage stages results in the titration and inactivation of a hypothetical cytoplasmic factor that inhibits onset of the MBT before the embryo attains the correct N/C ratio. However, it is unknown whether the control of the MBT requires the presence of specific genomic intervals and/or certain DNA sequences, or, conversely, depends upon total DNA content irrespective of specific sequences. Moreover, proper development requires that every cell in the embryo responds robustly and simultaneously to the N/C ratio, but no prior study has addressed how the N/C ratio ensures a robust response or to what degree the embryo is susceptible to variations in N/C values. The burst of zygotic transcription seen at the MBT is correlated with the elimination of maternal RNAs (Mathavan et al., 2005; Pilot et al., 2006). In zebrafish, zygotic activation and maternal degradation are coupled through the transcription of specific microRNAs that promote maternal RNA deadenylation and clearance (Giraldez et al., 2006). A similar role for zygotically expressed miRNAs has been postulated for maternal RNA degradation during Drosophila MBT (Bushati et al., 2008). However, no mechanistic links have yet been established between the alterations in cell cycle and the switch from maternally to zygotically driven developmental processes. It is unclear whether these changes in gene expression are controlled by the same processes that alter cell cycle downstream of the N/C measurement. Previous studies identified a small number of genes whose transcriptional activation appears to be controlled by the N/C ratio, as well as genes whose expression appears independent of that ratio (Grosshans et al., 2003; Grosshans and Wieschaus, 2000; Yasuda and Schubiger, 1991). No studies have characterized global transcriptional activation in the context of an altered N/C ratio, and thus it is uncertain how tightly general transcriptional activation is coupled to cell cycle control of the MBT. Here, we address these questions using an array of genetic tools available in Drosophila. By using compound chromosome rearrangements (Merrill et al., 1988), we found that cell cycle behavior does not require the presence of any specific genomic interval. We determined that the coordinated, robust initiation of MBT requires an N/C ratio above about 70% of that found in a normal embryo at cycle 14. Embryos whose DNA content is near this 70% threshold display patches of nuclei paused at either cycle 14 or cycle 15. Next, we compared the transcriptomes of MBTdelayed haploid embryos with those of wild-type diploid embryos. We found that most zygotic transcription, as well as the accompanying maternal RNA degradation, does not rely on the N/C ratio, but instead occurs at a strict time interval following fertilization, indicating the presence of a timing mechanism that operates independently of the N/C ratio. We also identified a small class of 88 zygotically active genes whose expression does depend on the N/C ratio. We found that in embryos with DN (...truncated)