Summary A conserved feature of the midblastula transition (MBT) is a requirement for a functional DNA replication checkpoint to coordinate cell cycle remodeling and zygotic genome activation (ZGA). We have investigated what triggers this checkpoint during Drosophila embryogenesis. We find that the magnitude of the checkpoint scales with the quantity of transcriptionally engaged DNA. Measuring RNA Polymerase II (Pol II) binding at 20-minute intervals over the course of ZGA reveals that the checkpoint coincides with widespread de novo recruitment of Pol II that precedes and does not require a functional checkpoint. This recruitment drives slowing or stalling of DNA replication at transcriptionally engaged loci. Reducing Pol II recruitment in zelda mutants both reduces replication stalling and bypasses the requirement for a functional checkpoint. This suggests a model where the checkpoint functions as a feedback mechanism to remodel the cell cycle in response to nascent ZGA.

During embryogenesis, the initial chromatin state is established during a period of rapid proliferative activity. We have measured with 3-min time resolution how heritable patterns of chromatin structure are initially established and maintained during the midblastula transition (MBT). We find that regions of accessibility are established sequentially, where enhancers are opened in advance of promoters and insulators. These open states are stably maintained in highly condensed mitotic chromatin to ensure faithful inheritance of prior accessibility status across cell divisions. The temporal progression of establishment is controlled by the biological timers that control the onset of the MBT. In general, acquisition of promoter accessibility is controlled by the biological timer that measures the nucleo-cytoplasmic (N:C) ratio, whereas timing of enhancer accessibility is regulated independently of the N:C ratio. These different timing classes each associate with binding sites for two transcription factors, GAGA-factor and Zelda, previously implicated in controlling chromatin accessibility at ZGA.DOI: http://dx.doi.org/10.7554/eLife.20148.001

Summary Membrane-less organelles are intracellular compartments specialized to carry out specific cellular functions. There is growing evidence supporting the possibility that such organelles form as a new phase, separating from cytoplasm or nucleoplasm. However, a main challenge to such phase separation models is that the initial assembly, or nucleation, of the new phase is typically a highly stochastic process, and does not allow for the spatiotemporal precision observed in biological systems. Here we investigate the initial assembly of the nucleolus, a membrane-less organelle involved in different cellular functions including ribosomal biogenesis. We demonstrate that the nucleolus formation is precisely timed in D. melanogaster embryos and follows the transcription of rRNA. We provide evidence that transcription of rRNA is necessary for overcoming the highly stochastic nucleation step in the formation of the nucleolus, through a seeding mechanism. In the absence of rDNA, the nucleolar proteins studied are able to form high concentration assemblies. However, unlike the nucleolus, these assemblies are highly variable in number, location and time at which they form. In addition, quantitative study of the changes in the nucleoplasmic concentration and distribution of these nucleolar proteins in the wild-type embryos is consistent with the role of rRNA in seeding the nucleolus formation.

In Drosophila, graded expression of the maternal transcription factor Bicoid (Bcd) provides positional information to activate target genes at different positions along the anterior-posterior axis. We have measured the genome-wide binding profile of Bcd using ChIP-seq in embryos expressing single, uniform levels of Bcd protein, and grouped Bcd-bound targets into four classes based on occupancy at different concentrations. By measuring the biochemical affinity of target enhancers in these classes in vitro and genome-wide chromatin accessibility by ATAC-seq, we found that the occupancy of target sequences by Bcd is not primarily determined by Bcd binding sites, but by chromatin context. Bcd drives an open chromatin state at a subset of its targets. Our data support a model where Bcd influences chromatin structure to gain access to concentration-sensitive targets at high concentrations, while concentration-insensitive targets are found in more accessible chromatin and are bound at low concentrations. This may be a common property of developmental transcription factors that must gain early access to their target enhancers while the chromatin state of the genome is being remodeled during large-scale transitions in the gene regulatory landscape.

SUMMARYIn vertebrates, canonical Wnt signaling controls posterior neural cell lineage specification. Although Wnt signaling to the neural plate is sufficient for posterior identity, the source and timing of this activity remain uncertain. Furthermore, crucial molecular targets of this activity have not been defined. Here, we identify the endogenous Wnt activity and its role in controlling an essential downstream transcription factor, Meis3. Wnt3a is expressed in a specialized mesodermal domain, the paraxial dorsolateral mesoderm, which signals to overlying neuroectoderm. Loss of zygotic Wnt3a in this region does not alter mesoderm cell fates, but blocks Meis3 expression in the neuroectoderm, triggering the loss of posterior neural fates. Ectopic Meis3 protein expression is sufficient to rescue this phenotype. Moreover, Wnt3a induction of the posterior nervous system requires functional Meis3 in the neural plate. Using ChIP and promoter analysis, we show that Meis3 is a direct target of Wnt/-catenin signaling. This suggests a new model for neural anteroposterior patterning, in which Wnt3a from the paraxial mesoderm induces posterior cell fates via direct activation of a crucial transcription factor in the overlying neural plate.

Chromatin immunoprecipitation (ChIP) is a powerful method for analyzing the interaction of regulatory proteins with genomic loci, but has been difficult to apply to studies on early embryos due to the limiting amount of genomic material in these samples. Here, we present a comprehensive technique for performing ChIP on blastula and gastrula stage Xenopus embryos. We also describe methods for optimizing crosslinking and chromatin shearing, verifying antibody specificity, maximizing PCR sensitivity, and quantifying PCR results, allowing for the use of as few as 50 early blastula stage embryos (approximately 5؋10 4 cells) per experimental condition. Finally, we demonstrate the predicted binding of endogenous ␤-catenin to the nodal-related 6 promoter, binding of tagged Fast-1/FoxH1 to the goosecoid promoter, and binding of tagged Tcf3 to the siamois and nodal-related 6 promoters as examples of the potential application of ChIP to embryological investigations.

Summary An emerging concept in development is that transcriptional poising pre-sets patterns of gene expression in a manner that reflects a cell’s developmental potential. However, it is not known how certain loci are specified in the embryo to establish poised chromatin architecture as the developmental program unfolds. We find that, in the context of transcriptional quiescence prior to the midblastula transition in Xenopus, dorsal specification by the Wnt/β-catenin pathway is temporally uncoupled from the onset of dorsal target gene expression, and that β-catenin establishes poised chromatin architecture at target promoters. β-catenin recruits the arginine methyltransferase Prmt2 to target promoters, thereby establishing asymmetrically dimethylated H3 arginine 8 (R8). Recruitment of Prmt2 to β-catenin target genes is necessary and sufficient to establish the dorsal developmental program, indicating that Prmt2-mediated histone H3R8 methylation plays a critical role downstream of β-catenin in establishing poised chromatin architecture and marking key organizer genes for later expression.

SUMMARY In most metazoans, early embryonic development is characterized by rapid mitotic divisions that are controlled by maternal mRNAs and proteins that accumulate during oogenesis [1]. These rapid divisions pause at the Mid-Blastula Transition (MBT), coinciding with a dramatic increase in gene transcription and the degradation of a subset of maternal mRNAs [2, 3]. In Drosophila, the cell cycle pause is controlled by inhibitory phosphorylation of Cdk1, which in turn is driven by down-regulation of the activating Cdc25 phosphatases [4, 5]. Here, we show that the two Drosophila Cdc25 homologues, String and Twine, differ in their dynamics and that, contrary to current models [4], their down-regulations are not controlled by mRNA degradation but through different post-translational mechanisms. The degradation rate of String protein gradually increases during the late syncytial cycles in a manner dependent on the nuclear-to-cytoplasmic ratio and on the DNA replication checkpoints. Twine, on the other hand, is targeted for degradation at the onset of the MBT through a switch-like mechanism controlled like String by the nuclear-to-cytoplasmic ratio, but not requiring the DNA replication checkpoints. We demonstrate that post-translational control of Twine degradation ensures that the proper number of mitoses precede the MBT.