Early development consists of a highly choreographed series of events controlled by temporally and spatially regulated batteries of genes. Although the sequence and nature of the events may vary between organisms, features such as the maternal-to-zygotic transition (MZT) where control of development is transferred from maternal to zygotic genes, and the establishment of gene networks initiated by master regulators, are common to all zygotes, pointing to their essential roles in embryogenesis.
One of the best-studied developmental systems is the Drosophila
embryo where transcription factor hierarchies act to pattern and subdivide the embryo along the anteroposterior (AP) and dorsoventral (DV) body axes. Only three hours (hrs) after fertilization at the height of the MZT, most of the ~6000 cells in the embryo have acquired their positional information and cell fates. At this time, the embryo has also completed cellularization, whereby each nucleus of the syncytial blastoderm becomes enclosed by cell membrane 
, and the processes of sex determination and dosage compensation are underway 
. Although much attention has focused on the gene networks that regulate these processes, less is known about how they are coordinated to occur in a temporally organized manner.
The recent discovery of the transcription factor Zld raised the possibility that a single factor could coordinately activate the early zygotic genome 
. Expression profiling studies of early embryos lacking maternal expression of zld
(henceforth referred to as zld−
) revealed that 70% of the genes normally activated between 1–2 hrs of development were down-regulated, including many genes required for cellularization, sex determination, and dorsal patterning 
. However, other early genes displayed more subtle changes in the absence of zld
. For example, activation of the ventral gene sna
was temporally delayed, but appeared to recover by nuclear cycle (nc) 14 
. Thus, Zld appeared to regulate early zygotic genes in different ways - some are completely dependent on Zld for activation, while others depend on Zld for proper timing of expression.
Zld binds in vitro
and related motifs referred to as TAGteam sites 
, which were first identified as conserved sequences over-represented in the regulatory regions of pre-cellular blastoderm genes 
. Indeed, the TAGteam sites are located upstream and often close to the transcription start site (TSS) of genes down-regulated in zld− 
. However, many genes with upstream TAGteam sites were unaffected in our profiling studies. They may not be expressed at 1–2 hrs, or they could have a maternal component masking the effect of Zld on their zygotic expression, or like sna
, they may have gone undetected in the profiling analysis due to more subtle effects in zld−
. Therefore, Zld may play a more extensive role in regulating early developmental genes than previously suggested.
To further investigate Zld targets, and possible mechanisms of their coordinated expression, we analyzed Zld binding across the genome in pre-cellular blastoderm embryos. These results, combined with our expression profiling studies, uncovered many new Zld targets, and demonstrated that Zld is responsible for timing the activation of genes across all three patterning systems, DV, AP and terminal. Our expression assays further showed that proper transcriptional onset is critical for the cascade of cross-regulatory interactions among patterning genes, and that changes in timing can lead to profound changes in positional information throughout the blastoderm. We found a remarkable overlap between Zld-bound regions and HOT (high occupancy transcription factor binding) regions, or hotspots, reported by the modENCODE consortium 
. The observation that Zld can be visualized in nuclei before other known transcription factors, and that the most over-represented motif in hotspots is the Zld binding site, hints at a role for Zld in marking, establishing, or maintaining hotspots.