Temporal Coordination of Gene Networks by Zelda in the Early Drosophila Embryo Chung-Yi Nien1., Hsiao-Lan Liang1., Stephen Butcher2, Yujia Sun1, Shengbo Fu1, Tenzin Gocha1, Nikolai Kirov1, J. Robert Manak2*, Christine Rushlow 1 * 1 Department of Biology, Center for Developmental Genetics, New York University, New York, New York, United States of America, 2 Departments of Biology and Pediatrics, Roy J. Carver Center for Genomics, University of Iowa, Iowa City, Iowa, United States of America Abstract In past years, much attention has focused on the gene networks that regulate early developmental processes, but less attention has been paid to how multiple networks and processes are temporally coordinated. Recently the discovery of the transcriptional activator Zelda (Zld), which binds to CAGGTAG and related sequences present in the enhancers of many early-activated genes in Drosophila, hinted at a mechanism for how batteries of genes could be simultaneously activated. Here we use genome-wide binding and expression assays to identify Zld target genes in the early embryo with the goal of unraveling the gene circuitry regulated by Zld. We found that Zld binds to genes involved in early developmental processes such as cellularization, sex determination, neurogenesis, and pattern formation. In the absence of Zld, many target genes failed to be activated, while others, particularly the patterning genes, exhibited delayed transcriptional activation, some of which also showed weak and/or sporadic expression. These effects disrupted the normal sequence of patterning-gene interactions and resulted in highly altered spatial expression patterns, demonstrating the significance of a timing mechanism in early development. In addition, we observed prevalent overlap between Zld-bound regions and genomic ‘‘hotspot’’ regions, which are bound by many developmental transcription factors, especially the patterning factors. This, along with the finding that the most over-represented motif in hotspots, CAGGTA, is the Zld binding site, implicates Zld in promoting hotspot formation. We propose that Zld promotes timely and robust transcriptional activation of early-gene networks so that developmental events are coordinated and cell fates are established properly in the cellular blastoderm embryo. Citation: Nien C-Y, Liang H-L, Butcher S, Sun Y, Fu S, et al. (2011) Temporal Coordination of Gene Networks by Zelda in the Early Drosophila Embryo. PLoS Genet 7(10): e1002339. doi:10.1371/journal.pgen.1002339 Editor: Gregory S. Barsh, Stanford University School of Medicine, United States of America Received August 19, 2011; Accepted August 29, 2011; Published October 20, 2011 Copyright: 2011 Nien et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by a grant from the National Institutes of Health (GM63024). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: chris.rushlow@nyu.edu (CR); john-manak@uiowa.edu (JRM) . These authors contributed equally to this work. Introduction 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 [1], and the processes of sex determination and dosage compensation are underway [2]. 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 [3]. Expression profiling studies of early embryos lacking maternal expression of zld (henceforth referred to as zld2) 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 pat- terning [3]. 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 [3]. 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 to CAGGTAG and related motifs referred to as TAGteam sites [3], which were first identified as conserved sequences over-represented in the regulatory regions of pre- cellular blastoderm genes [2,4]. Indeed, the TAGteam sites are located upstream and often close to the transcription start site PLoS Genetics | www.plosgenetics.org 1 October 2011 | Volume 7 | Issue 10 | e1002339