1613 RESEARCH ARTICLE INTRODUCTION During animal development, gene expression is highly regulated, both spatially and temporally. This regulation is primarily achieved at the transcriptional level by the sequence-specific binding of transcription factors (TFs). Such interactions are governed by gene regulatory networks, in which genes exert their effects in a sequential and combinatorial manner (Levine and Davidson, 2005). Remarkable progress has been made in reconstructing the gene regulatory networks responsible for developmental processes. For example, the expression profiles and regulatory networks involved in dorsoventral patterning and mesoderm specification of the fly embryo (Stathopoulos and Levine, 2005; Jakobsen et al., 2007; Li et al., 2008; Sandmann et al., 2006; Sandmann et al., 2007; Liu et al., 2009; Zinzen et al., 2009), endomesoderm specification in Xenopus and sea urchin embryos (Koide et al., 2005; Loose and Patient, 2004; Oliveri et al., 2008) and vulva development in Caenorhabditis elegans (Inoue et al., 2005; Ririe et al., 2008) have been reported. Significant progress has also been made towards understanding how tissues are specified in the early embryo of the simple chordate Ciona intestinalis (Imai et al., 2006). Since the fates of most cells are determined by the early gastrula stage in Ciona and most cells in the early embryo can be identified, gene regulatory networks can be analyzed at single-cell resolution. However, our knowledge of most of these networks is incomplete or provisional, particularly in chordates. Among the regulatory interactions in the Ciona embryo that have been revealed by gene knockdown and overexpression studies, only a small fraction have been examined at the cis-regulatory level (Bertrand et al., 2003; Brown et al., 2007; Corbo et al., 1997b; Di Gregorio et al., 2001; Oda-Ishii et al., 2005; Yagi et al., 2004). This is partly because an examination of cis-regulatory elements is very labor intensive. In addition, although most studies of cis-regulatory elements have identified elements that are qualitatively necessary and sufficient for recapitulating the endogenous gene expression patterns, they did not reveal the comprehensive binding profiles of TFs on cis-regulatory regions. Recent studies have revealed that weak or secondary cis- regulatory modules that have often been ignored in such analyses may or may not contribute to driving downstream genes in a quantitative respect (Segal et al., 2008; Zinzen et al., 2009). It therefore remains important to determine the complete binding profiles of TFs on the genome for an integrative understanding of gene regulatory networks in the Ciona embryo. Ciona tadpole larvae have a basic chordate body plan, with the notochord located in the center of the tail and flanked laterally by muscle, ventrally by endoderm and dorsally by the nerve cord (Satoh et al., 2003). The mesenchyme fills the space between the epidermal and endodermal tissues in the trunk. The fate of most cells is already specified before gastrulation is complete. The Ciona genome contains ~670 TF genes and their expression profiles during embryonic development have almost all been described at single- cell resolution (Imai et al., 2004; Miwata et al., 2006). Comprehensive functional assays examining regulatory genes expressed in the early embryo have revealed, at single-cell resolution, the molecular pathways that regulate early embryonic specification events (Imai et al., 2006). These studies identified 11 TF genes that play a central role in early regulatory networks. Here, we have examined their in vivo occupancies on genomic DNA and determined the architecture of their networks. Development 137, 1613-1623 (2010) doi:10.1242/dev.046789 © 2010. Published by The Company of Biologists Ltd 1 Department of Zoology, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto, 606-8502, Japan. 2 The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan. 3 Department of Biodiversity, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto, 606-8502, Japan. *Present address: Marine Genomics Unit, Okinawa Institute of Science and Technology Promotion Corporation, Uruma Okinawa, 904-2234, Japan † Author for correspondence (yutaka@ascidian.zool.kyoto-u.ac.jp) Accepted 9 March 2010 SUMMARY Precise spatiotemporal gene expression during animal development is achieved through gene regulatory networks, in which sequence-specific transcription factors (TFs) bind to cis-regulatory elements of target genes. Although numerous cis-regulatory elements have been identified in a variety of systems, their global architecture in the gene networks that regulate animal development is not well understood. Here, we determined the structure of the core networks at the cis-regulatory level in early embryos of the chordate Ciona intestinalis by chromatin immunoprecipitation (ChIP) of 11 TFs. The regulatory systems of the 11 TF genes examined were tightly interconnected with one another. By combining analysis of the ChIP data with the results of previous comprehensive analyses of expression profiles and knockdown of regulatory genes, we found that most of the previously determined interactions are direct. We focused on cis-regulatory networks responsible for the Ciona mesodermal tissues by examining how the networks specify these tissues at the level of their cis-regulatory architecture. We also found many interactions that had not been predicted by simple gene knockdown experiments, and we showed that a significant fraction of TF-DNA interactions make major contributions to the regulatory control of target gene expression. KEY WORDS: Ciona intestinalis, Gene regulatory network, Chromatin immunoprecipitation Genomic cis-regulatory networks in the early Ciona intestinalis embryo Atsushi Kubo 1 , Nobuhiro Suzuki 1 , Xuyang Yuan 2 , Kenta Nakai 2 , Nori Satoh 1, *, Kaoru S. Imai 3 and Yutaka Satou 1,† DEVELOPMENT