The formation of the Spemann–Mangold organizer in Xenopus is thought to be established by two major signaling pathways: Nodal/activin for mesendoderm induction and Wnt/b-catenin for dorsal determination. The initial state consists of maternal VegT

INTRODUCTION One of the main questions in the field of developmental biology is how are multiple developmental cues integrated stepwise with the transcriptional regulation of cell fate decisions. To date, a large number of cis-regulatory modules (CRMs) have been identified by promoter/enhancer analyses in many organisms, and conceptual gene regulatory networks have been created for various developmental processes (Davidson and Levine, 2008). However, little is known about the in vivo status of transcriptional regulation or the stepwise integration of developmental cues, at least in vertebrate embryogenesis. The formation of the Spemann–Mangold organizer in Xenopus is thought to be established by two major signaling pathways: Nodal/activin for mesendoderm induction and Wnt/b-catenin for dorsal determination. The initial state consists of maternal VegT (mVegT), a T-box transcriptional factor in the vegetal hemisphere (Lustig et al., 1996; Zhang and King, 1996), and nuclear b-catenin downstream of canonical Wnt signaling in the dorsal region (Blythe et al., 2010). At the blastula stage, mVegT and Wnt signaling coordinately upregulate nodal expression (Xnr5 cluster genes and Xnr6, called Xnr5 hereafter), which forms a Nodal gradient in a dorsoventral direction in the presumptive endoderm (Agius et al., 2000; Takahashi et al., 2000; Takahashi et al., 2006). In the dorsal region, high Nodal signaling and Wnt/b-catenin signaling are integrated to form the gastrula organizer. During this process, Nodal signaling induces endodermand mesodermspecific transcriptional activators, such as Mix1 (Rosa, 1989); zygotic VegT (zVegT) and organizer-specific transcription factors, such as Lim1/Lhx1 (Taira et al., 1992; Taira et al., 1994); FoxA4/XFKH1 (Dirksen and Jamrich, 1992); and Otx2/Xotx2 (Blitz and Cho, 1995; Pannese et al., 1995; Yamamoto et al., 2003). Wnt/b-catenin signaling induces expression of the organizerspecific transcriptional activators Siamois (Sia) and its paralog Twin (Lemaire et al., 1995; Laurent et al., 1997). In addition, the combination of these signaling proteins induces the production of a transcriptional repressor, Goosecoid (Gsc) (Watabe et al., 1995; Wessely et al., 2001). While the organizer is being formed, the secreted BMP antagonists Noggin, Chordin, Follistatin, Xnr3 and Cerberus emanate from the organizer region to induce neuroectoderm in the dorsal animal ectoderm and also dorsalize the mesoderm to further establish and maintain the organizer. In addition, Cerberus, which is expressed in the head organizer region, also inhibits Nodal and Wnt signaling to prevent their posteriorizing effects (Piccolo et al., 1999). Thus, the outline of organizer formation and neural induction has been established. To elucidate the molecular basis of organizer formation in Xenopus, several groups, including ours, have investigated the regulation of organizer genes such as (1) gsc by Nodal and Wnt signaling (Watabe et al., 1995), and (2) lim1 and hhex by Nodal signaling (Watanabe et al., 2002; Rankin et al., 2011). In addition, the regulation of target genes for Gsc and Lim1 have been studied, such as (3) wnt8 and brachyury (bra/Xbra) by Gsc (Latinkic et al., 1997; Latinkic and Smith, 1999; Yao and Kessler, 2001) and (4) gsc and cer by Lim1 and Mix1 (Latinkic and Smith, 1999; Mochizuki et al., 2000; Yamamoto et al., 2003). However, these 1Department of Biological Sciences, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. 2Graduate School of Biological Sciences, Nara Institute of Science and Technology (NAIST), 8916–5 Takayama, Ikoma, Nara 630-0192, Japan. 3JST, CREST, Graduate School of Biological Sciences, Nara Institute of Science and Technology (NAIST), 8916–5 Takayama, Ikoma, Nara 630-0192, Japan.