How multiple developmental cues are integrated on cis-regulatory modules (CRMs) 
for cell fate decisions remains uncertain. The Spemann-Mangold organizer in 
Xenopus embryos expresses the transcription factors Lim1/Lhx1, Otx2, Mix1, 
Siamois (Sia) and VegT. Reporter analyses using sperm nuclear transplantation 
and DNA injection showed that cerberus (cer) and goosecoid (gsc) are activated 
by the aforementioned transcription factors through CRMs conserved between X. 
laevis and X. tropicalis. ChIP-qPCR analysis for the five transcription factors 
revealed that cer and gsc CRMs are initially bound by both Sia and VegT at the 
late blastula stage, and subsequently bound by all five factors at the gastrula 
stage. At the neurula stage, only binding of Lim1 and Otx2 to the gsc CRM, among 
others, persists, which corresponds to their co-expression in the prechordal 
plate. Based on these data, together with detailed expression pattern analysis, 
we propose a new model of stepwise formation of the organizer, in which (1) 
maternal VegT and Wnt-induced Sia first bind to CRMs at the blastula stage; then 
(2) Nodal-inducible Lim1, Otx2, Mix1 and zygotic VegT are bound to CRMs in the 
dorsal endodermal and mesodermal regions where all these genes are co-expressed; 
and (3) these two regions are combined at the gastrula stage to form the 
organizer. Thus, the in vivo dynamics of multiple transcription factors 
highlight their roles in the initiation and maintenance of gene expression, and 
also reveal the stepwise integration of maternal, Nodal and Wnt signaling on 
CRMs of organizer genes to generate the organizer.