title: Siamois and Twin are redundant and essential in formation of the Spemann organizer.
abstract: The Spemann organizer is an essential signaling center in Xenopus germ layer patterning and axis formation. Organizer formation occurs in dorsal blastomeres receiving both maternal Wnt and zygotic Nodal signals. In response to stabilized βcatenin, dorsal blastomeres express the closely related transcriptional activators, Siamois (Sia) and Twin (Twn), members of the paired homeobox family. Sia and Twn induce organizer formation and expression of organizer-specific genes, including Goosecoid (Gsc). In spite of the similarity of Sia and Twn sequence and expression pattern, it is unclear whether these factors function equivalently in promoter binding and subsequent transcriptional activation, or if Sia and Twn are required for all aspects of organizer function. Here we report that Sia and Twn activate Gsc transcription by directly binding to a conserved P3 site within the Wnt-responsive proximal element of the Gsc promoter. Sia and Twn form homodimers and heterodimers by direct homeodomain interaction and dimer forms are indistinguishable in both DNA-binding and activation functions. Sequential chromatin immunoprecipitation reveals that the endogenous Gsc promoter can be occupied by either Sia or Twn homodimers or Sia-Twn heterodimers. Knockdown of Sia and Twn together, but not individually, results in a failure of organizer gene expression and a disruption of axis formation, consistent with a redundant role for Sia and Twn in organizer formation. Furthermore, simultaneous knockdown of Sia and Twn blocks axis induction in response to ectopic Wnt signaling, demonstrating an essential role for Sia and Twn in mediating the transcriptional response to the maternal Wnt pathway. The results demonstrate the functional redundancy of Sia and Twn and their essential role in direct transcriptional responses necessary for Spemann organizer formation.
authors.affiliation: Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine, 1110 Biomedical Research Building 2/3, 421 Curie Boulevard, Philadelphia, PA 19104-6058, USA. swbae@kcch.re.kr
authors.forename: Sangwoo
authors.initials: S
authors.lastname: Bae
authors.forename: Christine D
authors.initials: CD
authors.lastname: Reid
authors.forename: Daniel S
authors.initials: DS
authors.lastname: Kessler
mesh_terms: D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D019521:Body Patterning; D017124:Conserved Sequence; D004247:DNA / Q000235:genetics; D018507:Gene Expression Regulation, Developmental; D055785:Gene Knockdown Techniques; D051956:Goosecoid Protein / Q000235:genetics / Q000378:metabolism; D018398:Homeodomain Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D020897:Organizers, Embryonic / Q000196:embryology* / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D055503:Protein Multimerization; D012689:Sequence Homology, Nucleic Acid; D015398:Signal Transduction; D051153:Wnt Proteins / Q000235:genetics / Q000378:metabolism; D029867:Xenopus Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014982:Xenopus laevis / Q000196:embryology* / Q000235:genetics / Q000378:metabolism*
chemical_list: D051956:Goosecoid Protein; D018398:Homeodomain Proteins; C093133:SIA1 protein, Xenopus; D051153:Wnt Proteins; D029867:Xenopus Proteins; C109979:sia2 protein, Xenopus; D004247:DNA