title: Cooperative non-cell and cell autonomous regulation of Nodal gene expression and signaling by Lefty/Antivin and Brachyury in Xenopus.
abstract: Dynamic spatiotemporal expression of the nodal gene and its orthologs is involved in the dose-dependent induction and patterning of mesendoderm during early vertebrate embryogenesis. We report loss-of-function studies that define a high degree of synergistic negative regulation on the Xenopus nodal-related genes (Xnrs) by extracellular Xenopus antivin/lefty (Xatv/Xlefty)-mediated functional antagonism and Brachyury-mediated transcriptional suppression. A strong knockdown of Xlefty/Xatv function was achieved by mixing translation- and splicing-blocking morpholino oligonucleotides that target both the A and B alloalleles of Xatv. Secreted and cell-autonomous inhibitors of Xnr signaling were used to provide evidence that Xnr-mediated induction was inherently long-range in this situation in the large amphibian embryo, essentially being capable of spreading over the entire animal hemisphere. There was a greater expansion of the Organizer and mesendoderm tissues associated with dorsal specification than noted in previous Xatv knockdown experiments in Xenopus, with consequent exogastrulation and long-term maintenance of expanded axial tissues. Xatv deficiency caused a modest animal-ward expansion of the marginal zone expression territory of the Xnr1 and Xnr2 genes. In contrast, introducing inhibitory Xbra-En(R) fusion constructs into Xatv-deficient embryos caused a much larger increase in the level and spatial extent of Xnr expression. However, in both cases (Xatv/Xlefty-deficiency alone, or combined with Xbra interference), Xnr2 expression was constrained to the superficial cell layer, suggesting a fundamental tissue-specific competence in the ability to express Xnrs, an observation with direct implications regarding the induction of endodermal vs. mesodermal fates. Our experiments reveal a two-level suppressive mechanism for restricting the level, range, and duration of Xnr signaling via extracellular inhibition by Xatv/Xlefty coupled with potent indirect transcriptional repression by Xbra.
authors.affiliation: Department of Cell and Developmental Biology, Program in Developmental Biology, Vanderbilt University School of Medicine, 465 21st Avenue South, Nashville, TN 37232, USA.
authors.forename: Young Ryun
authors.initials: YR
authors.lastname: Cha
authors.forename: Shuji
authors.initials: S
authors.lastname: Takahashi
authors.forename: Christopher V E
authors.initials: CV
authors.lastname: Wright
mesh_terms: D017398:Alternative Splicing; D000818:Animals; D001483:Base Sequence; D019070:Cell Lineage; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D004707:Endoderm / Q000378:metabolism; D005091:Exons; D005326:Fetal Proteins / Q000378:metabolism / Q000502:physiology*; D005775:Gastrula / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental; D017403:In Situ Hybridization; D007438:Introns; D055458:Left-Right Determination Factors; D008648:Mesoderm / Q000378:metabolism; D008969:Molecular Sequence Data; D055457:Nodal Protein; D009841:Oligonucleotides / Q000737:chemistry; D014176:Protein Biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D020825:T-Box Domain Proteins / Q000378:metabolism / Q000502:physiology*; D013997:Time Factors; D016212:Transforming Growth Factor beta / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D015854:Up-Regulation; D014981:Xenopus; D029867:Xenopus Proteins / Q000378:metabolism; D000098908:Brachyury Protein
chemical_list: D017931:DNA Primers; D018076:DNA, Complementary; D005326:Fetal Proteins; D055458:Left-Right Determination Factors; D055457:Nodal Protein; D009841:Oligonucleotides; D020825:T-Box Domain Proteins; D016212:Transforming Growth Factor beta; D029867:Xenopus Proteins; D000098908:Brachyury Protein; C492176:lefty protein, Xenopus