Cooperative non-cell and cell autonomous regulation of Nodal gene expression and signaling by Lefty/Antivin and Brachyury in Xenopus.
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.
Cha
Young Ryun
YR
Department of Cell and Developmental Biology, Program in Developmental Biology, Vanderbilt University School of Medicine, 465 21st Avenue South, Nashville, TN 37232, USA.
Takahashi
Shuji
S
Wright
Christopher V E
CV
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
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