A 190 base pair, TGF-β responsive tooth and fin enhancer is required for stickleback Bmp6 expression.
The ligands of the Bone Morphogenetic Protein (BMP) family of developmental signaling molecules are often under the control of complex cis-regulatory modules and play diverse roles in vertebrate development and evolution. Here, we investigated the cis-regulatory control of stickleback Bmp6. We identified a 190bp enhancer ~2.5 kilobases 5' of the Bmp6 gene that recapitulates expression in developing teeth and fins, with a core 72bp sequence that is sufficient for both domains. By testing orthologous enhancers with varying degrees of sequence conservation from outgroup teleosts in transgenic reporter gene assays in sticklebacks and zebrafish, we found that the function of this regulatory element appears to have been conserved for over 250 million years of teleost evolution. We show that a predicted binding site for the TGFβ effector Smad3 in this enhancer is required for enhancer function and that pharmacological inhibition of TGFβ signaling abolishes enhancer activity and severely reduces endogenous Bmp6 expression. Finally, we used TALENs to disrupt the enhancer in vivo and find that Bmp6 expression is dramatically reduced in teeth and fins, suggesting this enhancer is necessary for expression of the Bmp6 locus. This work identifies a relatively short regulatory sequence that is required for expression in multiple tissues and, combined with previous work, suggests that shared regulatory networks control limb and tooth development.
Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, United States.
Priscilla A
PA
Erickson
Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, United States.
Phillip A
PA
Cleves
Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, United States.
Nicholas A
NA
Ellis
Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, United States.
Kevin T
KT
Schwalbach
Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, United States.
James C
JC
Hart
Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, United States. Electronic address: ctmiller@berkeley.edu.
Craig T
CT
Miller
D058500:Animal Fins / Q000196:embryology; D000818:Animals; D030801:Animals, Genetically Modified; D001483:Base Sequence; D001549:Benzamides / Q000494:pharmacology; D001665:Binding Sites / Q000235:genetics; D055418:Bone Morphogenetic Protein 6 / Q000096:biosynthesis / Q000235:genetics*; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics; D017124:Conserved Sequence / Q000235:genetics; D004149:Dioxoles / Q000494:pharmacology; D004742:Enhancer Elements, Genetic / Q000235:genetics*; D018507:Gene Expression Regulation, Developmental / Q000235:genetics; D006575:Heterocyclic Compounds, 3-Ring / Q000494:pharmacology; D008969:Molecular Sequence Data; D009805:Odontogenesis / Q000235:genetics; D018125:Receptors, Transforming Growth Factor beta / Q000037:antagonists & inhibitors / Q000235:genetics*; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D015398:Signal Transduction / Q000187:drug effects; D051900:Smad3 Protein / Q000235:genetics / Q000378:metabolism; D023701:Smegmamorpha / Q000196:embryology* / Q000235:genetics; D014070:Tooth / Q000196:embryology; D015027:Zebrafish / Q000196:embryology* / Q000235:genetics
C459179:4-(5-benzo(1,3)dioxol-5-yl-4-pyridin-2-yl-1H-imidazol-2-yl)benzamide; D001549:Benzamides; D055418:Bone Morphogenetic Protein 6; D004149:Dioxoles; D006575:Heterocyclic Compounds, 3-Ring; D018125:Receptors, Transforming Growth Factor beta; D051900:Smad3 Protein; C544261:XAV939
Bmp6; Bone morphogenetic protein; Enhancer; Stickleback; TGFß; Tooth development; Zebrafish