title: A Smad3 transgenic reporter reveals TGF-beta control of zebrafish spinal cord development.
abstract: TGF-beta (TGFβ) family mediated Smad signaling is involved in mesoderm and endoderm specifications, left-right asymmetry formation and neural tube development. The TGFβ1/2/3 and Activin/Nodal signal transduction cascades culminate with activation of SMAD2 and/or SMAD3 transcription factors and their overactivation are involved in different pathologies with an inflammatory and/or uncontrolled cell proliferation basis, such as cancer and fibrosis. We have developed a transgenic zebrafish reporter line responsive to Smad3 activity. Through chemical, genetic and molecular approaches we have seen that this transgenic line consistently reproduces in vivo Smad3-mediated TGFβ signaling. Reporter fluorescence is activated in phospho-Smad3 positive cells and is responsive to both Smad3 isoforms, Smad3a and 3b. Moreover, Alk4 and Alk5 inhibitors strongly repress the reporter activity. In the CNS, Smad3 reporter activity is particularly high in the subpallium, tegumentum, cerebellar plate, medulla oblongata and the retina proliferative zone. In the spinal cord, the reporter is activated at the ventricular zone, where neuronal progenitor cells are located. Colocalization methods show in vivo that TGFβ signaling is particularly active in neuroD+ precursors. Using neuronal transgenic lines, we observed that TGFβ chemical inhibition leads to a decrease of differentiating cells and an increase of proliferation. Similarly, smad3a and 3b knock-down alter neural differentiation showing that both paralogues play a positive role in neural differentiation. EdU proliferation assay and pH3 staining confirmed that Smad3 is mainly active in post-mitotic, non-proliferating cells. In summary, we demonstrate that the Smad3 reporter line allows us to follow in vivo Smad3 transcriptional activity and that Smad3, by controlling neural differentiation, promotes the progenitor to precursor switch allowing neural progenitors to exit cell cycle and differentiate.
authors.affiliation: Department of Biology, University of Padova, I-35121 Padova, Italy.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Casari
authors.affiliation: Department of Biology, University of Padova, I-35121 Padova, Italy.
authors.forename: Marco
authors.initials: M
authors.lastname: Schiavone
authors.affiliation: Department of Biology, University of Padova, I-35121 Padova, Italy.
authors.forename: Nicola
authors.initials: N
authors.lastname: Facchinello
authors.affiliation: Department of Biology, University of Padova, I-35121 Padova, Italy.
authors.forename: Andrea
authors.initials: A
authors.lastname: Vettori
authors.affiliation: University of Innsbruck, Austria.
authors.forename: Dirk
authors.initials: D
authors.lastname: Meyer
authors.affiliation: Department of Biology, University of Padova, I-35121 Padova, Italy.
authors.forename: Natascia
authors.initials: N
authors.lastname: Tiso
authors.affiliation: Department of Molecular Medicine, University of Padova, I-35121 Padova, Italy.
authors.forename: Enrico
authors.initials: E
authors.lastname: Moro
authors.affiliation: Department of Biology, University of Padova, I-35121 Padova, Italy. Electronic address: francesco.argenton@unipd.it.
authors.forename: Francesco
authors.initials: F
authors.lastname: Argenton
mesh_terms: D030201:Activin Receptors, Type I / Q000378:metabolism; D000818:Animals; D030801:Animals, Genetically Modified; D002453:Cell Cycle; D049109:Cell Proliferation; D018507:Gene Expression Regulation, Developmental; D017930:Genes, Reporter; D007150:Immunohistochemistry; D009474:Neurons / Q000378:metabolism; D010641:Phenotype; D011401:Promoter Regions, Genetic; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D000077293:Receptor, Transforming Growth Factor-beta Type I; D018125:Receptors, Transforming Growth Factor beta / Q000378:metabolism; D015398:Signal Transduction; D051900:Smad3 Protein / Q000235:genetics*; D013116:Spinal Cord / Q000196:embryology* / Q000502:physiology; D016212:Transforming Growth Factor beta / Q000378:metabolism*; D019076:Transgenes; D015027:Zebrafish; D029961:Zebrafish Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: D018125:Receptors, Transforming Growth Factor beta; D051900:Smad3 Protein; D016212:Transforming Growth Factor beta; D029961:Zebrafish Proteins; C497792:smad3a protein, zebrafish; C497793:smad3b protein, zebrafish; D017346:Protein Serine-Threonine Kinases; D030201:Activin Receptors, Type I; D000077293:Receptor, Transforming Growth Factor-beta Type I; C512426:acvr1ba protein, zebrafish
keywords: Proliferation; Reporter; Smad3; TGFbeta; Transcription; Transgenic; Zebrafish