PUBLICATION

A Smad3 transgenic reporter reveals TGF-beta control of zebrafish spinal cord development

Authors
Casari, A., Schiavone, M., Facchinello, N., Vettori, A., Meyer, D., Tiso, N., Moro, E., Argenton, F.
ID
ZDB-PUB-141007-1
Date
2014
Source
Developmental Biology   396(1): 81-93 (Journal)
Registered Authors
Argenton, Francesco, Casari, Alessandro, Facchinello, Nicola, Meyer, Dirk, Moro, Enrico, Schiavone, Marco, Tiso, Natascia, Vettori, Andrea
Keywords
Proliferation, Reporter, Smad3, TGFbeta, Transcription, Transgenic, Zebrafish
MeSH Terms
  • Animals
  • Cell Proliferation
  • Spinal Cord/embryology*
  • Spinal Cord/physiology
  • Protein Serine-Threonine Kinases/metabolism
  • Neurons/metabolism
  • Zebrafish
  • Genes, Reporter
  • Promoter Regions, Genetic
  • Smad3 Protein/genetics*
  • Cell Cycle
  • Zebrafish Proteins/genetics*
  • Zebrafish Proteins/metabolism
  • Immunohistochemistry
  • Phenotype
  • Transforming Growth Factor beta/metabolism*
  • Activin Receptors, Type I/metabolism
  • Gene Expression Regulation, Developmental*
  • Animals, Genetically Modified
  • Signal Transduction
  • Receptors, Transforming Growth Factor beta/metabolism
  • Transgenes*
(all 22)
PubMed
25286120 Full text @ Dev. Biol.
Abstract
TGF-beta (TGFβ) family mediated Smad signaling is involved in mesoderm and endoderm specification, 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.
Genes / Markers
Figures
Figure Gallery (16 images) / 2
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Expression
Phenotype
Mutations / Transgenics
Allele Construct Type Affected Genomic Region
ia15TgTransgenic Insertion
    ia16TgTransgenic Insertion
      ia18TgTransgenic Insertion
        ia50TgTransgenic Insertion
          m134
            Point Mutation
            ml2TgTransgenic Insertion
              ml14TgTransgenic Insertion
                sb1TgTransgenic Insertion
                  tt250
                    Point Mutation
                    1 - 9 of 9
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                    Human Disease / Model
                    No data available
                    Sequence Targeting Reagents
                    Target Reagent Reagent Type
                    smad2MO2-smad2MRPHLNO
                    smad3aMO1-smad3aMRPHLNO
                    smad3bMO3-smad3bMRPHLNO
                    smad4aMO2-smad4aMRPHLNO
                    smad4aMO3-smad4aMRPHLNO
                    smad4aMO4-smad4aMRPHLNO
                    smad4aMO5-smad4aMRPHLNO
                    1 - 7 of 7
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                    Fish
                    Antibodies
                    Name Type Antigen Genes Isotypes Host Organism
                    Ab2-smad3monoclonal
                      IgGRabbit
                      Ab4-h3polyclonal
                        IgGRabbit
                        1 - 2 of 2
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                        Orthology
                        No data available
                        Engineered Foreign Genes
                        Marker Marker Type Name
                        EGFPEFGEGFP
                        GFPEFGGFP
                        mCherryEFGmCherry
                        YFPEFGYFP
                        1 - 4 of 4
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                        Mapping
                        No data available