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BACKGROUND: Members of the T-box family of DNA-binding proteins play a prominent role in the differentiation of the three primary germ layers. VegT, Brachyury, and Eomesodermin function as transcriptional activators and, in addition to directly activating the transcription of endoderm- and mesoderm-specific genes, serve as regulators of growth factor signaling during induction of these germ layers. In contrast, the T-box gene, tbx2, is expressed in the embryonic ectoderm, where Tbx2 functions as a transcriptional repressor and inhibits mesendodermal differentiation by the TGFβ ligand Activin. Tbx2 misexpression also promotes dorsal ectodermal fate via inhibition of the BMP branch of the TGFβ signaling network.
RESULTS: Here, we report a physical association between Tbx2 and both Smad1 and Smad2, mediators of BMP and Activin/Nodal signaling, respectively. We perform structure/function analysis of Tbx2 to elucidate the roles of both Tbx2-Smad interaction and Tbx2 DNA-binding in germ layer suppression.
CONCLUSION: Our studies demonstrate that Tbx2 associates with intracellular mediators of the Activin/Nodal and BMP/GDF pathways. We identify a novel repressor domain within Tbx2, and have determined that Tbx2 DNA-binding activity is required for repression of TGFβ signaling. Finally, our data also point to overlapping yet distinct mechanisms for Tbx2-mediated repression of Activin/Nodal and BMP/GDF signaling.
Fig. 1. Tbx2 dorsalizes ventralmesoderm. RT-PCR analysis of marginal zone explants. Embryos were injected radially at early cleavage stages with 1 ng of tbx2; marginal zone explants were dissected at stage 10
Fig. 2. Tbx2 associates with R-Smads a). Tbx2 physically associates with Smad1. 1 ng myc-tbx2 RNA was injected at early cleavage stages. Pull-down of native Smad1 from injected embryos leads to co-immunoprecipitation of exogenous Tbx2. Normal rabbit IgG antibodies were used in parallel studies as a negative control. b Tbx2 physically associates with Smad2. 1 ng of myc-tbx2 and 1 ng of flag-smad2 were injected at early cleavage stages. Pull-down of Flag-Smad2 from injected embryos leads to co-immunoprecipitation of exogenous Tbx2. Normal rabbit IgG antibodies were used in parallel studies as a negative control c) Tbx2 represses Smad2-mediated mesoderm induction. Embryos were injected with tbx2 (1 ng), flag-smad2 (1 ng), or tbx2 (1 ng) and flag-smad2 (1 ng) at the two-cell stage and animal caps were explanted at stage 8.5. d Tbx2 physically associates with Smad4. 1 ng of myc-tbx2 and 1 ng of flag-smad4 were injected at early cleavage stages. Pull-down of Flag-Smad4 from injected embryos leads to co-immunoprecipitation of of exogenous Tbx2. Normal rabbit IgG antibodies were used in parallel studies as a negative control
Fig. 3. Depictions of Myc-tagged Tbx2 deletion constructs. “Myc†refers to a 6x Myc epitope tag. N and C refer to the N and C termini, respectively. S1 and S2 refer to the first and second spacer, respectively
Fig. 4. The Tbx2 Spacer 1 and T-box domains are sufficient for R-Smad association. a-e Smad1 physically associates with Tbx2 via the T-box. Embryos were injected at the two-cell stage with 1 ng of RNA synthesized from the indicated deletion construct. Pull-down of native Smad1 from injected embryos leads to co-immunoprecipitation of the exogenous deletion construct. Normal rabbit IgG antibodies were used in parallel studies as a negative control. f-j Smad2 physically associates with Tbx2 via the T-box. Embryos were injected at the two-cell stage with 1 ng of RNA synthesized from the indicated deletion construct and flag-smad2. Pull-down of Flag-Smad2 from injected embryos leads to co-immunoprecipitation of the exogenous deletion construct. Normal rabbit IgG antibodies were used in parallel studies as a negative control
Fig. 5. Effects of Tbx2 deletion constructs on the BMP/GDF and Activin/Nodal pathways a) Embryos were injected with 2 ng of tbx2 or tbx2S1T in the animal pole at the two-cell stage. Animal caps were explanted at stage 8.5. Activin was added (2.5 ng/ml) as indicated. b Embryos were injected with 2 ng of tbx2 or tbx2NS1T in the animal pole at the two-cell stage. Animal caps were explanted at stage 8.5. Activin was added (2.5 ng/ml) as indicated. c Embryos were injected with 2 ng of tbx2 or tbx2NS1T in the animal pole at the two-cell stage. Animal caps were explanted at stage 8.5. d Embryos were injected with 1 ng tbx2 or tbx2ΔN in the animal pole at the two-cell stage. Animal caps were explanted at stage 8.5. Activin was added (2.5 ng/ml) as indicated e) Embryos were injected with 1 ng tbx2 or tbx2TS2C in the animal pole at the two-cell stage. Animal caps were explanted at stage 8.5. Activin was added (2.5 ng/ml) as indicated
Fig. 6. Tbx2 does not decrease levels of C-terminal phosphorylation of Smad1 and Smad2 a) Embryos were injected with 1 ng tbx2 at the two-cell stage; animal caps were explanted at stage 8.5 and incubated in Activin (2.5 ng/ml), as indicated, until late gastrula stages. Uninjected embryos were used in parallel studies. b Embryos were injected with 1 ng tbx2 at the two-cell stage and animal caps were explanted at stage 8.5 and incubated until late gastrula stages. Uninjected embryos were used in parallel studies. c Quantification of data shown in B, indicating no change in Smad1 protein levels or phosphorylation of Smad1 as a result of ectopic tbx2 expression
Fig. 7. DNA binding is necessary for Tbx2 repressor activity. a RT-PCR analysis of animal cap explants injected with Tbx2 DNA binding mutant construct. Embryos were injected with 1 ng myc-tbx2R-A or myc-tbx2 at the two-cell stage; animal caps were explanted at stage 8.5 and incubated in Activin (2.5 ng/ml) where indicated until late gastrula stages. b Smad1 physically associates with Tbx2R-A. Embryos were injected at the two-cell stage with 1 ng of RNA synthesized from Myc-Tbx2R-A. Pull-down of native Smad1 from injected embryos leads to co-immunoprecipitation of exogenous Myc-Tbx2R-A. Normal rabbit IgG antibodies was used in parallel studies as a negative control. c Smad2 physically associates with Myc-Tbx2R-A. Embryos were injected at the two-cell stage with 1 ng myc-tbx2R-A and 1 ng flag-smad2. Pull-down of Flag-Smad2 from injected embryos leads to co-immunoprecipitation of exogenous Myc-Tbx2R-A. Normal rabbit IgG antibodies were used in parallel studies as a negative control
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