Figure 3. .
Functional analysis of TBX20 mutations. a, 293T cell–transfection assay measuring activation of the Nppa promoter in the presence of Tbx20c (short isoform lacking C-terminal trans activation and trans repression domains).16 b, COS cell–transfection assay measuring activation of the Nppa promoter in the presence of Tbx20a (full-length isoform), Nkx2-5, and Gata4, alone or in combination.16 Synergistic activation is seen only in the presence of all factors. c, Ability of WT and mutant Tbx20a proteins to disturb gastrulation movements in Xenopus laevis embryos after microinjection of respective mRNAs (1 ng) into fertilized eggs.16 WT protein likely disturbs gastrulation by dysregulating the function of endogenous T-box proteins, including brachyury and eomesodermin, and/or Gata factors, involved in mesoderm and endoderm formation. WT, I152M, and T209I are potent inhibitors, whereas Q195X is inactive. d, Comparison of the ability of Tbx20a WT and mutant proteins to activate expression of the endogenous Actc1 gene (encoding cardiac α-actin) in frog ventral-marginal-zone (VMZ) explants removed from gastrula embryos after microinjection of the indicated mRNAs into ventral cells of four-cell–stage embryos.16 Amounts of injected mRNA are indicated. Control tissue was the dorsal marginal zone (DMZ) that includes cardiac tissue. The histogram indicates normalized Actc1 levels, as determined by quantitative RT-PCR, with statistical significance (P) of indicated comparisons. e, Western blot (WB) showing Tbx20a protein expressed from injected mRNAs used in panel d, relative to levels of tubulin. Tbx20a proteins are linked to a C-terminal hemogluttinin (HA) epitope tag and are detected with anti-HA antibody, except for Tbx20a-Q195X, which lacks the tag and is detected with an anti-Tbx20 antibody.16