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Members of the T-box family of proteins play a fundamental role in patterning the developing vertebrate heart; however, the precise cellular requirements for any one family member and the mechanism by which individual T-box genes function remains largely unknown. In this study, we have investigated the cellular and molecular relationship between two T-box genes, Tbx5 and Tbx20. We demonstrate that blocking Tbx5 or Tbx20 produces phenotypes that display a high degree of similarity, as judged by overall gross morphology, molecular marker analysis and cardiac physiology, implying that the two genes are required for and have non-redundant functions in early heart development. In addition, we demonstrate that although co-expressed, Tbx5 and Tbx20 are not dependent on the expression of one another, but rather have a synergistic role during early heart development. Consistent with this proposal, we show that TBX5 and TBX20 can physically interact and map the interaction domains, and we show a cellular interaction for the two proteins in cardiac development, thus providing the first evidence for direct interaction between members of the T-box gene family.
Fig. 5. TBX5 and TBX20 morphants display dramatic morphological defects. Whole-mount in situ hybridization of cleared stage 36 embryos. (A-C) ANF whole-mount in situ hybridization. (D-F) XTnIc whole-mount in situ hybridization. (A,D) ControlMO. (B,E) TBX5MO and (C,F) TBX20MO.
Fig. 9. Tbx5 and Tbx20 synergistically act to regulate cardiac gene expression. (A-L) Embryos injected with the indicated morpholinos at the one-cell stage. (A-D) Nkx2-5 whole-mount in situ hybridization. (E-H) XANF whole-mount in situ hybridization. (I-L) XTnIc whole-mount in situ hybridization. (A,E,I) ControlMO, (B,F,J) TBX5MO injected at suboptimal dose, (C,G,K) TBX20MO injected at suboptimal dose, (D,H,L) TBX5MO and TBX20MO injected in combination at suboptimal doses. All embryo were cleared to reveal heart expression. (M) Statistics for embryos injected with suboptimal doses of TBX5MO and TBX20MO in combination with each other or with ControlMO. Hearts were judged as having defects if they displayed a pericardial edema, an unlooped heart tube or reduction in cardiac mass.
Fig. 3. TBX5 and TBX20 morphants fail to undergo looping and chamber formation and display reduced cardiac cell numbers. Cryosections of TBX5 and TBX20 morphant hearts taken at the anterior (outflow), middle (ventricular) and posterior (atrial) regions. (A-D) ControlMO, (E-H) TBX5MO and (I-L) TBX20MO. Sections stained for tropomyosin (red), DAPI (blue) and cardiac actin (green). (D,H,L) Same sections as B, F and J stained with cardiac actin. In the looped control heart, the middle ventricular section also contains the atrium. (M) Mean number of cells per heart obtained by cell counts of hearttissue in serial sections derived from a minimum of three embryos.
Fig. 4. Cardiac specification is unaltered in TBX5 and TBX20 morphants. Whole-mount in situ with Nkx2.5 on stage matched (A,D,G,J) ControlMO-, (B,E,H,K) TBX5MO- or (C,F,I,L) TBX20MO-derived embryos.
Fig. 7. Tbx5 and Tbx20 are not required for the expression of each other. Embryos injected at the one-cell stage with ControlMO, TBX5MO or TBX20MO. (A,C) Whole-mount in situ hybridization showing Tbx5 expression. (B,D) Whole-mount in situ hybridization showing Tbx20 expression.
Fig. S2. Tbx5 is expressed in the common cardinal and anterior hepatic veins. (A,B) Whole-mount in situ hybridization of Xenopus embryos at stage 36 (A) and 42 (B). Parasaggital section show expression in the myocardium (m), endocardium (e) and pericardium (p). (C,D) Oblique sections through a stage 36 embryo showing expression in the common cardinal (cc) and anterior hepatic veins (h).
Fig. S3. Tbx5 and Tbx20 morpholinos block the formation of a functional heart as assayed by benzidine staining of erythrocytes in stage 42 tadpoles. (A) Uninjected control, (B) TBX5MO injected at optimal dose, (C) TBX20MO injected at optimal dose, (D) TBX5MO and TBX20MO injected together at suboptimal doses. There is blood staining in the heart region (h) of control embryos but not in morphant embryos.
Fig. 2. Tbx5 and Tbx20 are required for proper cardiogenesis.
(A-F) Morpholino injected tadpoles at the indicated stages. Control morphant embryos (A,D), TBX5 morphant embryos (B,E) and TBX20 morphant embryos (C,F). Arrows indicate the heart region, arrowheads indicate the eye. (G) Chart displaying the percentage of morphants surviving and displaying cardiac abnormalities, as scored by the presence of an unlooped heart tube, a reduction in cardiac mass and the presence of a pericardial edema.
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