Horb ME and Thomsen GH (1999), Tbx5 is essential for heart development.

XB-ART-13363

Development 1999 Apr 01;1268:1739-51. doi: 10.1242/dev.126.8.1739.

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Tbx5 is essential for heart development.

Horb ME , Thomsen GH .

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Mutations in the Tbx5 transcription factor cause heart septal defects found in human Holt-Oram Syndrome. The complete extent to which Tbx5 functions in heart development, however, has not been established. Here we show that, in Xenopus embryos, Tbx5 is expressed in the early heart field, posterior to the cardiac homeobox transcription factor, Nkx2.5. During morphogenesis, Tbx5 is expressed throughout the heart tube except the anterior portion, the bulbus cordis. When Tbx5 activity is antagonized with a hormone-inducible, dominant negative version of the protein, the heart fails to develop. These results suggest that, in addition to its function in heart septation, Tbx5 has a more global role in cardiac specification and heart development in vertebrate embryos.

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Species referenced: Xenopus laevis
Genes referenced: myl2 nkx2-5 tbx5 tbxt vegt

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	Fig. 1. XTbx5 protein comparison. (A) A comparison of the predicted amino acid sequence of Xenopus, human and chick Tbx5. XTbx5 and hTbx5 share 67% amino acid identity overall, while XTbx5 and cTbx5 share 70% amino acid identity. The greatest degree of amino acid identity is found within the T domain, which is boxed. (B) Amino acid sequence alignment of the T domain of XTbx5 with other T domain proteins. The overall amino acid identity of each with the T domain of XTbx5 is: 98% human Tbx5, 91% mouse Tbx5, 98% chick Tbx5, 82% mouse Tbx4, 54% Brat, 53% Eom, 49% Xbra. Protein analysis was done using MacVector’s Clustal protein alignment software. Identical amino acids are shaded and gaps are represented as dashes. The amino acid sequences corresponding to the degenerate primers are indicated by arrows. GenBank accession number for Tbx5 clone is AF133036.
	Fig. 2. Expression of XTbx5 mRNA during organogenesis. (A) Stage 17 embryo, lateral view. The arrow shows faint expression of XTbx5 mRNA in the lateral mesoderm. (B) Stage 20 embryo, lateral view. Expression is detected in the eye (e) and the cardiac field (c). (C) Stage 25 embryo, lateral view. Expression of XTbx5 in the lateral mesoderm is greatest near the ventral midline and tapers off dorsally. (D) Ventral view of the same embryo in C. XTbx5 mRNA is not expressed across the ventral midline. (E) Stage 30 embryo, lateral view. Expression has moved further ventrally and has fused across the ventral midline, but some expression persists in lateral mesoderm. (F) Ventral view of the same embryo in E. Notice the heart-shaped expression along the ventral midline. (G) Stage 35 embryo. The expression of XTbx5 condenses in the lateral mesoderm, marking the sinus venosus (sv) (Nieuwkoop and Faber, 1967). (H) Dorsal-lateral view of a stage 35 embryo cleared in benzyl benzoate/benzyl alcohol (BBA). Notice that the expression of XTbx5 leaving the atrium branches onto either side of the embryos marking the developing sinus venosus. (I) Stage 40 embryo. The sinus venosus (sv) has moved further dorsally to lie just below the somites. Notice the lack of XTbx5 expression in the anterior portion of the pericardial cavity. (J) Stage 40 embryo in BBA. Notice the horseshoe-shaped expression marking the sinus venosus (sv). (K) XMLC2 expression, stage 30 embryo ventral view. Expression is seen further anteriorly than XTbx5 along the ventral midline and does not resemble the heart-shaped XTbx5 expression. (L) XMLC2 expression at stage 40. In contrast to XTbx5, XMLC2 is detected throughout the pericardial cavity. Abbreviations used in the figure are: atrium (a), bulbus cordis (bc), sinus venosus (sv) and ventricle (v). (M) Stage 28 embryo used to show the positions of the sections shown in N and O. (N) A section through the eye shows that XTbx5 is expressed only in the outer edge of the ciliary marginal zone (cmz), marking the stem cell population. There is also expression along the dorsal surface of the retina. (O) A section through the developing heart region shows that XTbx5 is expressed in the lateral and ventral mesoderm.
	Fig. 3. Serial sections through the heart of a stage 35 (A,B) and a stage 40 (C-L) embryo stained for XTbx5 mRNA expression. (A) Section through the ventricle of a stage 35 embryo. Expression is detected in both the endocardium and myocardium as well as in the tissue we identify as epicardium (ep), overlying the endoderm (en). (B) Section through the sinus venosus slightly posterior to the section in A. XTbx5 mRNA is detected in the ventral part of the sinus venosus. (CL) Selected serial sections through the heart of a stage 40 embryo proceeding from anterior to posterior. (C) Bulbus cordis (bc) (D) Bulbus cordis (bc) and ventricle (v). XTbx5 is expressed only in the ventricle. (E) Ventricle. Expression is also detected in the underlying epicardium (ep). (F,G) Sections through the atrium (a) as it turns dorsally. (H-L) Sections through the developing sinus venosus. The sections through the heart in C-L have been numbered going from anterior to posterior, beginning with the bulbus cordis and ending with the sinus venosus. The corresponding number for each section is printed in the lower right-hand corner of each panel. Each section is 14 mm thick. The bulbus cordis encompasses sections 1-8 (112 mm), the ventricle sections 5-16 (168 mm), the atrium sections 17-22 (84 mm) and the sinus venosus sections 23-46 (336 mm).
	Fig. 4. Double-labeled whole-mount in situ hybridization. (A,B) Expression of XTbx5 (blue) and XNkx-2.5 (purple). (A) At tailbud tadpole stage 25, cells expressing XNkx-2.5 are anterior to those expressing XTbx5. (B) At stage 30, expression of both overlaps in the developing heart in ventral, but not lateral domains. (C,D) Expression of XTbx5 (blue) relative to XMLC2 (purple). (C) Stage 30 embryo. Expression of both genes overlaps in the heart, while only XTbx5 expression is seen in the lateral mesoderm. (D) Stage 40 embryo. No XMLC2 expression is detected in the dorsal sinus venosus.
	Fig. 5. Construction and effectiveness of a hormoneinducible XTbx5 protein. (A) Schematic diagram of wildtype and dominant negative XTbx5 constructs. The T domain is in red, the engrailed repressor domain is depicted in black and the GR domain is in blue. (B) Wild-type stage 35 embryo with dexamethasone added at the 4 cell stage. (C) Stage 35 embryo injected with 1 ng of XTbx5-EnR mRNA into the dorsal marginal zone at the 4-cell stage. (D) Stage 35 embryo injected with 1 ng of XTbx5-EnR-GR mRNA and no dexamethasone added. (E) Stage 35 embryo injected with 1 ng of XTbx5-EnR-GR mRNA and dexamethasone added immediately after injections. (F) Stage 35 embryo injected with 1 ng of XTbx5-EnR-GR mRNA and dexamethasone added at stage 14/15. In B-F, the inset in the lower left corner shows a single example of a stage 17 embryo.
	Fig. 6. Embryonic phenotypes generated by a dominant negative XTbx5 protein. (A) Wild-type stage 45 embryo. Arrow points to the heart. Notice how the heart extends to the edge of the pericardial cavity and is near the ventral surface of the gut. (B-F) Typical phenotypes caused by XTbx5-EnR-GR. In each case, embryos were injected dorsally at the 4-cell stage with 1 ng of mRNA and treated with dexamethasone at stage 14/15. (B,C) Examples of the reduced heart phenotype. Arrow points to the small heart present in this class of embryo. (D-F) Examples of the beating nub or heartless phenotype. This phenotype is typified by the absence of a morphologically recognized heart. Sometimes, some small amount of beating tissue is present in the dorsal portion of the pericardial cavity. In B-F, notice the bloated pericardial cavity and improper gut formation in the injected embryos. In addition, the eyes are smaller in size. Arrowhead in B and E points to regions where blood cells have pooled.
	Fig. 7. Expression of heart markers in heartless embryos. (A,C,E) Stage 35 control embryos injected with 250 pg lacZ mRNA in the DMZ. (B,D,E) Stage 35 embryos injected with 1 ng of XTbx5- EnR-GR mRNA and 250 pg lacZ mRNA in the DMZ at the 4-cell stage and treated with dexamethasone at stage 14/15. (A,B) XMLC2 expression. (C,D) XNkx-2.5 expression. (E,F) Endogenous XTbx5 expression. Expression was scored at stage 35 in embryos injected with lacZ alone (A,C,E) or with lacZ + XTbx5-EnR-GR (B,D,F).