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First published online 22 October 2003
doi: 10.1242/dev.00797

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Tbx5 specifies the left/right ventricles and ventricular septum position during cardiogenesis

Jun K. Takeuchi^1,*,, Makoto Ohgi^1,*, Kazuko Koshiba-Takeuchi^1,, Hidetaka Shiratori², Ichiro Sakaki¹, Keiko Ogura³, Yukio Saijoh² and Toshihiko Ogura^3,

¹ Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5, Takayama, Ikoma, Nara 630-0101, Japan
² Division of Molecular Biology, Institute for Molecular and Cellular Biology, Osaka University, and Core Research for Evolutional Science and Technology Corporation, Osaka 565-0871, Japan
³ Department of Developmental Neurobiology, Institute of Development, Aging and Cancer, Tohoku University, Sendai 980-8575, Japan

View larger version (89K): [in a new window]   Fig. 1. (A,B) At stage 30, chick Tbx5 (A) is expressed in the developing left ventricle and Tbx20 (B) is expressed in the right ventricle. The ventricular septum is formed at the boundary of the two areas (arrowheads, A,B) that give rise to the left and right ventricles. (C,D) Expression of chick Nkx2.5 (C) and GATA4 (D) is observed throughout the developing heart ventricles. (E) A small indentation can be observed on the surface of the normal heart (arrowhead) at E5. This corresponds to the position of ventricular septum formation. (F) When Tbx5 was misexpressed, this indentation was not formed. (G) In electroporated hearts, Tbx5 was misexpressed ubiquitously, as confirmed by the GFP signals derived from the electroporated Tbx5-EGFP fusion gene. (H) At E8, the electroporated heart exhibited a round, smooth shape, whereas an indentation (arrowhead) was formed on the surface of the normal heart. (I) In electroporated hearts, GFP fluorescence signals were observed in the entire ventricle. No GFP signal was detected in the normal heart. (J) Normally, the chick atrial natriuretic factor (ANF) gene is expressed in the left ventricle. An indentation can be observed at the right border of ANF gene expression (arrowhead). When Tbx5 was misexpressed, the ANF gene was induced in the entire ventricle at E6 and (K) expression of Tbx20 gene disappeared from the right side where normally this gene is expressed.

View larger version (67K): [in a new window]   Fig. 2. (Top) Positions of sections A-O. (A-E) Serial sections of normal heart. The pulmonary artery and aorta are connected to the right and left ventricles, respectively. Both right and left ventricles develop normally with the extensive trabecular formation and a ventricular septum. Right and left atrio-ventricular canals are formed at the correct position. (F-J) An abnormal heart in which Tbx5 was misexpressed ubiquitously (Type 1). Ventricular septum formation was severely disturbed, resulting in a single ventricle. The ventricular wall was thin, and the trabeculae were coarse and rough. Both right and left atria were dilated with an atrial septum defect (ASD; arrowhead in I). The aorta and pulmonary artery were fused at their base and connected to the single ventricle, resulting in a double outlet left ventricle (DOLV; arrowhead in F). Arrowhead in J indicates the atrio-ventricular canal. (K-O) Another type of malformation was observed (Type 2) in which the left ventricle expanded and the right ventricle shrank. The relative sizes of these two ventricles indicate a shift of the ventricular septum formation to the right, although the trabecular formation and the thickness of ventricles were not affected. Conal septation/rotation defects were also observed (arrowhead in M and N). The atrial septum formed, but it was thin and membranous. (P) Illustrations of the induced malformations. Type 1: atrial and ventricular septum defects (ASDs and VSDs). Alterations in both the aorta and pulmonary artery (DOLV and conal septation/rotation defects). Thin ventricular wall, suggesting abnormal differentiation of cardiac muscle cells. Dilatation of atrium. Type 2: VSD with a shift of the position of septum, resulting in a small right ventricle and expanded left ventricle, the conal septation/rotation defects with coarctation and thin atrial septum.

View larger version (78K): [in a new window]   Fig. 3. (A) To obtain restricted expression of the transgene, two electrodes were placed in a cross configuration. With this arrangement, electric pulses hit only a limited region of the precardiac mesoderm. (B) Morphology of a heart, in which the Tbx5-EGFP expression construct was electroporated at stage 5. Abnormal indentation is shown by a red arrowhead. GFP fluorescence signals (C) are observed predominantly in the restricted part of the right ventricle (red arrowhead). (D) Morphology of the electroporated heart at E7 showing an abnormal broad indentation (red arrowhead) that shows GFP fluorescence (E, arrowhead). (F) Schematic representation of the experimental design. At stage 5, Tbx5-EGFP gene was electroporated at a restricted region of the rostral precardiac field. At stage 10, ectopic misexpression was evident in a rostral part of the prospective right ventricle, making an ectopic boundary of Tbx 5 expression. (G) At E7, when in situ hybridization using cANF probe was performed in electroporated heart, ectopic induction of this gene was evident in a restricted of the prospective right ventricle between two red arrowheads. Normal boundary of cANF expression is indicated by a yellow arrowhead. (H,I) At E5, serial sections showed that misexpression of Tbx5 resulted in (H) abnormal growth of trabeculae (red arrowheads). At the tip of each trabecula, ectopic BMP2 expression was induced, albeit weakly (red arrowheads). (I) cANF gene was ectopically induced in the right side of the abnormal trabecula growth (red arrowheads). Normal cANF expression is indicated by black arrowheads. Note that abnormal trabecula growth is evident between the ectopic cANF expression and the normal cANF expression. (J-N) At E6, serial sections were made and examined both histologically (Hematoxylin and Eosin staining) and by marker expression. In the Tbx5-misexpressed heart, an ectopic septum-like structure (IVS*) was observed (J) in addition to the normal septum (IVS). Higher magnification of this section revealed that trabeculae amalgamated to form both the ectopic and the normal septums (K). When in situ hybridization was carried out, BMP2 (L), Tll1 (M) and VEGF (N) genes were found to be expressed in the ectopic septum (red arrowheads) as in the normal septum (black arrowheads).

View larger version (47K): [in a new window]   Fig. 4. (A) The ß-MHC promoter drives expression of mouse Tbx5 in the entire ventricle. By contrast, the MLC-2 promoter targets expression of the chick Tbx5-EGFP fusion gene to the right side of heart. (B) In the normal heart, the prospective right ventricle develops as a bulge (black arrowhead). (C) When Tbx5 was misexpressed in the entire heart, this bulge was disrupted. The prospective left ventricle was connected to the conus cordis with a small junction indicated by a red arrowhead. (D-G) When the Tbx5-EGFP fusion gene was expressed by the MLC-2 promoter, GFP signals were observed on the right side of the heart (D,F) and a swelling was formed (E,G) at E1.5 on the prospective right ventricle (red arrowheads). (H) When chick Tbx5 was used to detect its expression in the heart, this transgene was found to be expressed along a gradient in the prospective right ventricle. (I) In some cases, a small protrusion was formed on the surface of the right ventricle (blue arrowhead).

View larger version (81K): [in a new window]   Fig. 5. (A,D,G) Expression of the eHAND (A), dHAND (D) and mouse Anf (G) genes in the normal heart. Both eHAND and mouse Anf are expressed in the left ventricle, whereas dHAND is expressed in the right. (J,M,R) These patterns of expression were also confirmed in tissue sections. (B,E,H) In the MHC-Tbx5 transgenic mice, both eHAND and mouse Anf were induced in the entire ventricle, whereas dHAND was repressed. (K,N,Q) This was also confirmed in tissue sections. Histological examination of these sections revealed that ventricular septum formation was disrupted with a tiny bulge at the right-most end (black arrowhead). (C,F,I) In the MLC-2v-Tbx5 transgenic mice, the right ventricle expanded (red arrowhead). In this region, both the eHAND and mouse Anf genes were induced, and the dHAND gene was repressed. (L,O,R) Histological analysis revealed the swelling of the right ventricle (red arrowhead in L). In this swelling, eHAND was induced in a gradient manner (red arrowhead in L) and was absent from a region near the septum. (O) In this small region, dHAND is expressed (red arrowhead), whereas it is repressed in the rest of right ventricle. (R) Mouse Anf was also induced in the right ventricle of this transgenic mouse.

View larger version (61K): [in a new window]   Fig. 6. Human ANF promoter-luciferase construct was transfected to COS7 cells along with various expression plasmids. Tbx5 and Nkx2.5 activated this promoter weakly (4.7- and 3.7-fold, respectively). As reported, co-expression of these factors synergistically activated this promoter (11.6-fold). Addition of zebrafish tbx20 expression vector repressed Tbx5-mediated activation (1.6-fold), although this did not affect Nkx2.5-mediated activation (5.2-fold). Interestingly, Tbx20 abrogated efficiently the synergistic activation by Tbx5 and Nkx2.5 (7.0-fold). When another heart-specific factor GATA4 was expressed, the ANF promoter was activated weakly (3.5-fold). When GATA4 and Tbx5 were coexpressed, robust activation was observed (25.3-fold). Tbx20 abrogated this strong synergistic activation efficiently (3.2-fold), whereas Tbx20 did not affect the ANF activation by GATA4 alone (2.5 fold). Tbx20 alone did not affect the ANF promoter activity (1.3 fold).

View larger version (56K): [in a new window]   Fig. 7. In the wild-type heart, Tbx5 is expressed in the prospective left ventricle (red), not in the prospective right ventricle (yellow). At the boundary of these Tbx5-positive and -negative regions, an interventricular septum was induced. (Type 1) When Tbx5 is misexpressed in the entire ventricle, this boundary is not formed and septum formation does not occur, resulting in a single ventricle. (Type 2) When Tbx5 expression expands to the right, leaving a small Tbx5-negative region, the ventricular septum is shifted to the right. In such hearts, the left ventricle expands, and the right ventricle shrinks. (Type 3) When misexpression of Tbx5 is more restricted to the right ventricle, an ectopic boundary of Tbx5-positive and -negative regions is formed, inducing the ectopic formation of the ventricular septum in the right ventricle. (Right panels) In zebrafish heart, tbx20 is expressed in the bulbus arteriosus (BA) and the ventricular wall near the BA. This gene shows a gradient of expression in the ventricle with strongest signals at the BA, and ending near the atrioventricular junction (AVJ). By contrast, expression of tbx5 is seen in the AVJ and the ventricle near the AVJ, but expression becomes faint near the BA. This indicates that tbx20 and tbx5 are expressed in a complementary fashion, but not discreetly. Expression patterns are basically same at 48 and 120 hours post fertilization (hpf).