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First published online 18 May 2005
doi: 10.1242/dev.01854

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Tbx20 is essential for cardiac chamber differentiation and repression of Tbx2

¹ Institut für Molekularbiologie, Medizinische Hochschule Hannover, 30625 Hannover, Germany
² Department of Anatomy and Embryology, Academic Medical Center, University of Amsterdam, 1105 AZ Amsterdam, The Netherlands
³ Department of Cell and Molecular Biology, Karolinska Institute, 17177 Stockholm, Sweden

View larger version (59K): [in a new window]   Fig. 1. Targeted disruption of the Tbx20 locus. (A) Schematic representation of the targeting strategy. Restriction map of the wild-type locus with boxes representing the first four exons of Tbx20; coding regions are shown in black, non-coding in white. Fragments used as RFLP probes are shown. The KpnI-EcoRV fragment designated as 5' detects the KpnI-RFLP shown in B. The HincII-RFLP shown in C is detected by the BamHI-KpnI fragment labelled as 3'. Only HincII sites relevant for RFLP analysis are shown. B, BamHI; E, EcoRI; H, HincII; K, KpnI; N, NcoI; RV, EcoRV; neo, loxP-flanked neomycin selection cassette. (B) Southern blot analysis of KpnI-digested genomic DNA extracted from E9.5 embryos derived from intercrosses of mice heterozygous for the mutant Tbx20 allele. Genotypes are indicated above each lane. The 4.8 kb and 8.2 kb bands represent the wild-type and the mutant allele, respectively. (C) Southern blot analysis of HincII-digested genomic DNA extracted from the same E9.5 embryos. Genotypes are indicated above each lane. The 10 kb and 8 kb bands represent the wild-type and the mutant allele, respectively. (D) In situ hybridization analysis of Tbx20 expression in wild-type (+/+) and Tbx20–/– (–/–) embryos at E8.5 using an antisense riboprobe against the T-box shows complete absence of Tbx20 mRNA in the mutant embryo.

View larger version (123K): [in a new window]   Fig. 2. Cardiac morphology and histology of embryos mutant for Tbx20. (A-I) External morphology of embryos and isolated hearts. (A-D) E8.5 heart regions. The wild-type heart (A,C) shows looping, the mutant heart (B,D) remains linear. (E) At E9.5, Tbx20 mutants are substantially smaller than their wild-type littermates, and exhibit bleeding and edema. Somites are smaller and irregularly organized, as is the neural tube. The first branchial arch is formed. Boxes indicate the heart regions. (F-I) Isolated hearts of E9.5 embryos. (F,H) Wild-type hearts show formation of chambers with right (rv) and left ventricle (lv) and an atrium (a), and of the atrioventricular canal (avc) and outflow tract (oft). (G,I) Mutant hearts feature an outflow tract, a primitive ventricle (pv) and an inflow tract from anterior to posterior. Anterior is up. Views are from the left-lateral side (A,B,E-G), or from the ventral side (C,D,H,I). (J-Q) Histological analysis of E9.5 embryonic hearts by Hematoxylin and Eosin staining of paraffin sections. Differentiation of cardiac tissue in endocardium (e), myocardium (m), cardiac jelly (cj) and endocardial cushion tissue (ect) can be seen in wild-type (J-L) and mutant hearts (M-Q). Trabeculated myocardium (tm) is only formed in the wild-type heart. Sections are sagittal, with anterior up and ventral to the left (J,K,M,N), or transverse, with right up and ventral to the left (L,O,P,Q). Transverse sections of the mutant heart are at the level of the inflow tract (O), primitive ventricle (P) and outflow tract (Q). pc, pericardium; pcv, pericardial cavity; peo, proepicardial organ. Genotypes are indicated in the figure.

View larger version (95K): [in a new window]   Fig. 3. Cardiac differentiation and patterning in Tbx20 mutant embryos. Analysis of molecular markers shows that cardiac differentiation and AP patterning of the heart tube occur normally in the Tbx20 mutant. Expression of pan-cardiac markers by in situ hybridization analysis (A-D) and ß-galactosidase activity staining of a lacZ reporter gene in the Tbx20 locus (E,F) in E9.5 hearts of wild-type (A-F) and Tbx20–/– embryos (A'-F'). In situ hybridization analysis of markers for AP patterning (G-K') and left-right asymmetry (L,L') at E8.5, in wild-type (G-L) and in Tbx20–/– (G'-L') hearts. Views are from the left lateral side (A,A',C,C',E,E') or from the ventral side (B,B',D,D',F,F',G-L'), with anterior up in all cases. Expression patterns are explained in the main text. Markers and genotypes are indicated in the figure.

View larger version (71K): [in a new window]   Fig. 4. Secondary heart field development in Tbx20–/– embryos. Whole-mount in situ hybridization analysis of Tbx20 and Mlc2a expression in wild-type embryonic hearts at E7.75 (A,C) and E8.0 (B,D); of markers for OFT (Wnt11) and OFT/RV (Hand2) in E9.5 hearts (E-F'), and of secondary heart field markers in E8.5 wild-type and mutant embryos. Images are magnifications of anterior body regions, including the heart, in views from the right lateral side (E-G',I,I',K-M'), or from the ventral side (A-D,H,H',J,J'). Anterior is always up. Markers and genotypes are as indicated in the figure, for details on expression, see main text.

View larger version (106K): [in a new window]   Fig. 5. Chamber formation in Tbx20 mutant hearts. Whole-mount in situ hybridization analysis of expression of cardiac chamber markers in wild-type and Tbx20–/– hearts at E9.5. All views are from the left lateral side, anterior up. Expression of markers for ventricular (arrows in wild-type embryos in A-F) and for atrial chamber myocardium (arrowheads in A-C) are missing in the corresponding stage-matched Tbx20 mutant hearts. Chisel is expressed very weakly in the primitive ventricle of the Tbx20–/– heart (arrow in B'). Vessel formation is affected in the mutant, as shown by Cx40 expression, which marks branchial arch vessels and dorsal aorta in the wild type (white arrows in C), but reveals only scattered cells in the branchial arch region in the mutant (white arrow in C'). Tbx3 expression is only found in the atrioventricular canal of the wild-type heart (arrow in G). (H,H') Tbx18 expression in the proepicardial organ (arrow) and the septum transversum (arrowhead) is unchanged in Tbx20–/– embryos. Note that expression of markers for pharyngeal mesoderm, Hey2 and Tbx3 (arrowheads in D and G) is preserved in Tbx20 mutants (arrowheads in D' and G'). Markers and genotypes are indicated in the figure.

View larger version (86K): [in a new window]   Fig. 6. Analysis of primary myocardium in Tbx20–/– hearts. Whole-mount in situ hybridization analysis in E7.5 to E9.5 embryos shows the presence of markers for primary myocardium (Tbx2, Bmp2) and endocardium (vinexin ) in the heart tube of Tbx20 mutant embryos. (G-H') Anti-PECAM immunohistochemistry for endocardial endothelium. Images are of whole embryos (A,A') and magnifications of anterior body regions, including the heart (B-N'), in views from the right lateral side (A-B',D,D',F-I',K-L',N,N'), or from the ventral side (C,C',E,E',J,J',M,M'). Anterior is up. Embryos are E7.5 (A,A'), E8.25 (B-C'), E8.5 (D-E',G,G',I-J',L-M') and E9.5 (F,F',H,H',K,K',N,N'). Arrow in A' marks Tbx2 expression in the cardiac crescent in Tbx20 mutant embryos. Arrows in all other figures point to the left ventricle that is free of hybridization signals for markers of primary cardiac phenotype. By contrast, corresponding mutant hearts show expression of these genes in the primitive ventricle (arrowheads in lateral views of E8.5 and 9.5 mutant hearts). Note the increase of Tbx2 expression (C') but decrease of Bmp2 expression (M') in the primitive inflow tract of the E8.5 mutant heart. In wild-type embryos, Tbx2 expression is restricted to the atrioventricular canal (avc) and the septum transversum (st). al, allantois. Markers and genotypes are indicated in the figure.

View larger version (93K): [in a new window]   Fig. 7. Apoptosis and proliferation in Tbx20–/– hearts. (A-D) TUNEL assay on transverse sections of E8.5 (A,B) and E9.5 (C,D) embryos does not detect a difference in apoptosis between hearts of Tbx20 mutant embryos (B,D) and wild-type littermates (A,C). However, note the increase in apoptosis in extracardiac tissues such as the neural tube (nt) in the Tbx20 mutant. White boxes highlight the heart regions. (E,F) Analysis of cell proliferation in transverse sections of the (ventricular) heart region of wild-type (E) and Tbx20 mutant (F) embryos at E8.75, by BrdU incorporation assay. A black box indicates the mutant heart in F. (G) Quantification of cell proliferation by the ratio of BrdU-positive cells to total cell number, the BrdU-labeling index, in the analyzed heart area. The labeling index is significantly reduced in the mutant heart (0.0298±0.0033) when compared with the wild type (0.1325±0.0089). avc, atrioventricular canal; lv, left ventricle; rv, right ventricle.