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First published online 2 June 2004
doi: 10.1242/dev.01174

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Tbx1 has a dual role in the morphogenesis of the cardiac outflow tract

¹ Program in Cardiovascular Sciences, Baylor College of Medicine, Houston, TX 77030, USA
² Center for Cardiovascular Development, Baylor College of Medicine, Houston, TX 77030, USA
³ Departments of Pediatrics (Cardiology), Baylor College of Medicine, Houston, TX 77030, USA
⁴ Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA
⁵ Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030, USA
⁶ Cardiovascular Research and Developmental Biology, The Hospital for Sick Children, University of Toronto, Toronto M5G 1X8, Canada
⁷ Department of Molecular and Medical Genetics, University of Toronto, Toronto M5G 1X8, Canada

View larger version (54K): [in a new window]   Fig. 1. Generation and phenotypic analysis of a hypomorphic Tbx1 mutant. (A) Gene targeting strategy used to generate the Tbx1neo allele. (B) Generation of the loxP-flanked (flox) Tbx1flox allele. (C) RT-PCR shows low amount of Tbx1 transcript expressed by the Tbx1neo allele. The RNA was extracted from whole E10.5 embryos. (D-F) Alignment of the OFT in E18.5 embryos; in a Tbx1+/+ embryo (D) the aorta (ao) communicates with the left ventricle (LV), in a Tbx1neo/neo mutant (E) with correct alignment, the truncus (T) communicates with both left and right ventricles (RV). However, in the Tbx1neo/neo embryo shown in F, the truncus communicates only with the RV (as in Tbx1–/– embryos, not shown). (G-I) Intracardiac ink injection of E11.5 embryos shows normal OFT alignment in a Tbx1+/+ embryo (G), lack of alignment in a Tbx1–/– embryo (I), and an intermediate phenotype in a Tbx1neo/neo embryo (H). av: atrioventricular valves; pt: pulmonary trunk; sv: semilunar valves; VSD: ventricular septum defect. Scale bars: 0.5 mm.

View larger version (64K): [in a new window]   Fig. 2. Fate map of Tbx1-expressing cells. (A) The gene targeting strategy utilized to generate the Tbx1mcm knock-in allele. (B) X-gal staining pattern generated by a Tbx1-lacZ knock-in allele (Tbx1+/–) in an E10.5 embryo. (C) X-gal staining pattern in a Tbx1mcm/+;R26R embryo at E10.5. (D) X-gal staining pattern in a Df1/Tbx1mcm;R26R embryo at E10.5 (Tbx1 homozygous mutant background). Small arrows in B-D indicate the OFT, the red arrowheads in C point to a superficial, ectodermal domain. (E) Dissected heart from a Tbx1+/– (lacZ knock-in) E10.5 embryo, and, F, from a Tbx1mcm/+;R26R E10.5 embryo. (G,H) Histological sections through the OFT at the same stage in Tbx1+/– (G) and Tbx1mcm/+;R26R (F) embryos at E10.5. Black arrows show blue cells localized in similar position in the two mutants, red arrows indicate blue cells detected only by cell fate mapping. (I,J) X-gal staining pattern generated by a Tbx1-lacZ knock-in allele (Tbx1+/–) in an E12.5 heart (I), compared with that in a Tbx1mcm/+;R26R embryo (J) at the same stage. (K-M) Comparison of the lacZ knock-in X-gal staining pattern (K, 37 somites) with that obtained by cell fate mapping in a heterozygous (L, 39 somites) and homozygous (M, 36 somites) Tbx1 mutant. The area in the black box in M is magnified (5x) in the inset at the bottom-left of M. K'-M' show magnified details of panel K, L and M, respectively. Red arrowheads indicate endothelial cells, black arrows OFT myocytes, and black arrowheads SHF cells. Scale bars: 1 mm in B-D, E,F and I,J; 100 µm in G,H; 200 µm in K-M; 50 µm in K'-M'.

View larger version (74K): [in a new window]   Fig. 3. Phenotype of conditional mutants Nkx2.5Cre/+;Tbx1flox/–. (A,B) Thymic hypoplasia in a conditional mutant (B) compared with wild-type embryo (A) at E18.5. Asterisks indicate thymic lobes. (C-E) Intracardiac phenotype in wild-type (C), conditional (D) and Tbx1–/– (E) mutants. Embryos in D and E have essentially identical phenotypes. C'-E' aortic arch and great artery patterning in wild-type (C'), conditional (D') and Tbx1–/– (E') mutants at E18.5. (F,G) Lateral views of ink-injected Tbx1+/– (F) and conditional mutant (G) embryos at E10.5. Note the absence of the 4th pharyngeal arch artery and reduced distance between the 3rd and 6th in the conditional mutant embryo. ao: aorta; rcc and lcc: right and left common carotid artery; rsa and lsa: right and left subclavian artery; pa: pulmonary arteries; pt: pulmonary trunk; RV: right ventricle; T: truncus arteriosus; VSD: ventricular septum defect. Scale bars: 1 mm in A-E and C'-E'; 200 µm in F,G.

View larger version (105K): [in a new window]   Fig. 4. Neural crest cell migration and AP septum phenotype in conditional mutants (E10-E10.5). (A-F) Fourth pharyngeal arch and neural crest migration phenotype in controls (Tbx1flox/+) (A,A',C,E) and conditional mutants (B,B',D,F). (A,B) Lateral view of wholemount immunohistochemistry with an anti-neurofilament M antibody (dorsal is left), note the abnormal branch of the glossopharyngeal nerve bundle (arrowheads in B,B'). The 4th pharyngeal arch is small with an apparently normal vagus nerve (arrows) but absent 4th pharyngeal arch artery (A',B'). A' and B' are coronal sections of the embryos shown in A and B, respectively. (C-D) Lateral view of wholemount RNA in situ hybridization with a neural crest marker (CrabpI) (dorsal is left), note the reduced staining at the 4th pharyngeal arch in the conditional mutant (compare with the region indicated by arrowhead in the wild-type embryo in C). (E,F) Radioactive in situ hybridization with the same probe on tissue sections of 32-somite embryos (E, wild type; F, conditional mutant). There is reduced expression of the neural crest marker and absence of the 4th PAA. (G,H) Partial overlap between Tbx1 expression (visualized by the Tbx1-lacZ knock-in pattern) (G) and Nkx2.5cre-induced recombination (visualized by the R26R reporter) (H) in the endoderm of the 3rd and 4th pharyngeal pouches (arrows), sagittal sections. (I,J) Coronal sections show absence of the AP septum (arrow) in a conditional mutant (J), compared with a control (Nkx2.5Cre/+;Tbx1+/–) embryo (I). (K,L) Sagittal sections, stained with X-gal, showing overlap between Tbx1 expression (visualized by the Tbx1-lacZ knock-in pattern, K) and Nkx2.5Cre-induced recombination (visualized by R26R reporter, L) in the endoderm lining the aortic sac adjacent to the AP septum (detail magnified in box, 5x). IV, fourth pharyngeal arch. P, pharynx. The developmental stages of embryos shown in G-L are in the range of 34-37 somites. Scale bars: 200 µm in A-D; 100 µm in the others.

View larger version (130K): [in a new window]   Fig. 5. Myocyte differentiation in OFT of conditional mutants. (A-C) Immunohistochemistry with an anti--smooth muscle actin antibody on coronal sections of E12.5 control (Tbx1flox/–) (A-C) and conditional mutant (A'-C') embryos at the level of the conus (A,A'), conal-truncal transition (B,B') and truncus (C,C'). Note the reduced immunoreactivity in conditional mutants (A'-C'), especially at the conal-truncal transition. Scale bars: 100 µm.

View larger version (62K): [in a new window]   Fig. 6. Chimera analysis and Fgf10 regulation by Tbx1. (A-B) Contribution of Tbx1–/– cells to OFT (arrow) and SHF (square box magnified in A') in a chimeric E10.5 embryo, compared with the same region of a Tbx1+/– chimera. Note that Tbx1–/– cells appear more intensely stained because they carry two lacZ knock-in alleles. (C) Tbx1 and Tbx5 activate the Fgf10 promoter carrying a wild-type (Fgf10Luc) but not mutant (Fgf10MutAluc) T-box binding element. Scale bars: 100 µm in A; 50 µm in A' and B. (D-F) RNA in situ hybridization on sagittal sections (E10.25) of control embryos (Tbx1flox/+) showing overlap of Tbx1 expression (D) and Fgf10 expression (E) in the SHF region (arrowheads). Fgf10 expression is reduced or absent in a conditional mutant (F). (G) Sagittal section (at the same stage) of an Nkx2.5Cre/+;R26R embryo showing extensive recombination that includes the region in which Tbx1 is expressed. (H) Schematic drawing of the proposed model for Tbx1 function in the SHF. Scale bars: 100 µm in D-G.