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First published online September 30, 2004
doi: 10.1242/10.1242/dev.01378

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Tbx2 is essential for patterning the atrioventricular canal and for morphogenesis of the outflow tract during heart development

¹ Department of Genetics and Development, College of Physicians and Surgeons of Columbia University, New York, NY 10032, USA
² Department of Biology, Washington University, St Louis, MO 63130, USA
³ Department of Molecular Biology, Lewis Thomas Laboratory, Princeton University, Princeton, NJ 08544, USA
⁴ Institute of Molecular Genetics and Development, Medical College of Georgia, Augusta, GA 30912, USA

View larger version (44K): [in a new window]   Fig. 2. Targeting strategy to generate the Tbx2tm1Pa allele. (A) Shown are the Tbx2 cDNA and the endogenous Tbx2 genomic locus, where black indicates untranslated regions and gray represents the T-box coding sequence. Two hundred and seven base pairs of exon 1 and all of exon 2 were targeted for deletion with a construct containing a neomycin-thymidine kinase selection cassette (neo-TK) flanked with loxP sites and a negative selection diphtheria toxin element (DTA) attached at the 5' end. A targeted line was electroporated in vitro with a Cre recombinase gene to excise the selection cassette and generate the final Tbx2tm1Pa allele with a 2.2 kb deletion. (B) Southern blot analysis, with the 5' internal probe (5' int) indicated in A, confirming the presence of the Tbx2tm1Pa allele in genomic XhoI digests of yolk sac DNA. The wild-type fragment is 5.1 kb, the mutant fragment is 2.8 kb. (C) PCR with the three primers indicated in A amplifies a 180 bp wild-type product and an 88 bp mutant product from yolk sac DNA. E, EcoRI; EV, EcoRV; N, NotI; X, XhoI.

View larger version (120K): [in a new window]   Fig. 1. Tbx2 expression during cardiogenesis, 8.5-9.5 dpc. (A) Right view of an 8.5 dpc embryo showing Tbx2 expression in the allantois (al) and cardiac crescent (cc). The line indicates the plane of the section shown in B. (B) Vibratome section of an 8.5 dpc embryo stained in whole-mount, showing expression in cardiac mesoderm (cm). (C) Ventral view of the head and heart of an 8.5 dpc embryo, showing Tbx2 expression in the otic placode (op), inflow tract (ift) and septum transversum (st). (D) Right view of a 9.5 dpc embryo showing previously unreported Tbx2 expression in somites (s) and nephrogenic mesoderm (nm). The line in the trunk indicates the plane of section in G. (E) Left view of the embryo shown in D. Labeled are the dorsal retina (dr), otic vesicle (ov), forelimb bud (fl) and previously unreported expression in the body wall of the caudal trunk and tail (bw). The line in the tail region indicates the plane of the section in F. (F) Vibratome section of a 9.5 dpc embryo showing specific expression in the nephrogenic mesoderm (nm). (G) Vibratome section of a 9.5 dpc embryo showing expression in pharyngeal arch mesenchyme (arrow), also the site of cardiac neural crest cells migrating into the outflow tract (oft). Note the absence of expression in the aorticopulmonary septum (arrowhead). (H) Right whole-mount view of a 9.5 dpc heart showing Tbx2 expression in the outflow tract (oft). (I) Left view of the heart shown in G, demonstrating Tbx2 expression in the atrioventricular canal (AVC) and septum transversum. (J) Ventral view of a dissected 9.5 dpc heart stained in whole-mount, showing Tbx2 expression in the outflow tract (oft). (K) Ventral view of the heart in J with the outflow tract removed, showing Tbx2 expression on the outer curvature of the AVC (arrowhead). (L) Dorsal view showing Tbx2 expression in the AVC and ventricular inner curvature (arrow). nf, neural fold; c, coelom; fg, foregut; nt, neural tube; da, dorsal aorta; hg, hindgut; rv, right ventricle; v, ventricle; a, atrium.

View larger version (76K): [in a new window]   Fig. 3. Abnormal atrioventricular morphology in Tbx2tm1Pa/Tbx2tm1Pa mutants (–/–), 9.5-10.5 dpc. (A-C) Left views of a wild-type embryo (A, +/+) and two homozygous mutants (B,C) with abnormal atrioventricular (AV) morphology at 9.5 dpc. (D-F) Enlarged images highlight the AV canal that in wild type is distinguishable by the presence of a morphological constriction, indicated with the yellow arrowhead (D). Homozygous mutants frequently lack this AV constriction (E) or exhibit an enlarged or dilated ventricle (F). Left views of a (G) normal heterozygous embryo and a (H) homozygous mutant at 10.5 dpc. White lines in G indicate the planes of section for histology in J-O. The homozygous embryo in H shows signs of circulatory distress. (I) Closer inspection of another affected homozygous mutant shows a lack of constriction at the AV canal (red arrowheads). (J-L) Transverse histology at the outflow tract (OFT) of wild-type and homozygous mutant embryos at 10.5 dpc. Homozygous mutants that appear morphologically normal by external criteria show normal OFT endocardial cushion development (K). Distressed homozygous mutants have small endocardial cushions and the OFT appears to be shortened (L). (M-O) Transverse histology at the AV canal of the same embryos shown in J-L. Some homozygous mutants have normal endocardial cushion formation (N). Distressed homozygous mutants show compromised cushion formation (O). v, ventricle; a, atrium; oft, outflow tract; rv, right ventricle; ec, endocardial cushion.

View larger version (71K): [in a new window]   Fig. 4. Outflow tract septation defects in Tbx2tm1Pa/Tbx2tm1Pa homozygous mutants (–/–), 11.5-12.5 dpc. (A) Left views of a normal heterozygote (+/–) and a homozygous mutant embryo. The mutant is suffering from circulatory distress. (B,C) Ventral view of hearts from the 12.5 dpc embryos in A (B) and a normal heterozygous 11.5 dpc heart (C) for comparison. The 12.5 dpc homozygous mutant aorta (red arrowhead) is abnormally positioned to the right relative to the pulmonary trunk (yellow arrowhead). (D-I) Transverse histology of wild-type (D-F, +/+) and homozygous mutant embryos (G-I) over a range of stages between 11.5 and 12.5 dpc. Homozygous mutant histology shows delayed outflow tract (OFT) septation into separate aortic (red arrowheads) and pulmonary (yellow arrowheads) outlets. (J,K) Transverse histology from 12.5 dpc wild-type (J) and homozygous mutant embryos (K). Homozygous mutants frequently show a misalignment where the aortic outlet is abnormally positioned with respect to the left ventricle. Normally at 12.5 dpc the aortic outlet is situated near the left ventricle, separated only by endocardial cushion (blue arrowhead in J). (L) The right 6th arch artery (green arrowhead) is often persistent in 12.5 dpc homozygous mutant embryos. ra, right atrium; la, left atrium; rv, right ventricle; lv, left ventricle; as, aortic sac; e, esophagus; da, descending aorta; dar, ductus arteriosus.

View larger version (114K): [in a new window]   Fig. 5. Chamber differentiation in the myocardium of the AVC in Tbx2tm1Pa/Tbx2tm1Pa mutants (–/–), 9.5 dpc. Various gene expression patterns in normal wild-type (+/+) or heterozygous (+/–) embryos (A,C,E,G,I,K,M,O,Q,S,U,W) compared with homozygous mutants (B,D,F,H,J,L,N,P,R,T,V,X). Nppa is ectopically expressed in myocardium at the atrioventricular canal (AVC) in homozygous mutants (B), as is Csl (D) and Cx40 (F). Some homozygous mutant embryos have an extended domain of Cited1 expression into the AVC (H). Tbx5 expression at the AVC is normal in homozygous mutants (J). MLC2v expression is normal (L), but abnormal atrial expression of ßMHC (N) is observed in homozygous mutants. eHAND expression is normal in homozygous mutants (P). Neural crest cell Crabp1 expression is normal in homozygous mutants (R). Expression of Islet1 and the Fgf10 enhancer-trap transgene 1v-nlacZ-24 are normal in 9.5 dpc homozygous mutants (T,V). 1v-lacZ-24 expression is also normal in homozygous mutants at 12.5 dpc.

View larger version (47K): [in a new window]   Fig. 6. Hindlimb-specific, bilateral distal digit duplication in Tbx2tm1Pa/Tbx2tm1Pa mutants (–/–). (A) Right view of a dead homozygous mutant embryo (with tail removed) collected at 14.5 dpc. (B) High magnification view of the embryo in A showing a distal duplication of digit IV in the hindlimb. (C) Dorsal view of an Alcian Blue-stained right hindlimb from a dead embryo dissected at 14.5 dpc, showing duplicated cartilage condensations (arrowheads) within the first phalangeal segment of digit IV.

View larger version (12K): [in a new window]   Fig. 7. Normal levels of p21, p19ARF, p16INK4a and p15INK4b expression in Tbx2tm1Pa/Tbx2tm1Pa mutants (–/–), 9.5-10.5 dpc. (A) Results of semi-quantitative RT-PCR expression analysis demonstrating no evidence of elevated p21 transcription in homozygous mutants versus either wild-type (+/+) or heterozygous (+/–) controls at 9.5 dpc, 10.5 dpc, or in cases with dissected 10.5 dpc heart and trunk (h). Mandible from a 14.5 dpc mouse embryo (m-14) serves as the positive control. (B) Results of semi-quantitative RT-PCR expression analysis demonstrating no evidence of elevated p19ARF or p16INK4a transcription in homozygous mutants versus wild-type controls at 9.5 dpc, 10.5 dpc or in cases with dissected 10.5 dpc heart and trunk (h). Adult testis (t) serves as the positive control. (C) Results of semi-quantitative RT-PCR expression analysis demonstrating no evidence of elevated p15INK4b transcription in homozygous mutants versus either wild-type or heterozygous controls at 9.5 dpc, 10.5 dpc, or in cases with dissected 10.5 dpc heart and trunk (h). Adult testis (t) serves as the positive control.