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First published online 20 April 2005
doi: 10.1242/dev.01799

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Murine T-box transcription factor Tbx20 acts as a repressor during heart development, and is essential for adult heart integrity, function and adaptation

Fiona A. Stennard¹, Mauro W. Costa^1,2, Donna Lai¹, Christine Biben¹, Milena B. Furtado¹, Mark J. Solloway¹, David J. McCulley³, Christiana Leimena¹, Jost I. Preis¹, Sally L. Dunwoodie^1,4, David E. Elliott^1,*, Owen W. J. Prall¹, Brian L. Black³, Diane Fatkin^1,4 and Richard P. Harvey^1,4,

¹ Victor Chang Cardiac Research Institute, St Vincent's Hospital, 384 Victoria Street, Darlinghurst 2010, New South Wales, Australia
² Laboratório de Cardiologia Celular e Molecular, Instituto de Biofisica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, 20941-000, Brazil
³ Cardiovascular Research Institute, University of California, San Francisco, CA 94143-0130, USA
⁴ Faculties of Medicine and Life Sciences, University of New South Wales, Kensington 2056, New South Wales, Australia

View larger version (40K): [in a new window]   Fig. 1. Generation of Tbx20 null mice. (A) Schematic representation of the wild-type Tbx20 locus (exons I-IV indicted as black boxes) above the Tbx20 targeting construct and targeted Tbx20Flox and Tbx20lacZ alleles. Arrows represent the direction of transcription of neomycin (neo) and lacZ genes. Neo is driven by the phosphoglycerokinase promoter. Arrowheads represent loxP sites. P1 and P2 indicate 5' and 3' Southern screening probes. (B,C) Validation of the primary targeting event in embryonic stem cells using Southern analysis of ScaI- and NcoI-digested DNA and probes P1 and P2. (D) PCR genotyping assay detecting wild-type (174 bp), Floxed (220 bp) and/or Cre-deleted (302 bp) Tbx20 alleles in mice or embryos of indicated genotypes. (E) RT-PCR analysis of RNA extracted from wild-type or Tbx20lacZ/lacZ embryonic hearts demonstrating deletion of exons I-III and preservation of exons IV-VI (read-through from lacZ cassette) in the mutant. (F-I) Expression of lacZ in Tbx20lacZ/+ embryos at E7.5-10.5. am, amniotic mesoderm; cp, cardiac progenitors; da, dorsal aorta; e, eye primordium; ecc, endocardial cushion; en, endothelium; h, heart; hb, hindbrain; ias, inter-atrial septum; la, left atrium; N, NcoI; neo, neomycin; nt, neural tube; pe, pharyngeal endoderm; ra, right atrium; S, ScaI; v, ventricle; wt, wild type; ysm, yolk sac mesoderm.

View larger version (123K): [in a new window]   Fig. 2. Abnormal heart morphogenesis in Tbx20lacZ/lacZ embryos. (A-D) Scanning electron micrographs of wild-type and Tbx20lacZ/lacZ embryos at E9.25. Relative scale bars are indicated. (E-J) Sections of E9.0 Tbx20lacZ/+ and Tbx20lacZ/lacZ embryos stained for lacZ. Avc, atrioventricular canal; fg, foregut; iv, inflow ventricle-like chamber; la, left atrium; lv, left ventricle; oft, outflow tract; ov, outflow ventricle-like chamber; rv, ventricle; sa, sinuatrium; wt, wild type.

View larger version (97K): [in a new window]   Fig. 3. Altered cardiac transcriptional program in Tbx20lacZ/lacZ embryos. (A-H) Whole-mount in-situ hybridization analysis of E8.25-9.5 wild-type or Tbx20lacZ/lacZ embryos showing expression of genes indicated. Arrows in B indicate cranial limit of Tbx5 expression. Arrows in E indicate cells of the primary heart field. (I) Tbx20lacZ/+ and Tbx20lacZ/lacZ embryos bearing the Mef2c-AHF-hPLAP transgene, stained for alkaline phosphatase activity. oft, outflow tract; wt, wild type.

View larger version (149K): [in a new window]   Fig. 4. Disrupted cardiac pre-pattern and Tbx2 expression in Tbx20lacZ/lacZ embryos. (A-M) Whole-mount in-situ hybridization analysis of wild-type (wt), Tbx20lacZ/lacZ, Nkx2-5GFP/GFP or compound Tbx20lacZ/lacZ/Nkx2-5GFP/GFP embryos using indicated probes. Panel C shows sections of whole-mount embryos at the level of the sinuatrium. sa, sinuatrium.

View larger version (95K): [in a new window]   Fig. 5. Tbx20 expression is repressed by Nrg1. (A-D) Panels show whole-mount in-situ hybridizations using probes indicated on E9.5 control embryos compared with embryo explants cultured from E8.5-9.5 in control medium or medium plus 10-9 mol/l Nrg1.

View larger version (100K): [in a new window]   Fig. 6. Arrested development of yolk sac vasculature in Tbx20 null embryos. (A,B) LacZ expression in yolk sac flat mounts showing lack of mature vessels in Tbx20lacZ/lacZ embryos, as seen in wild type. (C,D) Sections of E9.5 Tbx20lacZ/+ or Tbx20lacZ/lacZ yolk sacs showing expression of lacZ in the mesodermal layer, but not in blood cells. (E,F) Tbx20 is expressed in yolk sac mesoderm and vessel derivatives of Tbx20lacZ/+ embryos from E11.5-14.5. Note that expression was never observed in the endodermal layer or hematopoietic cells. (G,H) Whole-mount immunohistochemical detection of Pecam1, a marker of mature endothelial cells at E9.5. (I,J) TUNEL showing increased apoptosis in the mesodermal layer of E9.5 mutant yolk sacs compared with wild type. (K,L) Transmission electron microscopy of E9.5 yolk sacs showing an example of perforation of the endothelial layer (red arrowheads) in the mutant. (M) RT-PCR analysis of E9.0 yolk sacs (n=3) for markers of hemangioblast specification (Tal1), angioblast specification (Kdr) and remodeling (Angpt1, Tek) and vessel maturation (Acta2, Gja5). Scale bar: 5 µm. e, endoderm; ec, endothelial; wt, wild type.

View larger version (68K): [in a new window]   Fig. 7. Dilated cardiomyopathy in adult Tbx20lacZ/+ and Tbx20lacZ/+/Nkx2-5lacZ/+ mice. (A) Expression of LacZ in adult Tbx20lacZ/+ and Nkx2-5lacZ/+ hearts. (B) Dilated right ventricle in Tbx20lacZ/+/Nkx2-5lacZ/+ heart. (C-F) Transverse sections of a wild-type heart and one Tbx20lacZ/+/Nkx2-5lacZ/+ heart that showed right ventricular dilation (haemotoxylin and eosin staining). Arrows indicate fibrosis. (G) Northern analysis of stress and hypertophy markers in three mice of each genotype from the echocardiographic cohort. LV, left ventricle; RV, right ventricle; wt, wild type.