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First published online 15 June 2005
doi: 10.1242/dev.01887

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Congenital heart disease reminiscent of partial trisomy 2p syndrome in mice transgenic for the transcription factor Lbh

Karoline J. Briegel^1,*,, H. Scott Baldwin², Jonathan A. Epstein³ and Alexandra L. Joyner¹

¹ Howard Hughes Medical Institute and Developmental Genetics Program, Skirball Institute of Biomolecular Medicine, New York University School of Medicine, New York, NY 10016, USA
² Departments of Pediatrics and Cell and Developmental Biology, Vanderbilt University Medical Center, Nashville, TN 37232, USA
³ Cardiovascular Division, University of Pennsylvania, Philadelphia, PA 19104, USA

View larger version (26K): [in a new window]   Fig. 1. Chromosomal locations of mammalian LBH loci. The positions of mouse Lbh on chromosome 17E2 and human LBH on chromosome 2p23.3 are shown relative to linkage markers and neighboring genes within the same syntenic region. The scheme shows a physical linkage map of mouse chromosome 17 (in centiMorgans; cM), whereas the map of human chromosomal region 2p23-21 represents the actual gene sequence on the chromosome 2 contingent in 100 kilobase (kb) increments. Mouse Lbh maps either proximally or distally of linkage marker D17Mit92 with a LOD score of 20.0. Bars indicate chromosomal segments triplicated in individuals with partial trisomy 2p syndrome with CHD (reviewed by Lurie et al., 1995).

View larger version (72K): [in a new window]   Fig. 2. Generation of Carp-Lbh transgenic mice. (A) Schematic of the Carp-Lbh transgene. A 2.5 kb promoter of Carp drives cardiomyocyte-specific expression of a Flag-tagged Lbh transgene. Other elements of the transgenic construct are a ß-globin intervening region (IVS), a 3' untranslated region (UTR) consisting of part of the lacZ gene and a SV40 polyA (pA) site. P1 and P2, probes used for RNA in situ hybridization shown in C-H. (B) A 6-month-old transgenic (Tg) and a wild-type (wt) littermate. Note hunched body posture and lethargy of the Tg animal. (C-H) Comparison of endogenous Lbh expression in wt (C,E,G) with expression of the Lbh transgene in Tg embryos (D,F,H) at embryonic day 9.5 (E9.5) and E12.5 by RNA in situ hybridization using probes P1 or P2 as indicated. Right and left views of E9.5 whole mount embryos (C-F; line no. 23, see Table 1), and transverse cardiac sections of E12.5 embryos (G,H; line no. 25). Note overexpression (red arrows) in outflow tract (oft) and left ventricle (lv; D), as well as ectopic expression (thick red arrows) of the transgene in the atria of Tg mice (F,H); asterisks indicate lack of endogenous and transgenic Lbh expression in the conotruncal endocardial cushions in both wt (G) and Tg (H). at, common atrium; avc, atrioventricular canal; lb, limb bud; la, left atrium; ra, right atrium; rv, right ventricle; so, somites; sv, sinus venosus.

View larger version (78K): [in a new window]   Fig. 3. Phenotypic analysis of right ventricular outflow tract (rvot) defects in adult Carp-Lbh transgenic mice. (A,B) In situ lateral view of the great arteries of 6-month-old wild-type (wt) and transgenic (Tg) mice, and (C,D) transverse histological sections through the rvot of 3-month-old wt and Tg hearts stained with Hematoxylin and Eosin show pulmonary atresia (arrow) in the Tg hearts (B,D). (E,F) Cross sections of the hearts shown in A and B and close-ups on right ventricular (rv) cardiomyocytes (insets) depict biventricular hypertrophy (asterisks) in the Tg (F). (G,H) Polymer casts of 5 months-old wt and Tg mice. Mixing of pulmonary (blue) and systemic (red) blood in the ascending aorta (arrowhead) and pulmonary stenosis (arrow) are evident in the Tg (H). (I,J) Diagrams of a wt and a Tg heart with Tetralogy of Fallot (TOF) showing the association of pulmonic stenosis (PS), an overriding aorta, a patent ductus arteriosus (PDA), a ventricular septum defect (VSD) and rv hypertrophy. ao, aorta; la, left atrium; lv, left ventricle; pa, pulmonary artery; ra, right atrium.

View larger version (79K): [in a new window]   Fig. 4. Inflow tract and cardiac laterality defects in adult Carp-Lbh transgenic mice. (A,B) Posterior view of 3-month-old wild-type (wt) and transgenic (Tg) hearts. Note abnormal pulmonary venous return (black arrow) in the Tg. (C,D) Transverse Hematoxylin and Eosin-stained sections of the hearts shown in (A,B). Note the dilated atria and ectopic veins (arrowheads) in the Tg. (E,F) Anterior in situ view of 5-month-old wt and Tg hearts. Dextrocardia (white arrow) and dextroposition of the vasculature (asterisk) were observed in the Tg heart only. (G,H) Frontal Hematoxylin and Eosin-stained sections of the hearts shown in E,F, showing the rightward inclination of the apex in the Tg heart (black arrowhead). (I,J) Close-ups of left ventricular (lv) myocardium, revealing cardiomyocyte hypoplasia in mispositioned Tg heart. ao, aorta; la, left atrium; pa, pulmonary artery; ra, right atrium; rv, right ventricle.

View larger version (68K): [in a new window]   Fig. 5. Cardiomegaly in postnatal day 15 (P15) Carp-Lbh transgenic mice. Hearts of wild-type (wt; A,D,G) and transgenic (Tg; B,C,E,F,H,I) littermates at P15. (A-C) Frontal Hematoxylin and Eosin-stained sections at the levels of the atrioventricular valves. The Tg#1 heart is hyperplastic (B,E), whereas the Tg#2 heart is hypoplastic (C,F). Double-headed arrow marks the thickness of the intraventricular septum (IVS). lv, left ventricle; rv, right ventricle. (D-F) Higher magnification of hearts shown in (A-C) with close-up on IVS. Note reduced cardiomyocyte size and fusion in Tg#2 (F). (G-I) Adjacent histological sections from wt and Tg hearts shown in A-F stained with an anti-phospho-histone H3 antibody, demonstrating ventricular hyperproliferation of Tg#1 (H). (J) Average numbers of positive cells in selected areas (n=3) of the RV, IVS and LV myocardium on three different sections of each genotype per 100 DAPI-positive cells are shown. *P=0.016 versus wt.

View larger version (125K): [in a new window]   Fig. 6. Histological analysis of neonatal and embryonic Carp-Lbh transgenic hearts. (A-F) Frontal Hematoxylin and Eosin-stained sections of postnatal day 0 (P0) wild-type (wt) and transgenic (Tg) hearts. (A,B) Sections of entire hearts at level of pulmonary valves (pv) showing ventricular hypertrophy (double headed arrow) and dilation of the right atria (ra) in the Tg (B). (C,D) Close-up of the right ventricular outflow tract (rvot) below the pv, showing an obstruction of the pulmonary infundibulum in Tg mice (D, arrow) with excessive mesenchymal valve tissue and myocardial cells (# and arrowhead, respectively, in inset). (E,F) Close-up of the atrial septae. Note the patent foramen ovale in the Tg (asterisk). (G-L) Transverse Hematoxylin and Eosin-stained sections of E13 wt and hemizigous Carp-Lbh Tg hearts. Ectopic OFT valve formation (pound sign) in the right ventricle (rv), left ventricular (lv) hyperplasia (insets), levocardia (G,H), and a VSD (asterisk) are present in the Tg (I,J). (K,L) Sections at the level of the great arteries. Arrowhead indicates double right outlet ventricle (DORV) in the Tg heart. ao, aorta; aov, aortic valve; la, left atrium; lv, left ventricle; os, ostium secundum; pa, pulmonary artery; sp, septum primum.

View larger version (72K): [in a new window]   Fig. 7. Molecular analysis of embryonic Carp-Lbh transgenic hearts. (A-H) In situ hybridization showing downregulation of Anf mRNA (white arrow) in Carp-Lbh transgenic hearts. Serial sections of E9.5 wild-type (wt; A,C,E,G) and transgenic (Tg; B,D,F,H) hearts were hybridized with radioactive probes for Nkx2.5 (A,B), Tbx5 (C,D), Gata4 (E,F) and Anf (G,H). (I,J) Lbh inhibits activation of Anf-human growth hormone (Anf-hGH) reporter by cardiac transcription factors in NIH 3T3 cells. (I) Dose-dependent suppression of Anf activation by Nkx2.5, Tbx5 and Gata4 with increasing amounts of Lbh expression plasmid. Values are expressed as percentage of total Anf-hGH reporter transactivation by Nkx2.5, Tbx5 or Gata4 respectively. *P<0.001 (n=3) versus Nkx2.5; **P<0.001 (n=3) versus Tbx5; ***P<0.05 (n=3) versus Gata4. (J) Inhibitory effect of Lbh on synergistic transactivation of Anf by Nkx2.5 and Tbx5. *P=0.008 (n=4) versus Nkx2.5 + Tbx5.