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First published online March 4, 2005
doi: 10.1242/10.1242/dev.01696


Development 132, 1337-1348 (2005)
Published by The Company of Biologists 2005


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Cited2 is required both for heart morphogenesis and establishment of the left-right axis in mouse development

Wolfgang J. Weninger1,*,{dagger}, Kylie Lopes Floro2,*, Michael B. Bennett3, Sarah L. Withington2, Jost I. Preis2, Juan Pedro Martinez Barbera4, Timothy J. Mohun3 and Sally L. Dunwoodie2,5,{dagger}

1 Integrative Morphology Group, Department of Anatomy, University of Vienna, Waehringerstrasse 13, A-1090 Vienna, Austria
2 Developmental Biology Program, The Victor Chang Cardiac Research Institute, 384 Victoria Street, Darlinghurst, NSW 2010, Australia
3 Developmental Biology Division, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK
4 The Neural Development Unit, The Institute of Child Health, 30 Guilford Street, London WC1N 1EH, UK
5 St Vincent's Clinical School, and School of Biotechnology and Biomolecular Sciences University of New South Wales, Kensington, NSW, Australia



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Fig. 1. Cited2 is differentially expressed in the heart and elsewhere during development. (A) Anterior view of a late headfold stage embryo (7.5 dpc) showing expression in the ventral node (arrow), cardiac crescent and blood islands. (B) Ventral view of a flatmounted four-somite stage embryo (8 dpc) showing expression in the cardiac crescent and in the caudal lip and pit of the node (arrow). (C) Ventral view of a flatmounted six-somite stage embryo (8 dpc) showing expression in the cardiac crescent, anterior lateral mesoderm (black arrow), paraxial mesoderm (white arrow) and node. (D) Anterior view of a six-somite stage embryo showing elevated expression in the cardiac crescent, anterior lateral mesoderm and trunk paraxial mesoderm. (E,F) Transverse sections through D at the levels indicated showing elevated expression in the cardiac mesoderm (arrow) and low-level expression throughout. (G) Lateral and dorsal view of 21-somite stage embryo (9.5 dpc) showing elevated expression in the forebrain-midbrain boundary, branchial arches 1 and 2, heart, and somites. (H) Dorsal view of the embryo in G showing expression in the hindbrain (rhombomeres 3, 5, 6, 7) and adjacent paraxial mesoderm. (I-K) Frontal sections through a 21-somite stage embryo at the levels indicated in G. (I) Expression is elevated in the ventricular myocardium and trabeculae of the outflow tract and presumptive right ventricle; (J) in the myocardium and, to a lesser extent, the endocardium and endocardial cushion cells in the outflow tract and atrioventricular canal; and (K) in the aortic sac, presumptive atria and sinus venosus (inflow tract). (L) Lateral view of 40-somite stage embryo (10.5 dpc) showing expression in the outflow tract, atrioventricular canal (black arrow), branchial arch 4 (white arrow), central nervous system, somites and limbs. (M,N) Sections through a 40-somite stage embryo at the levels indicated in L, showing: expression in the (M) outflow tract and, to a lesser extent, in the compact myocardium and trabeculae of the ventricles; and expression in the (N) myocardium adjacent to the endocardial cushion of the atrioventricular canal. (O) Ventral and (P) dorsal views of a heart dissected from an embryo at 13.5 dpc showing expression predominantly in the outflow tract (O) and inflow region (P). (Q) Frontal section through a dissected heart at the same stage as shown in O,P showing expression in the septum primum (black arrow), around the vena cava (asterisk) and in the endocardial cushions of the atrioventricular canal (white arrow). Cited2 expression was assayed by detecting ß-galactosidase activity from the Cited2-lacZ allele in embryos heterozygous (A,B,G,H,L,O,P) or homozygous (I,J,K,M,N,Q) for the allele, or via Cited2 RNA hybridization (C-F). The colorimetric reaction used to identify Cited2 transcripts was extended for the embryo in D, in order to reveal low levels of Cited2 expression. cc, cardiac crescent; oft, outflow tract; ift, inflow tract; v, ventricle; avc, atrioventricular canal; as, aortic sac. Scale bar: 135 µm in A,B; 260 µm in C,D,H; 35 µm in E,F; 280 µm in G; 50 µm in I-K; 550 µm L; 94 µm in M,N; 390 µm O,P; 310 µm Q.

 


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Fig. 2. Cited2-null hearts display cardiac looping defects and atrial isomerism. (A-C) Scanning electron micrographs showing ventral views of hearts at 9.5 dpc (23 somites). The head, tail and the pericardium were removed. (A) Cited2 wild-type heart showing a normal D (rightward) loop. (B) Cited2-null heart showing an abnormal D loop. The left ventricle is ventrally displaced and occupies a more central position than normal. (C) Cited2-null heart showing an abnormal L (leftward) loop. (D-F) Right atrial isomerism is observed in Cited2-null embryos with abnormal heart loops. Ventral views of hearts from 10.5 dpc show expression of the MLC3F-nlacZ-2E transgene. (D) A Cited2 wild-type heart shows a normal D (rightward) loop and asymmetric MLC3F-nlacZ-2E transgene. (E) A Cited2-null heart shows a normal D loop and asymmetric MLC3F-nlacZ-2E transgene. (F) A Cited2-null heart shows an L (leftward) loop and strong bilateral expression of the MLC3F-nlacZ-2E transgene, indicating right atrial isomerism. Arrows indicate orientation of the outflow tract. A, atrium; R L, right-left axis. Scale bar: 100 µm.

 


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Fig. 3. Cited2-null embryos have abnormal expression of left sided determinants. RNA in situ hybridization of embryos and hearts hybridized with probes that recognize Nodal (A-C), both Lefty1 and Lefty2 (D-I), and Pitx2 (J-R) transcripts. Nodal is expressed in the left lateral mesoderm (arrow) and node of six- to seven-somite stage Cited2 wild-type (A) and heterozygous (B), but not in LPM of Cited2-null (C) embryos. (D-I) A combined Lefty1/2 probe shows that Lefty1 and Lefty2 are expressed in the left prospective floor plate of the neural tube (white arrow), and left LPM (black arrow) respectively, in seven- to nine-somite stage Cited2-wild-type (D,G) and heterozygous (E,H) embryos. In Cited2-null embryos (F,I), Lefty2 expression is not detected, whereas the expression of Lefty1 is restricted to the posterior floor plate. (J-L) Pitx2 is expressed in left lateral mesoderm (arrow) of 12- to 14-somite stage Cited2 wild-type (J) and heterozygous (K), but not Cited2-null (L) embryos. (M-R) At 13.5 dpc, Pitx2 is expressed in the ventral myocardium of the right ventricle, throughout the left atrium and in ventrally viewed veins of Cited2 wild-type embryos (M,P) and in null embryos that lack a laterality defect (N,Q). Pitx2 is not expressed in the hearts of Cited2-null embryos that have a right isomeric phenotype (O,R). In some Cited2-null hearts, a novel domain of Pitx2 expression was observed in dorsal ventricular cells (Q; arrow). Ventral view (A-C,J-O), lateral view (D-F), posterior view (G-I), dorsal view (P-R). Scale bar: 235 µm in A-C; 175 µm in D-I; 365 µm in J-L; 335 µm in M-R.

 


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Fig. 4. Cited2-null embryos show a range of pulmonary and cardiac defects. Two dimension EFIC sections at 14.5 dpc through corresponding planes of a Cited2-null embryo (A-C) and a wild type (D-F) embryo. Cited2-null embryos show symmetrical arrangement of lung lobes, similar symmetry is evident in the size, topology and branching of the left and right principal bronchus (PB) and lobar bronchi (LB). Cited2-null embryos show cardiac defects that include a ventricular septal defect (arrows in A and B) and symmetrical right and left atrial appendages (aa) adjoining a median chamber (asterisk in B). Histological sections of 14.5 dpc Cited2-null embryos (G,H) show subtypes of atrioventricular junction (J) malformations: (G) single atrioventricular ostium opening solely into the right ventricle, (H) single atrioventricular ostium opening equally into both ventricles because of the large ventricular septal defect. The median atrial chamber is indicated by an asterisk. (I) Retro-esophageal right subclavian artery (arrowhead). E, esophagus; T, trachea; DA, descending aorta; VS, ventricular septum; R, right; L, left. Scale bar: 500 µm in A-F; 500 µm in G-I.

 


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Fig. 5. Three-dimensional models of the bronchial system and great arteries, illustrating the right isomeric phenotype of Cited2-null embryos at 14.5 dpc. Models viewed from ventral (A,B) or left and right sides (C,D). (A,A') Differences in topology, size, course and branching pattern of the right (yellow) and left (red) bronchial trees in the normal embryo, as well as in the arrangement of the blood vessels (brown). The Cited2-null embryo shows a symmetrical bronchial tree (B,B'), profound differences in the relationship between the bronchi and pulmonary arteries; transposition of the great arteries and interruption of the aortic arch between the origin of the left common carotid artery (CC) and the left subclavian artery (SA). (B,D) The pulmonary trunk, pulmonary arteries, ductus arteriosus, descending aorta, and left subclavian artery are shown in magenta; the aorta, coronary arteries, brachiocephalic trunk, and left common carotid artery are shown in gold. AA, ascending aorta; AR, aortic arch; DA, descending aorta; PT, pulmonary trunk; PA, pulmonary artery; DB, ductus arteriosus; CA, coronary artery; BT, brachiocephalic trunk; T, trachea; LB, lobar bronchus.

 


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Fig. 6. Three-dimensional models (A-C) and labeled diagrams (A'-C') showing atrial malformations of Cited2-null embryo hearts at 14.5 dpc. The models show the dorsal side of the atria (orange) and associated blood vessels (blue). (A,A') Normal, asymmetric atrial appendages. (B,B') Cited2-null hearts show symmetrical atrial appendages, each resembling a normal right atrium. The left superior vena cava (LSC) of the Cited2-null heart does not undercross the common space formed by pulmonary veins (PV): the symmetric arrangement of the pulmonary veins and the additional blood vessel (asterisk) to the right of the inferior vena cava (IVC). (C,C') Dorsal atrial wall from the ventral aspect of a Cited2-null heart, after removal of the ventricles and atrioventricular junction from the model. Between the left and right atrial appendages, there is a median chamber (MC, purple) that is incompletely separated from them by ridges protruding from the dorsal side (arrowheads), this MC receives all veins. RA, right atrial appendage; LA, left atrial appendage; RSC, right superior vena cava; O, orifice of the lung vein.

 


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Fig. 7. Ventricular septal defects in Cited2-null embryos. (A) Three-dimensional model of a 14.5 dpc Cited2-null heart, viewed from the ventral side. (B) Ventral aspect, after removal of the ventral and lateral walls of the ventricles, up to (but not including) the ventricular septum (VS). The ventral and lateral walls of the atrial chambers have also been removed. The right ventricle has only a small volume. (C,C') Model of (B) after 90° rotation (viewed from the left). A ventricular septal defect (VSD) is evident beneath the outflow tract, above the endocardardial cushion material (green, asterisk). A second VSD can be seen beneath the free margin of the septal leaflet (yellow) of the single atrioventricular valve. RA, right atrium; LA, left atrium; RV, right ventricle; LV, left ventricle.

 


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Fig. 8. Malformations in the atrioventricular junction of Cited2-null embryos. (A) Semi-transparent view of a 14.5 dpc Cited2-null heart, annotated in B. The cranial parts of the ventricles and atria have been removed from the model. A single atrioventricular ostium opens solely into the left ventricle (LV), guarded by leaflets (V) of a single atrioventricular valve. (C) An equivalent view obtained by modeling a normal heart. (D,D') Atrioventricular junction of a Cited2-null embryo, seen from the atrium after removal of the dorsal atrial wall. The atrioventricular valve has only one leaflet (brown, asterisk) and the opens into the left ventricle (arrowhead). (E,E') Atrioventricular junction of a Cited2-null heart, viewed from ventral side after removal of the apical parts of the ventricle walls and the ventricle septum (VS). The right (RV) and left (LV) ventricle differ in volume and there is only a single opening of the atrioventricular ostium (yellow, arrowhead) into the LV. LA, left atrium; RA, right atrium; MC, median chamber of atrium; PV, pulmonary vein; LSC, left superior vena cava; IVC, inferior vena cava; R L, right-left axis.

 


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Fig. 9. Cited2-null embryos have reduced cell density of the atrioventricular cushions. Hematoxylin and Eosin-stained sections of Cited2 wild-type (A,E) and null embryos (B-D,F-H) at 10 dpc. Sections were cut at a 45° angle (midway between frontal and transverse). (E-H) Higher magnifications of the boxed regions in A-D, showing the reduced cell density within the atrioventricular cushions in the Cited2-null hearts. Scale bar: 285 µm in A-D; 75 µm in E-H.

 

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© The Company of Biologists Ltd 2005