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First published online 20 April 2005
doi: 10.1242/dev.01827

Development 132, 2463-2474 (2005)
Published by The Company of Biologists 2005
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Tbx20 dose-dependently regulates transcription factor networks required for mouse heart and motoneuron development

Jun K. Takeuchi1,2,3, Maria Mileikovskaia4, Kazuko Koshiba-Takeuchi1,2,3, Analeah B. Heidt5, Alessandro D. Mori1,2,3,6, Eric P. Arruda1,2,3,6, Marina Gertsenstein4, Romain Georges7,8, Lorinda Davidson9, Rong Mo2, Chi-chung Hui2,6, R. Mark Henkelman9,10, Mona Nemer7,8, Brian L. Black5, Andras Nagy4,6 and Benoit G. Bruneau1,2,3,6,*

1 Cardiovascular Research, The Hospital for Sick Children, Toronto, ON, M5G 1X8, Canada
2 Developmental Biology, The Hospital for Sick Children, Toronto, ON, M5G 1X8, Canada
3 The Heart and Stroke/Richard Lewar Centre of Excellence, University of Toronto, Toronto, ON, M5S 1A8, Canada
4 Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, ON, M5G 1X5, Canada
5 Cardiovascular Research Institute and Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94143-0130, USA
6 Department of Molecular and Medical Genetics, University of Toronto, Toronto, ON, M5S 1A8, Canada
7 Unité de recherche en développement et différenciation cardiaques, Institut de recherches cliniques de Montréal (IRCM), Montréal, QC, H2W 1R7, Canada
8 Programme de biologie moléculaire, Faculté des études supérieures, Université de Montréal, Montréal, QC, H3C 3J7, Canada
9 Mouse Imaging Centre, The Hospital for Sick Children, 555 University Avenue, Toronto ON, M5G 1X8, Canada
10 Department of Medical Biophysics, University of Toronto, Toronto, ON, M5S 1A8, Canada



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  		Fig. 1. Expression of Tbx20 in mouse development. Tbx20 is expressed in cardiac progenitors at E7.75 (A), including a medial domain of cells corresponding to a similar domain (B) of expression of Nkx2-5 (red arrowheads). These medial domains are a part of the anterior heart field (AHF), and overlap with expression of Isl1 (C). Sections of the embryos shown in A-C are shown in A'-C'. Sections of embryos at E8.5 are shown in A''-C''; arrowheads show overlapping Tbx20, Nkx2-5 and Isl1 expression. (D-F) Dynamic expression of Tbx20 at E10.5 (D-D''), E12.5 (E-E'') and E16.5 (F-F''). At E10.5 (D-D''), Tbx20 is expressed in the myocardium and cushion of the outflow tract (ot), in the myocardium and endocardium of the right ventricle (rv), and in the atria (la), with weaker expression in the left ventricular (lv) myocardium. Asterisk indicates the interventricular septum. Tbx20 is expressed most strongly in the endocardial cushions (red asterisks) at E12.5 (E-E''); at E16.5 (F-F''), its expression is primarily confined to cushion derivatives such as the pulmonary valve (pv) and the aortic valve (av). (G,H) Tbx20 expression in motoneurons (asterisk and ochre arrowhead) and dorsal aorta (red arrowhead) is lost in Shh-/- embryos at E9.5. Whole mounts are shown in G, while transverse sections are shown in H.

 


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  		Fig. 2. Restricted expression of Tbx20 isoform a. (A) cDNAs encoding Tbx20 isoforms a-d [adapted, with permission, from Stennard et al. (Stennard et al., 2003Go)]. Location and size of the in situ probes are indicated, along with nucleotide positions along Tbx20 cDNA. (B) In situ hybridization using Tbx20 (left) or Tbx20a (right) in situ probes on E9.5, E11.0 and E12.0 embryos. Apparent Tbx20a staining in brain at E9.5 is due to artefactual probe trapping. lv, left ventricle; rv, right ventricle; ot, outflow tract.

 


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  		Fig. 3. Efficient Tbx20 mRNA knockdown in embryonic stem (ES) cells (A) and ES cell-derived embryos (B,C). (A) Quantitative RT-PCR of Tbx20 mRNA levels in wild-type ES cells (wt) or ES cells stably transfected with constructs expressing Tbx20 shRNA a or b. Tetraploid aggregations were performed with ES cells with a 95% reduction in Tbx20 mRNA levels (b12), an 85% reduction in Tbx20 mRNA levels (a5, a6, b2) or a 65% reduction in Tbx20 mRNA levels (a4, b3). (B) Western analysis of protein extract from whole E9.5 embryos derived from lines a4, a6 and b12, and from wild-type embryos. (C) In situ hybridization for Tbx20 on wild-type, a6 and b12 embryos.

 


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  		Fig. 4. Altered cardiac morphology in Tbx20 knockdown embryos. (A-C) Bright-field frontal views of E9.25 embryos. (A) Wild-type, (B) b12 knockdown and (C) a6 knockdown. Insets in B,C show the absence of EGFP signal in the embryo. (D-U) Rendered optical projection tomography of embryos stained for Actc and Mybpc3 to label cardiac myocytes. Embryo is rendered translucent white; heart is red (D-F,J-L,P-Q); chamber fill is yellow (G-I,M-O,S-U). Views are from the right (D,G,J,M,P,S), front (E,H,K,N,Q,T) or left (F,I,L,O,R,U) sides. (D-I) Wild-type embryo; (J-O) b12 knockdown embryo; (P-U) a6 knockdown embryo. a, atrium; lv, left ventricle; ot, outflow tract; rv, right ventricle. White arrowhead indicates unusual bulging of the rv.

 


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  		Fig. 5. Histological analysis of altered cardiac morphology in Tbx20 knockdown embryos at E9.5. (A,D,G,J) Wild-type embryos. (B,E,H,K) Tbx20 b12 knockdown; B,E and H,K correspond to two distinct embryos. (C,F,I,L) Tbx20 a6 knockdown. The outflow tract and right ventricle (ot/rv) are combined in b12 embryos. Epithelial to mesenchymal transformation (EMT) of both the outflow and AV cushions is impaired (arrowheads in F,K; see ot in C) compared with wild-type embryos (asterisks in G, arrowhead in J). (J) Higher magnification of the atrioventricular canal in wild type compared with a b12 knockdown (K) embryo, showing the lack of EMT in the b12 embryo (arrowhead). (L,M) Higher magnification of left ventricular wall of a6 (L) and wild-type (M) embryos, showing thinned ventricular wall in a6 (compare red brackets). a, atrium; lv, left ventricle; ot, outflow tract; rv, right ventricle.

 


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  		Fig. 7. Altered cardiac gene expression in Tbx20 knockdown embryos at E9.0. Expression of Nkx2-5 (A), Tbx5 (B), Nppa (C), Gata4 (D), Hand1 (E), Bmp4 (F), Mef2c (G), Isl1 (H), Pitx2 (I) and Hand2 (J) by whole-mount in situ hybridization of E9.0 wild-type (WT), b12 knockdown (b12) and a6 knockdown (a6) embryos are shown.

 


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  		Fig. 6. Cardiac defects resulting from a mild knockdown of Tbx20 resemble human congenital heart defects. (A) External view of E12.5 wild type and a4 knockdown embryos, showing exencephaly in the a4 knockdown embryo. (B-D) Bright-field view of E12.5 hearts from wild-type (B), and a4 (C) and b3 (D) knockdown embryos. The right ventricle exhibits hypoplasia and there is abnormal septation of the outflow tract in a4 and b3 knockdown embryos. (E,F) Rendered optical projection tomography (OPT) of wild-type (E) and a4 knockdown (F) embryos. (E,F) Surface rendered views of OPT scan (left), followed by rendered chamber fills of OPT scans (middle; atrial chambers are purple, ventricular chambers are dark red, pulmonary artery is blue and aorta is red); and lateral view of the chamber fills (right), with the atria removed. The outflow tracts are spiral and straight in the wild-type and knockdown embryo, respectively. (G-N) Histology of wild-type (G-J) and a4 knockdown (K-N) embryos. There is a lack of outflow valves in L, and very primitive aortic and tricuspid valves in M,N. ao, aorta; av, aortic valve; la, left atrium; lv, left ventricle; mv, mitral valve; pa, pulmonary artery; PTA, persistent truncus arteriosus; pv, pulmonic valve; ra, right atrium; rv, right ventricle; tv, tricuspid valve.

 


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  		Fig. 8. Abnormal motoneuron differentiation in Tbx20 knockdown embryos. (A) Expression of Tbx20 and motoneuron markers Isl2, Hb9 and Isl1 in sections of E9.5 embryos at the thoracic level. Isl2 and Hb9 expression is decreased in Tbx20 knockdown embryos (siTbx20) compared with wild-type embryos (wt). (B) Normal dorsoventral patterning of the spinal cord in Tbx20 knockdown embryos, as shown by expression of Irx3 and Pax6. (C) Expression of Tbx20, Isl1, Isl2 and Hb9 in overlapping domains in the spinal cord at E9.

 


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  		Fig. 9. Tbx20 cooperatively activates transcription. (A) Activation of various reporter constructs by Tbx20, Tbx5 or Tbx1 expression constructs co-transfected into 10T1/2 cells. Reporter constructs used are Nkx2-5-luciferaseFL (Nkx-up), Pitx2-luciferase (Pitx2), Fgf10-luciferase (Fgf10), Myl7-luciferase (b-MHC), Gja5-luciferase (Cx40) and Nppa-luciferase (ANF). (B) Activation of the Mef2c-lacZ #3 reporter by Isl1, Gata4, Nkx2-5 or Foxh1 expression constructs, with (+, ++) or without (–) a Tbx20 expression construct. Red bars indicate 250 ng of the indicated expression construct, blue bars indicate 500 ng of the expression construct. +, 250 ng Tbx20 expression construct; ++, 500 ng Tbx20 expression construct. (C) Alignment of mouse (mm) and human (hs) sequence of part of the Nkx2-5 enhancer that comprises the Isl1-binding site (Isl1, red) and the Gata-binding site (Gata, blue). (D-H) Transgenic mouse embryos (E9.75) carrying the Nkx2-5-lacZ transgene (Nkx2-5 wt, D,F), or the Nkx2-5-lacZmutIsl1 transgene (Nkx2-5 Isl1 mut, E,G) shown as whole-mounts (D,E) or sections of the whole-mount stained embryos (F,G). Arrowhead shows expression in pharynx. For Nkx2-5 wild type, nine out of 12 Southern blot-positive embryos expressed lacZ strongly in the pattern shown; for Nkx2-5 Isl1 mut, 11 out of 14 Southern blot-positive embryos expressed lacZ weakly in the pattern shown, three were completely negative. (H) Activation of the Nkx2-5-luciferase FL reporter by Tbx20, Gata4, Isl1 or activated Alk3 (aAlk3) expression constructs, co-transfected singly or in combination in 10T1/2 cells. (I) Co-immunoprecipitation shows interactions between Isl1 and Myc-Tbx20 (m-T20), but not FLAG-Gata4 (F-G4), myc-Tbx1 (m-T1) or FLAG-Tbx5 (F-T5). Bottom two panels show 10% input.

 

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© The Company of Biologists Ltd 2005