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First published online April 25, 2008
doi: 10.1242/10.1242/dev.016147

Development 135, 1887-1895 (2008)
Published by The Company of Biologists 2008
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Intracardiac septation requires hedgehog-dependent cellular contributions from outside the heart

Matthew M. Goddeeris1,*, Silvia Rho1,2, Alexandra Petiet3, Chandra L. Davenport1, G. Allan Johnson3, Erik N. Meyers1,2,{dagger} and John Klingensmith1,2,{ddagger}

1 Department of Cell Biology,, Duke University Medical Center, Durham, NC, USA.
2 Department of Pediatrics, Duke University Medical Center, Durham, NC, USA.
3 Center for In Vivo Microscopy, Duke University Medical Center, Durham, NC, USA.


Figure 1
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  		Fig. 1. DM contributions to atrial septation are marked by Mef2C-AHF-Cre. (A-F) Mef2C-AHF-Cre mediated R26R expression. Whole-mount (A) and sagittal section (B) at E10.5 demonstrates DM expression leading into the atria. (C) At E11.5 DM expression is continuous with the DMP in the atria. (D) Transverse E12.5 section demonstrates interface between the DMP and AV cushion-derived CMM. (E,F) Transverse sections at E14.5 show expression in DMP and PAS but not in AV cushion derived tissues. (G,H) Comparison of Mef2C-AHF-Cre; R26R transverse sections and MRM slices enabled 3D MRM reconstruction of the heart. (G) Sample color-labeled transverse slice demonstrating labeling scheme used for renderings. (H) 3D reconstruction depicts heart from a dorsal-lateral perspective with AV cushion-derived tissue labeled yellow and DMP labeled green. A, atria; Ao, aorta; CMM, central mesenchymal mass; DM, dorsal mesocardium; DMP, dorsal mesenchymal protrusion; IAVC, inferior atrioventricular cushion; L, lung; LA, left atria; LBPa, left branch pulmonary artery; LV, left ventricle; MV, mitral valve; OFT, outflow tract; PA, pharyngeal arch; PAS, primary atrial septum; RA, right atria; RBPa, right branch pulmonary artery; RV, right ventricle; SAVC, superior atrioventricular cushion; SM, splanchnic mesoderm; TV, tricuspid valve.

 

Figure 2
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  		Fig. 2. Shh-/- mutant AVSD and defective DMP development. Transverse sections of E13.5 WT (A) and Shh-/- mutant (B) demonstrate a typical AVSD (asterisk) characterized by a large atrial septal defect and abnormal AV valve openings (scale bar, 500 µm). (C,D) Sagittal sections of Shher-Cre; R26R embryos show lack of heart expression, but foregut expression (FE) is juxtaposed to the DM. (E) Ptch1lacZ is observed in the DM, splanchnic mesoderm and DMP at E11.5 (sagittal section). (F) OFT and atrial DMP expression are the only areas of the heart where Hh activity is detected at E11.5 (wholemount, dorsal view). (G) E10.5 WT Ptch1lacZ embryo shows expression in the DMP (wholemount, right view,). (H) E10.5 Shh-/- mutants largely lack Ptch1lacZ expression in atria (asterisk). A, atria; FE, foregut endoderm; GE, gut endoderm; IAVC, inferior atrioventricular cushion; LE, lung endoderm; LA, left atrium; LV, left ventricle; MV, mitral valve; PAS, primary atrial septum; RA, right atrium; RV, right ventricle; SAVC, superior atrioventricular cushion; SM, splanchnic mesoderm; TV, tricuspid valve; V, ventricle.

 

Figure 3
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  		Fig. 3. AV cushion formation is normal in Shh-/- mutant embryos. Wild-type (A) and Shh-/- (B) E10.5 AV cushion sagittal sections stained with PECAM1 (blue) and nuclear marker (green) demonstrated no significant change in morphology or total mesenchyme per cushion (quantitated in C; wild type and Shh-/- n=4). Identical analysis at E11.5 found no significant change in total mesenchyme cells or cushion volume (D; wild type and Shh-/- n=3). A, atria; V, ventricle; IAVC, inferior atrioventricular cushion; SAVC, superior atrioventricular cushion.

 

Figure 4
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  		Fig. 4. The AV cushions do not require Hh signaling for normal AV septation. (A) TnT-Cre is expressed in the myocardium as detected by R26R β-galactosidase activity in whole-mount (A) or in sagittal section (A') at E10.5. (B) Ablation of Smo with TnT-Cre results in normal AV septation (E18.5). (C) Tie2-Cre is expressed in all endothelial cells and derivatives, including AV cushion mesenchyme (C,C'). (D) Tie2-Cre; Smoflox/- embryos have normal AV septation (E18.5). (E,F) Simultaneous ablation of Smo using both Tie2-Cre and TnT-Cre (reported in E,E') does not induce AV septation defects (F, E14.5 transverse section). A, atria; IAVC, inferior atrioventricular cushion; LV, left ventricle; MV, mitral valve; RV, right ventricle; SAVC, superior atrioventricular cushion; TV, tricuspid valve; V, ventricle.

 

Figure 5
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  		Fig. 5. Conditional ablation of Smo using Mef2C-AHF-Cre results in AVSD. E14.5 transverse sections of (A) WT (Mef2C-AHF-Cre; R26R) and (B) Mef2C-AHF-Cre; R26R; Smoflox/- mutant embryos. Loss of Hh receptiveness results in loss of the DMP (arrowhead in B). The central mesenchymal mass on the ventricular septum (asterisk) is more rounded with less distinct AV septal valve leaflets in mutants. (C,D) The disparity in septal valve development becomes more pronounced by E16.5. Mural leaflets (arrows in D) appear unaffected in mutants, while septal leaflets (asterisks in D) are missing. Multiple views of wild type (E,E') and conditional mutant (F,F') MRM renderings demonstrate loss of the DMP in addition to a single OFT vessel in mutants (purple). Atrial walls have been subtracted in E and F to allow visualization of wedge-shaped DMP (green). A section of the dorsal wall has been subtracted in E' and F' to demonstrate relative size of DMP to other septal structures. Ao, aorta; LA, left atria; LV, left ventricle; MV, mitral valve; Pa, pulmonary artery; PAS, primary atrial septum; RA, right atria; RV, right ventricle; TV, tricuspid valve. Color scheme: red, aorta; brown, ventricles; yellow, AV cushion tissue; blue, pulmonary artery; purple, atria; DMP, green.

 

Figure 6
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  		Fig. 6. Deficient Hh activity results in abnormal DM development and reduced cell motility. Mef2C-AHF-Cre; Smoflox/-;R26R mutant embryos and controls are compared. β-galactosidase staining at E10.5 (A,B) demonstrates relatively normal DM levels in mutant (B). Staining at E11.5 (C,D) demonstrates loss of DMP (asterisk) while some atrial myocardial and PAS staining is maintained. (E-H) Lysotracker Red cell death analysis demonstrates no detectable increase in mutant embryos (F,H) when compared with controls (E,G) at E10.5 or E11.5. (I,J) Sagittal sections stained for MF-20 (red) and nuclei (green) demonstrate normal myocardial staining in the DMP and absence of MF-20 staining in DM in E11.5 wild type (I) compared with abnormal MF-20 staining within the DM of Mef2C-AHF-Cre; Smoflox/- embryos (J). MF-20 channel only (I',J') shows ectopic staining. (K) Significantly decreased DM migration is observed with 10 µm cyclopamine treatment compared with media alone or DMSO controls. A, atria; IAVC, inferior atrioventricular cushion; PAS, primary atrial septum; RV, right ventricle; SAVC, superior atrioventricular cushion. *P=0.02 (media versus cyclopamine), *P=0.001 (DMSO versus cyclopamine).

 

Figure 7
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  		Fig. 7. Model of DMP development in mutant backgrounds. Shh has multiple roles in AV septation, depicted by schematic sagittal sections at E11.5. (A) Shh produced by foregut endoderm (blue) signals to adjacent DM (green) allowing for mesenchymal movement into the atria and subsequent formation of the DMP. DM-derived endocardium (dark green) and splanchnic mesoderm (red) are also added to the atria at this time. (B) Global loss of Shh results in a failure of DMP development as well as abnormal AV cushion shape, probably owing to OFT and right ventricle shortening. (C) Loss of receptiveness for Hh signaling in the myocardium, endocardium and mesenchyme derived from the endocardium (TnT-Cre; Tie2-Cre; Smoflox/-) does not recapitulate the Shh-null AVSD phenotype. (D) While Shh is maintained in Mef2C-AHF-Cre; Smoflox/- mutants, lack of receptiveness in DM leads to failure of these cells to develop into the DMP.

 

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© The Company of Biologists Ltd 2008