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First published online November 28, 2005
doi: 10.1242/10.1242/dev.02156

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Bmp2 is essential for cardiac cushion epithelial-mesenchymal transition and myocardial patterning

Institute of Biosciences and Technology, Texas A&M University System Health Science Center, 2121 West Holcombe Boulevard, Houston, TX 77030, USA

View larger version (60K): [in a new window]   Fig. 1. Bmp2 expression pattern during cardiogenesis. Whole-mount in-situ hybridization with Bmp2 exon3 probe shows Bmp2 expression pattern (A-I). At 7.5 dpc, Bmp2 expression can be visualized bilaterally in the primitive heart tube (A, indicated by arrows). Side view (B) and frontal view (C), indicating that at 8.5 dpc Bmp2 is expressed in the sinus venosus. At 9.5 dpc, both lateral view (D) and frontal view (E) showed that Bmp2 is highly expressed in the myocardium of the AV canal, as well as lower level of expression in the outflow tract (indicated by arrows). (F,G) Side view and frontal view, showing that at 10.5 dpc Bmp2 expression is maintained in the myocardium of the AV cushion region and in the outflow tract (arrows). At 11.5 and 12.5 dpc, Bmp2 expression decreased significantly, as denoted by arrows (H,I). A, atrium; LA, left atrium; LV, left ventricle; OFT, outflow tract; RA, right atrium; RV, right ventricle; V, ventricle.

View larger version (61K): [in a new window]   Fig. 2. Nkx2.5Cre activity and tissue-specific inactivation of Bmp2 in mouse embryos. (A) At 8.5 dpc, Nkx2.5Cre activity is visualized throughout the heart (ventral view in A). (B) At 9.5 dpc, parasagittal sections indicated there is intense ß-gal activity in both myocardium (arrow) and endocardium (arrowhead). (C-J). In-situ analysis using Bmp2 exon 3 as a probe, showing that at 8.5 dpc Bmp2 is expressed in the sinus venosus in the wild-type embryo (arrow, C) and is still expressed in the mutant (arrow, D). At 9.0 dpc (E,F) and 9.5 dpc (G,H,I,J), Bmp2 is highly expressed in the myocardium of the control AV canal (arrow, E,G,I) but is absent in the Bmp2 CKO mutant (arrow, F,H,J). I and J are parasagittal sections of embryos in G and H. (K,L) Immunohistochemistry of phospho-Smad1/5/8, effectors of Bmp signaling, indicating Bmp-responding cells in the endocardium (denoted by arrow) and myocardial cells (arrowhead) of the AV canal at 9.5 dpc (K). In the Bmp2 CKO mutant, the phospho-Smad 1/5/8 signal is reduced in both cell populations (L). a, atrium; ba, branchial arch; fn, fronto-nasal process; hf, head fold; sv, sinus venosus; v, ventricle.

View larger version (84K): [in a new window]   Fig. 3. Severe AV cushion defects in Bmp2 CKO mutant embryos. Parasagittal sections through heart of wild type (A,B) and Bmp2 CKO mutant (C,D) at 9.5 dpc, showing that mesenchymal cells are absent in the AV cushion (arrows). Panels B and D are high power images of A and C. (E,F) Parasagittal sections at 10.5 dpc, showing lack of AV cushion mesenchyme in the Bmp2 CKO mutant (arrows). Also note the severely compromised myocardium in the mutant heart. (G-J) In-situ hybridization indicated that Msx2 is expressed in the AV myocardium and migrating mesenchymal cells (arrow, G), but its expression is diminished in the mutant (arrow, H). Panels I and J are parasagittal sections of embryos in G and H, showing lack of Msx2 expression in the mutant (arrows). (K-N) Has2, encoding an extracellular matrix protein, is present in the AV myocardium (arrow) and migrating mesenchyme (arrowhead) in the 9.5 dpc wild-type embryos (K,M) but is reduced in the Bmp2 CKO myocardium (L,N). M and N are parasagittal sections of K and L. a, atrium; ba, branchial arch; v, ventricle.

View larger version (89K): [in a new window]   Fig. 4. Defective EMT in the Tie2Cre; Bmpr1a n/f mutant embryos. (A,B) Histologic analysis of wild-type (A) and Tie2Cre; Bmpr1a n/f mutant (B) embryos, showing lack of migrating mesenchymal cells in the AV cushion region at 9.5 dpc (arrows). (C,D) Immunohistochemistry of phospho-Smad1/5/8, showing Bmp-responsive endocardium in the control embryo (arrows in C). In the mutant, endocardium with no phospho-Smad1/5/8 (arrows in D) or reduced phospho-Smad1/5/8 is shown (D, arrowhead). (E-N) Whole-mount in-situ hybridization analysis with Twist1 (E,F), VE-cadherin (G-J), Notch1 (K,L) and Snai1 (M,N). Genotypes are shown, and arrows denote hybridization signal. a, atrium; ba1, first branchial arch; ba2, second branchial arch; v, ventricle.

View larger version (115K): [in a new window]   Fig. 5. Defective endocardium in Bmp2 CKO mutant embryos. Immunohistochemistry of NFATc1 in wild-type (A) and Bmp2 CKO mutant (B) embryos at 8.5 dpc. At 9.5 dpc, there is upregulation of NFATc1 expression in the AV cushion endocardium (C) in wild-type embryos but in the Bmp2 mutant many of the endocardial cells are negative and there is no regional specificity of the signal (D). Arrows denote positively stained cells. Whole-mount in-situ hybridization followed by sectioning for some probes with Twist1 (E-H), Msx1 (I-L), Smad6 (M,N), VE-cadherin (O,P), Notch1 (Q-T) and Snai1 (U,V) probes at 9.5 dpc in wild-type and Bmp2 CKO mutant embryos. Arrows denote positive hybridization signal or areas where signal is up- or downregulated in the mutant embryo. Probes and genotypes are labeled. a, atrium; ba1, first branchial arch; v, ventricle.

View larger version (23K): [in a new window]   Fig. 6. Tbx expression in Bmp2 CKO mutant embryos at 9.5 dpc. Tbx2 mRNA expression is located in the myocardium of the AV canal in wild-type embryos (A) but is not present in the mutant (B, indicated by arrow). Other Tbx genes, including Tbx5 and Tbx20, are expressed in the myocardium of the atrium and ventricle in wild-type embryos (C,E) and also in the Bmp2 CKO mutant embryos (D,F). Arrows denote hybridization signal. a, atrium; ba, branchial arch; v, ventricle.

View larger version (101K): [in a new window]   Fig. 7. AV myocardium defect in the Bmp2 CKO mutants at 9.5 dpc. In wild-type embryos, chamber-specific genes, including Anf (A,C), chisel (E,G) and connexin 40 (I,K), are expressed in the myocardium of the atrium and ventricle but not in the AV myocardium (denoted by arrows). In the Bmp2 CKO mutant, the expression of Anf (B,D), chisel (F,H) and connexin 40 (J,L) is expanded into the AV canal myocardium (denoted by arrows). Tgfß2 is expressed in the AV myocardium in wild-type embryos (M) but is significantly reduced in the mutant, as indicated by arrows (N). Lef1 is also expressed in the AV myocardium in wild-type embryos (O) but is significantly reduced in the mutant (P, indicated by arrow). a, atrium; v, ventricle.

View larger version (60K): [in a new window]   Fig. 8. Summary of Bmp2 function in the AV canal. Bmp2 signals to the cushion endocardium through Bmpr1a to promote expression of genes that support EMT, such as Twist and NFATc1. Bmp2 also induces expression of Smad6, an inhibitory Smad, to limit the extent of EMT in the forming AV cushion. An additional function of Bmp2 is to induce expression of Has2, thereby accentuating cardiac jelly deposition. Finally, within the AV myocardium, Bmp2 signaling is required for Tbx2 expression, which normally represses expression of chamber-specific genes in the AV canal. Arrows denote genetic, not necessarily direct, relationships.

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