Distinct roles of Wnt/beta-catenin and Bmp signaling during early cardiogenesis -- Klaus et al. 104 (47): 18531 Data Supplement - HTML Page - index.htslp -- Proceedings of the National Academy of Sciences

Klaus et al. 10.1073/pnas.0703113104.

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SI Figure 6
SI Figure 7
SI Figure 8
SI Figure 9
SI Figure 10
SI Materials

SI Figure 6

Fig. 6. Canonical Wnt and Bmp signaling are activated in the developing heart of mice. (A) Scheme indicates FHF (red) and SHF (green) in a transverse section plane of mouse embryos at E8.35. [Scheme reproduced and modified with permission from ref. 1 (Copyright 2005, Nat Rev Genet).] (B-F) Immunofluorescence analysis of b-gal (green), Isl1 (red) and pSmad1/5/8 (blue) on a transverse section of Axin2^lacZ/+ mice. (C-F) The magnification of the dotted square in B shows coexpression of b-gal and Isl1 (arrowheads in C and D) and of pSmad1/5/8 and Isl1 in SHF cells (arrowheads in E and F), and additional expression of pSmad1/5/8 in the FHF (arrows in E and F). nf, neural fold; pV, primitive ventricle.

1. Buckingham M, Meilhac S, Zaffran S (2005) Nat Rev Genet 6:826-835.

SI Figure 7

Fig. 7. Conditional ablation of the Bmp receptor 1a prevents cardiac differentiation of the FHF. (A-D) Whole-mount in situ hybridization for BmpR1a in controls shows the expression within the cardiac crescent at the zero- to three-somite stage and in the SHF at six- and eight-somite stages (arrows). (E-P1) Frontal views of whole-mount in situ hybridizations for Nkx2-5, MLC2a, eHand, Mef2c, and Isl1 in controls (E, E1, G, I, K, M, O, and O1) and mutants (F, F1, H, J, L, N, P, P1) at crescent (E-J) and looping (K-P1) stages. Note that Nkx2-5 is almost normally expressed and cardiac differentiation is blocked at the two- to four-somite stage, whereas Isl1 and Mef2c are still expressed in the SHF of MesP1-cre; BmpR1a^lox/lox mice.

SI Figure 8

Fig. 8. Cardiac crescent morphogenesis in MesP1-cre; b-catenin^lox/lox and MesP1-cre; b-catenin^loxEx3/+ embryos. (A-C1) Immunofluorescence for b-catenin (green) and nuclei (blue) on transverse sections at the 5-6-somite stage. (A1-C1) Magnifications of dotted squares in A, B, and C show loss of b-catenin in FHF (arrow) and SHF (arrowhead) in MesP1-cre; b-catenin^lox/lox embryos and nuclear localization of b-catenin in FHFs and SHFs (arrow and arrowhead in C1) in MesP1-cre; b-catenin^loxEx3/+ embryos. (D-I) Transverse sections of whole-mount in situ hybridization for Bmp4 and Isl1 at the five- to six-somite stage showing the loss Bmp4 in MesP1-cre; b-catenin^lox/lox embryos and the overexpression of Bmp4 and Isl1 in the SHF of MesP1-cre; b-catenin^loxEx3/+ embryos. (J-O) Whole-mount in situ hybridization for Tbx-5 and eHand in control, MesP1-cre; b-catenin^lox/lox and MesP1-cre; b-catenin^loxEx3/+ embryos, showing no difference in their expression.

SI Figure 9

Fig. 9. Conditional loss-of-function of b-catenin affects expression of Bmps during SHF formation. (A-L1) Whole-mount in situ hybridization for Gata4, MLC2a, Nkx2-5, Bmp7, Bmp4 and Bmp2 in controls (A, C, E, G, I, and K) and MesP1-cre; b-catenin^lox/lox (B, D, F, H, J, and L) embryos at the seven- to eight-somite stage. (E1, F1; K1, and L1) Transverse sections of the outflow tract region of whole-mount in situ hybridizations for Nkx2-5 and Bmp2 in controls and MesP1-cre; b-catenin^lox/lox mutants. Note the loss of Bmp4 expression and the overexpression of Bmp2 in the outflow tract region of MesP1-cre; b-catenin^lox/lox embryos. OFT, outflow tract; fg, foregut; nt, neural tube.

SI Figure 10

Fig. 10. Conditional loss-of-function of b-catenin affects SHF proliferation. (A) Scheme indicates FHF (red) and SHF (green) in three transverse section planes of mouse embryos at E8.35 that were used for quantification. (B-D) Immunofluorescence analysis of pHH3 (green), Isl1 (red), and nuclei (blue) on a transverse section (highlighted in red in A) of control, b-catenin loss- and gain-of-function mice at E8.35. Note the reduction of pHH3-positive cells (green) and the equal amount of Isl1-positive cells (red) in MesP1-cre; b-catenin^lox/lox mice (C) in comparison with controls (B). (E) Scheme indicates FHF (red) and SHF (green) in six transverse section planes of mouse embryos at E8.5 that were used for quantification. (F and G) Immunofluorescence analysis of pHH3 (green), Isl1 (red), and nuclei (blue) on a transverse section (highlighted in red in E) of control and b-catenin loss-of-function mice at E8.5. Note the reduction in Isl1-positive cell (red) and the comparable amount of pHH3 positive cells (green) in MesP1-cre; b-catenin^lox/lox mice.

SI Materials

Antibodies and cDNAs.
Additional primary antibodies were used for the SI figures: goat-anti-b-galactosidase (Biogenesis) and rabbit-anti-phospho Smad1/5/8 (Cell Signaling Technology). Additional anti-sense transcripts of mouse cDNAs were used for the SI figures: Bmp2 (NM_007553.2); Bmp7 (NM_007557.2); BmpR1a (NM_009758); and Gata6 (NM_010258).