DEV02344 Supplementary Material

Files in this Data Supplement:

• Supplemental Table - Adobe PDF

• Supplemental Figure 1 -

  Fig. S1. Semi-quantitative RT-PCR analysis of the cardiogenic genes, Mlc2a, Mlc2v, Mhca, Mhcb, Nkx2.5, Mef2c, Gata4, neuregulin 1 type I, neuregulin 1 type III, Erbb4, Ccnd11, jumonji (Jarid2) and Snail. Total RNA was isolated from the atria and ventricles of E10.5 hearts. Although Mhcb is induced in the atrium of the NCID1-activated heart, no other significant changes were observed between wild-type and NICD1-activated hearts.

• Supplemental Figure 2 -

  Fig. S2. Sections showing Pecam staining patterns, which indicate the presence of endocardial cells in the interventricular septum (IVS) of the NICD1-activated heart. The sections were prepared from the same samples shown in Fig. 2I (wild-type) and J (NICD1-activated), but the sectioning planes are different. (A,B) The wild-type heart. (C,D) The NICD1 activated heart.

• Supplemental Figure 3 -

  Fig. S3. Hematoxylin and Eosin staining of serial sections reveals that ectopic cells are present in the AV cushions of NICD1-activated heart at E9.5. Transverse sections of the wild-type (A) and NICD1-activated (B) hearts are shown from an anterior (1) to posterior (8) direction. Ectopic cells are indicated by arrows. Abbreviations: R, right side, L, left side; PV, primitive ventricle; AVC, atrioventricular canal; OFT, outflow tract. Scale bars: 200 μm.

• Supplemental Figure 4 -

  Fig. S4. Expression of Notch1, Hesr1, Wnt2, Bmp6 and Jag1 in wild-type (A,C,E,G,I) and NICD1-activated hearts (B,D,F,H,J) at E8.5. Whole mount in situ hybridization analyses in E8.5 embryos revealed the expression of Notch1 in endocardium (A), Hesr1 in endocardium, in addition to the sinus venosus and myocardium of the outflow tract (C), Wnt2 in the sinus venosus in addition to the of the outflow tract (E) and Bmp6 in endocardium and in the endothelium of the sinus venosus (G). Jag1 is expressed in the sinus venosus at E8.5 (I). The expression of each of these genes is induced in the atrium and ventricle of the NICD1-activated heart (D,F,H,J) in addition to the sites that are normally observed. Scale bar: 500 μm in J.

• Supplemental Figure 5 -

  Fig. S5. Reporter analysis of Wnt signaling using TOP-Gal transgenic mice. lacZ staining of E10.5 embryos revealed reporter activity in the somites, limb bud and otic vesicles (arrow, ot) but not in the hearts of either wild-type (A,C) or NICD1-activated embryos (B,D). Scale bars: 1 mm in B; 500 μm in D.