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First published online 22 March 2006
doi: 10.1242/dev.02344

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Activation of Notch1 signaling in cardiogenic mesoderm induces abnormal heart morphogenesis in mouse

Yusuke Watanabe^1,*,, Hiroki Kokubo^1,2,*, Sachiko Miyagawa-Tomita³, Maho Endo¹, Katsuhide Igarashi⁴, Ken ichi Aisaki⁴, Jun Kanno⁴ and Yumiko Saga^1,2,

¹ Division of Mammalian Development, National Institute of Genetics, Yata 1111, Mishima 411-8540, Japan.
² Department of Genetics, The Graduate University for Advanced studies, Yata 1111, Mishima 411-8540, Japan.
³ Department of Pediatric Cardiology, The Heart Institute of Japan, Tokyo Women's Medical University, 8-1 Kawada-cho, Shinjyuku-ku Tokyo162-8666, Japan.
⁴ Cellular and Molecular Toxicology Division, National Institute of Health Sciences, 1-18-1 Kamiyohga, Setagaya-ku Tokyo 158-8501, Japan.

View larger version (157K): [in a new window]   Fig. 1. Notch activation in the mouse cardiac cell lineage. (A-D) Notch1 expression revealed by whole-mount in situ hybridization at E10.5. In wild-type embryos (A,B), it is difficult to detect Notch1 expression in the heart at the whole-mount level, whereas in NICD1-activated hearts (C,D), Notch1 is strongly induced in whole heart which shows an abnormal morphology. The heart regions are magnified (B,D). (E-L) Immunohistochemical analysis of cleaved-Notch1 (NICD1 Ab) at E10.5. NICD1 signals are only observed in the nuclei of endocardial cells in the wild-type heart (E-H), whereas the signals are observed in both endocardial and myocardial cells in NICD1-activated hearts (I-L). The images of other sections were generated to indicate that only endocardial cells are NICD1-positive in the AV cushion of the wild-type embryonic heart (H). Arrows indicate endocardial cells. Arrowheads indicate myocardial cells. Abbreviations: LA, left atrium; RA, right atrium; LV, left ventricle; RV, right ventricle. Scale bars: 200 µm in E,I; 50 µm in F-H,J-L.

View larger version (145K): [in a new window]   Fig. 2. Morphological and histological defects induced by NICD1-activation. (A,B) Sections stained with Hematoxylin and Eosin show normal EMT in the AV cushion (brackets) and trabecular development in wild-type hearts (A), whereas the trabeculae are not formed normally in NICD1-activated hearts (B). Ectopic cell masses were also evident (arrowheads in B), the interventricular septum is right-shifted (arrow) and trabeculation occurs in the AV myocardium (asterisk in B) in NICD1-activated hearts. (C,D) Bmp10 expression was observed in the trabecular cells in both the wild-type (C) and NICD1-activated (D) ventricles. (E-J,O,P) Immunohistochemistry using anti-myosin (E,F,O), anti--smooth muscle actin (Sma) (G,H,P) and anti-Pecam (I,J) antibodies reveal that ectopic cell masses (arrowheads) are present in the cushion tissue in NICD1-activated hearts (F,H,J,O,P), but are never observed in wild-type hearts (E,G,I). (M,N) Hematoxylin and Eosin staining at E9.5 also shows ectopic cell masses in the cushion of NICD1-activated heart (arrowheads in N) but not in wild-type heart (M). (K,L) In wild-type myocardium (K), myofibrils with sarcomere structures (Z bands are indicated by black arrowheads) are observed by transmission electron microscopy, but only thin myofibrils without a proper sarcomere structure (no Z band indicated by while arrowheads) are formed in NICD1-activated hearts (L). Embryo samples were prepared at either E10.5 (A-L) or E9.5 (M-P). Serial sections were used for E,G,I (wild-type), F,H,J (NICD1-activated) and N-P (NICD1-activated). Abbreviations: LA, left atrium; RA, right atrium; LV, left ventricle; RA, right ventricle; Nu, nucleus. Scale bars: 200 µm in A-J,M-P; 1 µm in K,L.

View larger version (117K): [in a new window]   Fig. 3. Molecular characterization of defects in NICD1-activated hearts. The differences between the right and left ventricles were examined by whole mount (A,B,E,F) or section (C,D,G,H) in situ hybridization using probes Cited1 (A-D) or Hand1 (E-H). To demarcate the boundaries between both the atrial and ventricular chambers and the AV canal, chamber markers Anf (I,J), Smpx (K,L) and AV canal markers Bmp2 (M,N) and Tbx2 (O,P) were used as probes. Boundaries between the chambers and AV canal became less evident in the NICD1-activated heart (indicated by arrows in J and L). AV canal marker expression is also downregulated in the NICD1-activated hearts (indicated by brackets in M-P). Abbreviations: LA, left atrium; RA, right atrium; LV, left ventricle; RA, right ventricle. Scale bars: 200 µm in C,G.

View larger version (66K): [in a new window]   Fig. 4. Expression changes in the Hes and Hesr gene families at E10.5. (A) Semi-quantitative RT-PCR analysis showing upregulation of Hesr1, Hes1 and Hes5 in NICD1-activated hearts. Hesr1 is ectopically induced at particularly high levels in the ventricle. In situ hybridization analysis also revealed the ectopic induction of Hesr1 in the ventricle and AV canal of NICD1-activated hearts (C), whereas Hesr1 is expressed only in the atrium in wild-type hearts (B). In contrast to Hesr1, Hesr2 is expressed in the ventricle in wild-type hearts (D) and its distribution is not altered in NICD1-activated hearts (E). Abbreviations: LA, left atrium; RA, right atrium; LV, left ventricle; RA, right ventricle. Scale bar: 500 µm in E.

View larger version (109K): [in a new window]   Fig. 5. NICD1-activated hearts in a Hesr1-null background at E10.5. Hesr1-null mice do not show any detectable defects (A,C,E) and NICD1-activation in Hesr1-null induced heart defects (B,D) is similar to NICD-activation in Hesr1-positive mice. Hematoxylin and Eosin analysis also revealed impaired trabeculation, the appearance of cell masses in the AV cushion (arrowhead) and a right-shifted interventricular septum (arrow). However, the AV myocardium is recovered (brackets) (F). Abbreviations: LA, left atrium; RA, right atrium; LV, left ventricle; RA, right ventricle. Scale bar: 1 mm in B; 500 µm in D; 200 µm in F.

View larger version (93K): [in a new window]   Fig. 6. Upregulation of Wnt2, Bmp6, Tnni2 and Jag1 in NICD1-activated hearts at E10.5. (A) RT-PCR analysis was performed using RNA from the ventricle with the AV canal. Expression of Wnt2, Bmp6, Tnni2 and Jag1 is upregulated in NICD1-activated hearts. (B-I') Whole-mount and section in situ hybridization confirmed the upregulation of these genes (B,B',D,D',F,F',H,H', wild-type hearts; C,C',E,E',G,G',I,I', NICD1-activated hearts). The upregulation is also observed in NICD1-activated hearts in a Hesr1-null background (J-M). Abbreviations: LA, left atrium; RA, right atrium; LV, left ventricle; RA, right ventricle.