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First published online January 26, 2007
doi: 10.1242/10.1242/dev.02777

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Hesr1 and Hesr2 regulate atrioventricular boundary formation in the developing heart through the repression of Tbx2

¹ Division of Mammalian Development, National Institute of Genetics, 1111 Yata, Mishima Shizuoka 411-8540, Japan.
² Department of Genetics, The Graduate University for Advanced studies, 1111 Yata, Mishima Shizuoka 411-8540, Japan.
³ Department of Pediatric Cardiology, The Heart Institute of Japan, Tokyo Women's Medical University, 8-1 Kawada-cho, Shinjyuku-ku, Tokyo 162-8666, Japan.
⁴ National Institute for Basic Biology, 38 Nishigonaka, Myodaiji, Okazaki Aichi 444-8585 Japan.

View larger version (124K): [in this window] [in a new window]   Fig. 1. The possible involvement of Hesr1 and Hesr2 in AV boundary formation. (A-F) Expression patterns of Hesr1 and Hesr2 in wild-type mouse embryos at E9.5 revealed by whole-mount in situ hybridization. The specific expression of Hesr1 in the atria (A,D) and of Hesr2 in the ventricles (B,E) can be seen, but neither gene is found to be expressed in the AV canal (AVC) (C,F). The heart regions in A-C are magnified in D-F, respectively. (G-J,L-S) The expression patterns of AV boundary markers in wild-type (G,L,P), Hesr1-null (H,M,Q), Hesr2-null (I,N,R) and Hesr1-Hesr2 double-KO (J,O,S) mouse embryonic hearts at E9.5. To demarcate the boundaries between both the atrium and ventricular chambers and the AV canal, the AV myocardial markers Bmp2 (G-J) and Tbx2 (P-S), and the chamber marker Anf (L-O), were used as probes. (K) The ratio of the AV canal area was calculated by determining the Bmp2 expression area in comparison with the entire heart area from a lateral side view (n=4, P<0.005, t-test). The brackets in G-J and L-P indicate the AV canal. The arrowheads in Q-S indicate the affected AV boundaries. The gene probes used (upper right) and genotypes examined (lower right) are indicated in each panel. LA, left atrium; LV, left ventricle.

View larger version (112K): [in this window] [in a new window]   Fig. 2. Misexpression of Hesr1 and Hesr2 in the entire embryonic mouse heart at E9.5. (A-E) The expression of Hesr1 (A,E) and Hesr2 (B,D) was examined by whole-mount in situ hybridization of double-transgenic mouse embryos. The embryos were produced by crossings between Mesp1-Cre and either CAG-lox-CAT-lox-Hesr1 (A,D) or CAG-lox-CAT-lox-Hesr2 (B,E) mice. (C) Expression levels of Hesr1 and Hesr2 in two independent lines for each gene derived from these misexpressing mouse embryonic hearts (n=10). Both Hesr1 and Hesr2 are strongly induced (2.5-5.5 fold) compared with the wild-type heart. (F-H) Sections stained with Hematoxylin and Eosin showing a normal morphology for the atrium, AV canal (bracket), and the ventricle in the wild-type embryonic heart (F). The width of the AV canal (bracket) is smaller in the Hesr1-ME heart (G). The atrium and ventricle seem to be directly connected (arrowhead) in the Hesr2-ME heart (H). (I-K) Analysis of cardiomyocytes in the left ventricles of the mouse embryonic heart by transmission electron microscopy. Myofibrils were observed to be thin and disorganized in the Hesr1-ME (J) and Hesr2-ME (K) hearts, compared with wild type (I). In the Hesr2-ME hearts, Z bands (arrowhead) were sometimes barely discernible (asterisks). Scale bars: 100 µm in F-H; 1 µm in I-K. RA, right atrium; RV, right ventricle; LA, left atrium; LV, left ventricle.

View larger version (108K): [in this window] [in a new window]   Fig. 3. Defects in AV boundary formation in both the Hesr1- and Hesr2-misexpressing heart. Chamber identities were examined by whole-mount in situ hybridization of E9.5 embryos. Hand1 (A-C) and Irx4 (D-F) were used as markers for the ventricular chamber. Cx40 (G-I) was used to demarcate both the atrial and ventricular chambers. Boundaries between the chambers and AV canal are less evident in the Hesr1-ME heart (B,E,H) and appear to be lost in the Hesr2-ME heart (C,F,I), as compared with the wild-type embryo (A,D,G). The bracket in D indicates the AV canal (AVC) and the arrowheads in E,F,H,I indicate the AV boundaries for the atrium and/or ventricle.

View larger version (88K): [in this window] [in a new window]   Fig. 4. The gene expression pattern underlying the regulation of AV canal formation and the possible regulation of Tbx2 by both Hesr1 and Hesr2. (A-F) Expression of the AV myocardium-specific markers, Bmp2 (A-C) and Tbx2 (D-F), were examined in wild-type (A,D), Hesr1-ME (B,E) and Hesr2-ME (C,F) mouse embryos (E9.5) by in situ hybridization. The reduction or lack of an AV canal was evident in these misexpressing hearts (B,C,E,F). (G-N) X-Gal staining of transgenic embryos containing either a 2.5 kb (Tbx2-Xho-nlacZ) (G,H) or 6 kb (Tbx2-D3-nlacZ) (I-N) upstream region of Tbx2 in wild-type (G-J), Hesr1-ME (K,L) or Hesr2-ME (M,N) mouse hearts. The heart regions shown in G,I,K,M are magnified in H,J,L,N, respectively. The brackets in A,B,D indicate the AV canal and the arrowheads in C,E,F indicate the AV boundaries. LA, left atrium; LV, left ventricle. (O) Schematic of the luciferase or LacZ reporter constructs harboring the Tbx2 upstream regions. A region containing Smad-binding sites is present 2.9 kb upstream from the first ATG of the Tbx2 gene. (P) Reporter assay using the Tbx2-D3 luciferase construct (shown in P) in NIH3T3 cells. Luciferase activity was assessed with or without the empty vector (pCDNA), 6xMyc-Hesr1 or 6xMyc-Hesr2, in the presence or absence of the constitutive active (CA) or kinase-dead (KD) form of Alk3 and Smad5.

View larger version (118K): [in this window] [in a new window]   Fig. 5. The expression of Hesr1 and Hesr2 is unaffected in Tbx2-misexpressed, Notch2-KO or Notch2-activated hearts. Hesr1 and Hesr2 expression was examined in Tbx2-ME (A,B), Notch2-KO (D,E) and Notch2-ICD-ME (G,H) embryos (E9.0). (A,B) The expected expression patterns of Hesr1 (A) and Hesr2 (B) were observed in the Tbx2-ME hearts. A normal expression pattern for Hesr1 (D,G) and Hesr2 (E,H) was observed in Notch2-KO (E9.5) and Notch2-ICD-ME (E9.0) embryos. (C) Tbx2 was detectable throughout the entire heart in the Tbx2-ME embryo. (F,I) Notch2 expression was detectable using a probe directed against the Notch2-ICD (I), but not with a probe for the extracellular domain (ECD) of Notch2 (F). A, atrium; V, ventricle; LA, left atrium; LV, left ventricle.

View larger version (18K): [in this window] [in a new window]   Fig. 6. Model of the role of Hesr1 and Hesr2 in AV boundary formation. Hesr1 and Hesr2 are specifically expressed in the atrium and ventricle but not the AV canal. The AV canal is specified by Bmp2 expression, which induces Tbx2 expression. Bmp2 signals might diffuse to the chamber area and thus induce Tbx2 in the chamber cells. However, because of Hesr1 and Hesr2 expression in the atrium and ventricle, the undesirable expression of Tbx2 is suppressed in the chamber area, which leads to clear boundary formation between the AV canal and each chamber. Tbx20 is a possible upstream factor in this process as it is known to be required for expression of Hesr1 and Hesr2. Unknown factor (X) could induce Bmp2 expression, and possibly be regulated by Hesr2. Therefore, Hesr1 and Hesr2 determine the AV boundary by restricting the expression of Tbx2 to the AV myocardium.

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