Table 1.
Figure 1.
Analysis of DNA, protein, and morphological defects in the l11Jus8 mutant mouse.
(A) Three coding mutations found in the 35 Mb candidate intervals on mouse chromosome 11 are located in the Med31, Nf1 and Erbb2 genes. Only recombinant embryos with the C57BL/6 homozygous DNA fragment inherited from the mutagenised parent which contained the Erbb2 mutation were lethal at weaning, whereas embryos with homozygous Med31 and Nf1 and heterozygous Erbb2 mutations were viable. Straight and dotted lines depict homo- and heterozygous DNA, respectively. (B) Confirmation of the T98,433,986G base change in the Erbb2 gene in l11Jus8 mutants by Sanger sequencing. (C) Phenotype of wild type, homozygous l11Jus8 and Erbb2M802R mutant embryos and hearts. Scale bars: 2 mm for embryos, 500 µm for hearts. (D) Erbb2 protein structure: FLD - Furin-Like Domain, STKD - Serine/Threonine-Tyrosine Kinase Domain. Partial sequence of the STK domain (60 amino acids around the mutation point) is shown for mouse (M), human (H), chick (C) and zebrafish (Z) Erbb2 protein. Asterisks indicate conserved amino acids, colon and period indicate conservation between groups of strongly and weakly similar properties, respectively. Arrowhead demarcates the location of the amino acid change. (E) Predicted changes in the protein structure of the Erbb2M802R conserved kinase catalytic domain based on the reported crystal structure of the Erbb2 protein. (F) Top panel: expression of the Erbb2 mRNA in atria (A) and ventricles (V) of hetero- and homozygous l11Jus8, and embryonic erythrocyte (blood) samples. Prpf8 mRNA expression is used as a positive control for cDNA isolation in blood sample. Bottom panel: Erbb2 expression in the yolk sack (YS) of homozygous l11Jus8 embryo, “-RT” control, genomic DNA (gen) control. A no template negative control (neg) is shown in both panels. (G) Erbb2 immunohistochemistry in heterozygote and l11Jus8 atria (Atr) and ventricles (Ven). Embryonic erythrocytes within the cardiac chambers are indicated with an arrowhead. Scale bar = 200 µm.
Figure 2.
Effect of the homozygous Erbb2M802R mutation on heart morphology in l11Jus8 hearts.
(A–L) Representative H and E stained coronal sections of E12.5 hearts. (A–B) Atrioventricular cushions, (C–D) dorsal outflow tract, (E–F) ventricles, (G–H) atria. Arrows in (G–H) point to the atrial wall. AVC, atrioventricular cushion; OFT, outflow tract; Ven, ventricle; Atr, atrium; Ep, epicardium; En, endocardium; My, myocardium. (I–J) Longitudial atrial and (K–L) ventricular sections; homozygous l11Jus8 heart had distended atria. Embryos in I–L are littermates. Magnified areas in (I–J) show the developing pectinate muscles (arrows). (M–N) Activated Caspase 3 (Cas3) staining of atrial wall at E11.5. Inset shows positive activated Caspase3 staining from trigeminal ganglion on same embryo section. (O–P) Propidium Iodide (PI) labelling of the necrotic cells in E12.5 hearts. RA, right atrium; LA, left atrium. Arrowheads point to the unspecific PI staining resulting from cells of the OFT damaged during dissection. Scale bars: 200 µm in A–H and M–N, 50 µm in I–L.
Figure 3.
Optical mapping of the cardiac electrical activity obtained with di-4-ANEPPS voltage sensitive dye.
(A–B) and (C–F) show reading of E11.5 and E12.5 hearts, respectively. The location from which the electrical reading was taken is shown with lines onto the cardiac dissection image. Black arrowheads point to matched atrial and ventricular signals. (D) White arrowhead indicates absent signal in left ventricle corresponding to a present signal in left atria (black arrowhead), indicative of AV block. (E–F) White arrowheads indicate absent atrial signals corresponding to present ventricular signals (A block), a phenotype only present in mutant samples. Asterisks on RA and LA traces in (F) indicate signal described in Figure 5C. RA, right atrium; LA, left atrium; LV, left ventricle; RV, right ventricle.
Table 2.
Numbers of distended hearts, hearts with atrioventricular block (AVb), atrial block (Ab), and atrial signal conduction gaps in optical mapping experiments.
Figure 4.
Mapping of electrical signal propagation in E11.5 hearts.
(A) Pattern of wild type electrical signal propagation in the E11.5-12.5 heart. Different colours encode the time scale of the signal conduction; each colour represents a one-millisecond interval with black as the initial time point and fuchsia as the final time point. (B–C) Derivatives of the signal recorded in wt and homozygous mutant l11Jus8 E11.5 hearts. Black arrowheads point to the peaks used for map generation. (D–F) Optical maps showing spatio-temporal propagation of the electrical signal through the atria. Asterisk on (F) points to the gap in normal electrical activity in homozygous mutant l11Jus8 atria. (G–I) Optical maps showing electrical signal propagation through the ventricles. Maps are similar for all genotypes. SAN, sinoatrial node; RA, right atrium; LA, left atrium; LV, left ventricle; RV, right ventricle; AVC, atrioventricular canal; IVG, interventricular groove.
Figure 5.
Mapping of electrical signal propagation in E12.5 hearts.
(A–C) Derivatives of the signal recorded in wt and homozygous l11Jus8 E12.5 hearts. Black arrowheads point to the present peaks used for map generation, white arrowheads point to the missing peaks. Asterisk on (C) depicts the missing derivative for signal present on Figure 3F. The derivatives used in each map image are indicated on the electrical traces. (D–F) Optical maps showing spatio-temporal propagation of the electrical signal through the atria. Asterisks indicate gaps in normal electrical activity. White arrow shows the direction of the signal propagation in wild type and homozygous l11Jus8 atria. (G–I) Optical maps showing electrical signal propagation through the ventricles. Maps are similar for all genotypes. Each colour (D–I) represents a one-millisecond interval with black as the initial time point and fuchsia as the final time point. RA, right atrium; LA, left atrium; LV, left ventricle; RV, right ventricle.