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Fig. 4. Bundle-branch patterning defects in Tbx5^del/+ mice. (A) Schematic representation of dissection of left-bundle-branch conduction system showing the plane of dissection (dashed line) and specimen orientation (arrow). The left ventricular free wall and mitral valve were removed to view the interventricular septum from the left. ß-Galactosidase expression marked the left-bundle-branch conduction system in minK^lacZ/+ (B,C) and Tbx5^del/+/minK^lacZ/+ (D,E) mouse hearts. The atrioventricular node (black arrow), atrioventricular bundle (red arrowhead) and left bundle branch (black arrowhead) were visible on the surface of the muscular interventricular septum. The broad left bundle branch in newborn minK^lacZ/+ mice (B) matured into a narrow fascicle with a well-defined atrioventricular bundle (red arrowhead) in adult minK^lacZ/+ mice (C). Comparable maturation did not occur in Tbx5^del/+/minK^lacZ/+ mice (D,E) and adult Tbx5^del/+/minK^lacZ/+ mice (E) retained the broad band of specialized conduction cells, without a discrete atrioventricular bundle. (F) Schematic representation of dissection of right-bundle-branch conduction system showing the plane of dissection (dashed line) and specimen orientation (arrow). The right ventricular free wall and tricuspid valve were removed and the interventricular septum viewed from the right. ß-Galactosidase expression marked the right bundle branch in minK^lacZ/+ (G,H) and Tbx5^del/+ minK^lacZ/+ (I,J) mouse hearts. The atrioventricular node (black arrow) and right bundle branch (black arrowhead) were visible on the surface of the muscular interventricular septum. In newborn minK^lacZ/+ mouse hearts (G), the right bundle branch was visible as a poorly defined band of cells loosely associated with the septal band and anterior papillary muscle of the right ventricle. In adult minK^lacZ/+ mouse hearts (H), the right bundle branch was well defined by a thin band of cells running along the inferior aspect of the septal band and onto the anterior papillary muscle of the right ventricle. In 9/20 newborn (I) and 7/15 adult (J) Tbx5^del/+/minK^lacZ/+ mouse hearts, the right bundle branch was absent from the right ventricular septal surface, with conduction cells present only along the crest of the interventricular septum demarcating the atrioventricular bundle. (K) Schematic representation of dissection of the interventricular septum for images in (L,M). In-situ hybridization with a connexin 40 probe in wild-type (L) and Tbx5^del/+ (M) mouse hearts. Connexin 40 expression was observed in the atrioventricular bundle (arrow), and left and right bundle branches (arrowheads) in the wild-type mouse heart (L) and in the atrioventricular bundle (arrow) and left bundle branch but not the right bundle branch in the Tbx5^del/+ mouse heart (M). In 3/5 newborn Tbx5^del/+/minK^lacZ/+ mouse hearts, the right bundle branch could not be identified.

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