Figure 4. In vivo expression directed by the R2R3 Hcn4 enhancer in the post-natal AV bundle and its interface with the AV Node.

(A–E) A postnatal (P5) R2R3-LacZ heart was stained with bluo-gal, imaged with optical projection tomography in whole-mount, then sectioned and counterstained with hematoxylin-eosin. (A) An image from a 3D reconstruction of optical projection tomograms shows a discrete band of enhancer activity along the anterior-posterior axis of the AV junction. Dashed lines indicate the positions of the coronal sections shown in panels B-E proceeding from anterior-posterior (1–4). The band of reporter activity begins below the annulus fibrosis (af) anteriorly at the crest of the interventricular septum (ivs) just below the aortic valve (ao) (B). As it moves posteriorly (C,D) expression band proceeds superiorly and penetrates the af, eventually terminating within it. In section 4 (E), the solid black line demarcates the area of the compact atrioventricular node (cavn) + inferior nodal extension (ine) and the solid white line demarcates the atrioventricular bundle (avb) + lower nodal cells (lnc) where reporter activity was present. (F) Post-natal hearts were also sectioned and counterstained with trichrome to visualize fibrous tissue (green). A close-up view of the area of bluo-gal is shown to contain abundant insulating collagen fibers (light blue), consistent with its identity as the avb. (G,H) Immunohistochemical analysis of an R2R3-LacZ section from an adult heart at the level of the avb (outlined, dashed line) showed mutual exclusion of β-gal and Cx43, but co-expression of β-gal and Cx40 in the avb. (I) A more posterior section at the level of the cavn (outlined, solid white) showed β-gal expression within the Cx40+ lnc domain (outlined, dashed line) that comprises the interface between the avb and the avn. Abbreviations: A, anterior; P, posterior; Cr, cranial; Ca, caudal; lv, left ventricle; ra, right atrium; rv, right ventricle; tv, tricuspid valve; mv, mitral valve.