Figure 3

Gjd^{3′UTR-IRES-CreEGFP/+}; R26R^tdTomato/+ mice exhibit robust reporter expression in the atrioventricular node (AVN). (a) High-power confocal image of consecutive heart sections from a P0 Gjd3^{3′UTR-IRES-CreEGFP/+}; R26R^tdTomato/+ mouse were stained as follows: (i) Hcn4 (green), (ii) tdTomato (red), (iii) Merged (Hcn4/tdTomato overlap), and (iv) Acetylcholinesterase (AChase). (b) (i–iii) Consecutive heart cryosections from a P0 Gjd3^{3′UTR-IRES-CreEGFP/+}; R26R^tdTomato/+ mouse were co-stained with mitotic marker phospho-Histone 3 (pH3) and pan-cardiac marker sarcomeric α-actinin. Amongst the three AVN sections, only one pH3⁺tdTomato⁺ cells was identified (iii) (see inset and yellow arrow). (iv) In contrast, multiple pH3⁺ nuclei were identified in the ventricular apex (see inset). (c) Sections were obtained from a Gjd3^{3′UTR-IRES-CreEGFP/+}; R26R^tdTomato/+ mouse at P28 and stained for Cx30.2 (green) and Hcn4 (magenta). (i) In the distal AVN, almost complete overlap of tdTomato, Cx30.2, and Hcn4 was observed. (ii) In the nodal extension, similar overlap was seen. (d) (i) At P28, tdTomato reporter expression was also observed in cells along the surface of the heart. ii) PECAM-1 staining of cryosections confirmed co-localization of tdTomato in endothelial cells at the left ventricular (LV) free wall. (iii) Similarly, tdTomato marked endothelial cells at the LV apex based on PECAM-1 co-localization. Nuclei were counterstained with DAPI (blue). Scale bars: as shown for ((a–d(ii,iii)) 100 µm; (d(i)) 500 µm.