Figure 1.

HCN4 expression during embryogenesis (A) In situ hybridization analysis reveals HCN4 is expressed in the developing head and along the dorsal midline at the completion of neurulation embryos (NF stage 21 lateral and frontal views). At early tailbud stages, HCN4 expression is observed primarily in the craniofacial region, in 2 stripes along the neural tube, and in the developing somites. This expression pattern is maintained as development progresses (NF stage 25, lateral and anterior/ventral views). At NF stage 26, HCN4 mRNA begins to be expressed on the left side of the developing heart field (arrow; lateral and ventral views). Expression becomes bilateral beginning at NF stage 27/28 (arrows; lateral and ventral views) but remains more highly expressed on the left side of the body. Scale bars = 200μm. (B) As cardiogenesis progresses during tadpole stages (NF stages 29–39, lateral and ventral views, arrows denote heart), HCN4 expression is diffusely present in the developing myocardium (outlined in yellow dashed lines/circles), but the highest expression is in the septum transversum where other proepicardial genes are expressed (Tbx18, NF stage 34). Scale bars = 200μm. (C,D) Histological sections confirm HCN4 expression in the cardiac mesoderm at tailbud stages, and in the myocardium and proepicardial region in tadpole stages. HCN4 expression can also be observed in the notochord and somites. Red lines in schematic diagrams denote the plane of section. Sections = 10μm, scale bars = 100μm. (E) Immunohistochemistry shows HCN4 protein is expressed at the completion of cardiac looping (NF stage 35) and is expressed throughout the entire ventricle of mature hearts (NF stage 46). Another important cardiac isoform, HCN2, is also found throughout the ventricle of mature hearts, similar to what has been described in mammalian systems. Transverse sections = 15 µm, scale bars = 100 µm. Schematic images are modified from Xenbase.²⁰