Fig. 2.

Double staining of wildtype and Shox2 ^−/− embryos. A Double whole-mount in situ hybridization with corresponding sections (50 μm) on E12.5 (a, b) and E13.5 (c, d) wildtype and Shox2 ^−/− mouse hearts using Isl1 and Hcn4 RNA probes. Murine Isl1 (blue staining) and Hcn4 (red staining) are expressed in the SAN (a, a’, c, c’) of the developing heart in wildtype embryos (indicated by black arrowhead). In the Shox2 ^−/− mouse heart Isl1 expression is completely absent in the SAN (b, b’, d, d’), which represents the overlapping expression domains of Shox2 and Isl1 in the wildtype heart. Note that Hcn4, another marker of the SAN region, is still expressed in Shox2 ^−/− hearts, even if the expression is somewhat reduced (b, b’, d, d’). B Transverse sections (10 μm) of E11.5 wildtype (a–c; g–i) and Shox2 ^−/− (d–f; j–l) embryos were stained with Isl1 (red) and the SAN marker Hcn4 or Tbx18 (green). Nuclei are counterstained with Hoechst (blue). Isl1 and Hcn4 expression clearly overlaps in the SAN region of wildtype embryos (a; white arrowhead). Hcn4 is still detectable in the SAN of Shox2 ^−/− embryos (f), whereas no Isl1 staining is visible in this region (e). Isl1 and Tbx18 expression clearly overlaps in the SAN head of wildtype embryos (g; white arrowhead). Tbx18 is still detectable in the SAN of Shox2 ^−/− embryos (l), whereas no Isl1 staining is visible in this region (k). RA right atrium, LA left atrium, RV right ventricle, LV left ventricle, SAN sinoatrial node. Scale bars: a–f 1,000 μm; g–l 500 μm