Loss of Isl1 disrupts formation of the dorsal pyloric outer longitudinal muscle. (A) Immunofluorescence of Isl1 and α-SMA in Isl1F/+and Isl1MCM/Del embryonic pylorus at E18.5. Loss of Isl1 resulted in nearly complete loss of α-SMA-positive cells in the dorsal pyloric OLM (asterisks). Yellow dotted lines mark the epithelial basement membrane and white dotted lines indicate ICM and OLM boundary. Red staining is Isl1, green staining is α-SMA, and DAPI nuclear counterstaining (DNA) is blue. Scale bars: 50 μm. (B) α-SMA immunofluorescence of Isl1F/+and Isl1MCM/Del embryonic pylorus at E18.5. Compared with Isl1F/+embryos, the pyloric sphincter constriction was wider in Isl1MCM/Del animals. Sphincter constriction measurements are shown. Yellow bars demarcate the pylorus and highlight the marked difference in width between Isl1F/+and Isl1MCM/Del samples. Data are mean ± SEM (n = 6 mice per group), *P <0.05 (Student’s t-test). Scale bars: 50 μm. α-SMA, α-smooth muscle actin; ICM, inner circular muscle; OLM, outer longitudinal muscle.