Ectopic smooth muscle develops at the periphery of the lung. (A-D) IHC for smooth muscle markers SM22α and SMA indicates smooth muscle development around the periphery of the Ezh2mesoderm-KO lung at E18.5 (B, arrowheads). Inset shows morphological differences between control blood vessel (C, outlined by the dashed line) and Ezh2mesoderm-KO ectopic smooth muscle (D). A, airway; V, blood vessel. (E,F) OPT on whole-mount IHC for SM22α with E-cadherin counterstain to outline lung endoderm. Insets of right and left lung lobes reveal the patterning of ectopic smooth muscle in Ezh2mesoderm-KO lungs. (G-L) IHC for SM22α and SMA throughout lung development shows that ectopic smooth muscle development initiates by E14.5 (H,K) and expands between E14.5 and E16.5 (I,L, arrowheads in inset). Scale bars: 20 µm in A,C; 500 µm in E,F; 100 µm in G-L.