Immunofluorescence of (i–iii) Nkx2-5flox/flox (WT) or (iv–vi) CAGGCre-ER™;Nkx2-5flox/flox (N25 CKO) pylorus (after two days of intraperitoneal tamoxifen injections) harvested at (A) E16.5 or (B) E18.5: (i,iv) DAPI; (ii,v) αSMA; or (iii,vi) merged. (A) In N25 CKO mice at E16.5, cell mass and αSMA expression is reduced in the dorsal OLM (asterisks). (B) At E18.5, the dorsal OLM in WT mice has expanded further and shows increased αSMA expression (Bi–iii). Deletion of Nkx2-5 beginning at E16.5, when the dorsal OLM is already αSMA positive (Aii–iii), leads to its regression (Biv–vi). Stomach is left; duodenum is right; and, dorsal is top. Green lines mark the epithelial basement membrane, and white lines separate ICM and OLM. Asterisk = dorsal pyloric OLM. Scale bars = 100 μm.