Fig. 3. Expansion of smooth muscle layers and enteric motoneurons in conditional mutant stomach. (A-D) Embryos were obtained by mating with COUP-TFII floxed homozygous males and Nkx3-2^Cre/+; COUP-TFII^flox/+ females. COUP-TFII floxed homozygote served as controls, and Nkx3-2^Cre/+; COUP-TFII^flox/flox were designated as conditional mutants. Histological analysis of stomach dissected from E12.5 controls (A) and conditional mutants (B) showed that the epithelium of the conditional mutant is thicker (marked by the black arrows) in Hematoxylin and Eosin stained sagittal sections. At E13.5, the smooth muscle layers of the mutant stomach are disorganized in comparison with the controls, as seen by -smooth muscle actin immunostaining (brown) (compare C with D). (E-H) -smooth muscle actin staining of sagittal sections of E16.5 stomach. Smooth muscle cells were immunoassayed for -smooth muscle actin (green), and counterstained with propidium iodide (red). White arrows indicate the extension of -smooth muscle actin staining in the submucosal mesenchyme of the conditional mutant (F). (E,G,H) The thickened circular smooth muscle layer formation was observed in both the fore-stomach (F) and the hind-stomach (H) of the conditional mutant in comparison with the control (E,G). (I,J) Semi-thin section semi-thin sections of the stomachs from E15.5 embryos were examined. The circular smooth muscle layer of the conditional mutant stomach (J) is disorganized in comparison with the control (I). (K,L) PGP9.5 staining of E16.5 stomach. Enteric neurons were stained by protein gene product 9.5 (PGP9.5) antiserum (brown) and counterstained with Hematoxylin. (M,N) TUJ1 staining of E13.5 stomach. Anti-TUJ1 antibody was employed in immunostaining. An increase of TUJ1-positive cells is shown in the conditional mutant (N) in comparison with the littermate control (M).