Figure 6. Differentiation of visceral smooth muscle.

A–D: At E6, faint staining for α-SMA and γ-SMA defined the outer circular muscle layer. Asterisks represent SMA-negative mesenchymal cells bordering the outer circular muscle layer. E–H: Robust staining for α-SMA marked the outer circular and outer longitudinal muscle layers. γ-SMA was observed in the outer circular but not the outer longitudinal layer. SMA-negative submucosal mesenchyme was still present (asterisk). I–L: By E14, the inner circular layer (α-SMA-positive, weak γ-SMA) was evident. Asterisk denotes SMA-negative submucosal mesenchyme. M–P: At E16, four muscle layers were present including the inner longitudinal layer. All layers stained for both α-SMA and γ-SMA. Double asterisks denote submucosal neuronal plexus. Limited SMA-negative submucosal mesenchyme was present (asterisk). Arrowhead in M indicates SMA-positive staining within the villi. Q–T: In the adult intestine, the four visceral smooth muscle layers were directly subjacent to the lamina propria (arrow) with no intervening submucosal mesenchyme. The outer circular layer was α-SMA-negative. Scale Bars: 50µm (A, E, I, M, Q) and 10µm (B–D, F–H, J–L, N–P, R–T). En, endoderm; IC, inner circular; IL, inner longitudinal; LP, lamina propria; L, lumen; M, mesenchyme; Mes, mesothelium; Mu, mucosa; OC, outer circular; OL, outer longitudinal; V, villi.