Figure 3. Differentiation of outer and inner longitudinally oriented smooth muscle layers is required for development of the zigzags and villi, respectively.

(A) Left - Experimental schematic of tissue separation along the longitudinal axis used to measure longitudinal compression ratio. The muscle (green) from a strip of tissue is dissected away from the mesenchyme (blue)/endoderm (red) and the resulting lengths are compared. Right – Graph of separated muscle layers relative to mesenchyme and attached endoderm before (E12) and after (E13, E14, E15) longitudinal muscle layer forms. (B) Separation of endoderm from mesenchyme and muscle at E14 does not abolish zigzag pattern. (C) Top left panel - Experiment schematic of E12 gut cultured for 48 hours. Bottom - E12 guts cultured in DMSO alone or with either 10μm AG1295 or 10μm FK506 for 48 hours. Middle panels show luminal views, and bottom panels show longitudinal sections labeled with DAPI (blue) and SMA (green). Arrowhead denotes absence of muscle layer. Top right - Quantification of compression from E14 longitudinal muscle characterized by the ratio of the length of the control cultured segments to those lacking muscle. (D) Top left– Experiment schematic of E15 gut cultured for 48 hours. Bottom - Fresh E17 gut or E15 guts cultured in DMSO alone or with either 10μm AG1295 or 10μm FK506 for 48 hours. Middle panels show luminal views and bottom panels show longitudinal sections, labeled as in 3C. Arrowhead denotes absence of muscle layer. Top right - Quantification of compression from E16 longitudinal muscle, as in 3C. (n > 3 for all culture experiments, error bars represent one SD. Scale bars = 20μm)