Twist1 has similar spatiotemporal expression profiles as Snai2 in the intestine. A: Twist1 mRNA level peaks at the climax (Stage 62). Quantitative RT-PCR analysis of Twist1 mRNA level in intestine from tadpoles at indicated stages, normalized against the control gene EF1a. B-G: In situ hybridization reveals connective tissue-specific expression of Twist1 in intestine. Note that Twist1 mRNA level peaked in the connective tissue at the climax (St61-62). H-I: Twist1 is not expressed in the muscle layers. In situ hybridization sections of Twist1 at the stage 61 were double-stained with smooth muscle actin antibody (red) to labeled the muscle layers (Ms). Ty: typhlosole, the single epithelial fold in in premetamorphic tadpole intestine where connective tissue is abundant; Lu: lumen; Ep: epithelium. Note that Twist1 labeled cells are localized between epithelium and muscle layers.