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With the aid of a dissecting microscope, the extraembryonic tissue was manually removed from embryos in phosphate buffered saline (PBS). To dissociate the germ layers, embryos were subsequently placed in 500 uL of 1% trypsin in Hanks solution. After 30 minutes of incubation on ice, the digestion was stopped with the addition of 1 mL of 20% fetal bovine serum (FBS) in Hanks solution plus a small amount of DNase I powder. Tungsten needles or fine forceps were used to tease away the whole ectoderm from other two germ layers. Then the whole endoderm was cut into foregut and hindgut regions down the line indicated in the image. The hindguts were collected in tubes containing Trizol.