Trypsin solution

The dissected tooth germs from the lower jaw of EGFP positive embryos were put into the Hank’s solution (Sigma Aldrich). The Hank’s solution was replaced by 1% trypsin solution (Difco Laboratories) in 4°C for one to two hours (according to the developmental stage of embryos) to dissociate the epithelium from the mesenchyme. Dissociated epithelia were documented in the Stop solution (20% FCS - Sigma Aldrich) using the inverted fluorescent microscope Leica AF6000 (Leica Microsystems GmbH, Germany). Shh expression domains were determined according to the green fluorescence in the cells actually expressing Shh.

To supplement the results obtained using WISH with intermediate stages, epithelia of the developing lower incisor germs of EGFP positive embryos were dissociated (112 Eda−/− and 109 Eda+/+ mouse embryos were used in total). Incisor regions of the lower jaws were dissected and placed into Hank´s solution. The Hank´s solution was replaced by 1% trypsin solution (Difco Laboratories) at 4°C to dissociate the epithelium from the mesenchyme. Dissociated epithelia were documented in the stop solution (20% FCS in Hank´s solution, Sigma Aldrich) using the inverted fluorescence microscope Leica AF6000 (Leica Microsystems GmbH, Wetzlar, Germany). Shh expression was determined according to the green fluorescence.