Normal sheep serum

The standard Western blotting protocol (BioRad) was used with the following specifications. The utricles containing sensory epithelia, transitional epithelia, and underlying mesenchyme were isolated by microdissection and lysed in 50 μL RIPA lysis buffer for 30 min at 4 ˚C and sonicated thrice at low power for 10 s each with the sample kept on ice between the sonications. The total protein concentration in each sample was determined by the BCA assay (Thermo Fisher). A NuPAGE 12% Bis-Tris Protein Gel (Thermo Fisher) was used to resolve the proteins in 5 μg of each sample. The proteins were transferred to a nitrocellulose membrane (BioRad) and blocked for 1 hr at room tempirature in a 5% solution of skim-milk powder (Sigma-Aldrich) in tris buffer (BioRad) with 0.1% Tween 20 (Sigma-Aldrich). After the primary antibodies—rabbit anti-Yap (Cell Signaling) and rabbit anti-H3 (Millipore)—had been reconstituted at 1:10000 in tris buffer blocking solution containing 0.1% Tween 20% and 5% normal sheep serum (Sigma-Aldrich), the membrane was incubated over night at 4 ˚C. After 5 30 min washes at room tempirature in TBST, the anti-rabbit HRP secondary antibody (Millipore) was applied in TBST for 1 hr at room temperature. Horseradish-peroxidase activity was detected with the Amersham ECL Western Blotting System (GE Healthcare Life Sciences).

Digoxigenin-labelled RNA probes were prepared with the MAXIscript Kit (Thermo Fisher Scientific, Waltham, MA, USA), in accordance with the manufacturer’s instructions. The DNA fragment of Fgf4 (NM_010202, located between 117 and 731) was amplified by polymerase chain reaction (PCR), using primers shown in Supplementary Table S1. The fragment was subcloned into the pGEM-T vector (Promega, Madison, WI, USA) and used to generate sense and antisense probes. Bioengineered tooth germs after 5-day organ culture were fixed in 4% PFA overnight at 4 °C, treated with 6% H₂O₂ and 0.1% Tween 20 in PBS at room temperature, and incubated with 10 µg/mL proteinase K (Roche, Mannheim, Germany) for 3 min at 30 °C. After post-fixation and prehybridization, samples were hybridized overnight at 70 °C with digoxigenin-labelled RNA probes. Samples were blocked with 10% normal sheep serum (Sigma) and then incubated with anti-digoxigenin antibody conjugated with alkaline phosphatase (Roche), overnight at 4 °C. Nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate (Roche) were used for signal detection.

For Lcp1 immunostaining, larvae were anesthetized with 200 μg/ml tricaine and then immediately fixated in 4% paraformaldehyde in PBS (phosphate-buffered saline, pH 7.2) for 16 h at 4 °C. After fixation, the larvae were rinsed in PBS-DTx (phosphate-buffered saline with 0.5% DMSO and 0.3% Triton X-100) and treated with proteinase K (10 μg/ml in PBS-DTx; Roche) for 10 min at 37 °C. The larvae were blocked in 5% normal sheep serum (Sigma-Aldrich) in PBS-DTx for 2 h at room temperature, incubated with Lcp1/L-Plastin antibody (a gift from Dr. Anna Huttenlocher, University of Wisconsin, USA) in 1:1000 dilution at 4 °C overnight and subsequently incubated with Alexa-488 conjugated secondary antibody (1:200; Invitrogen) for 2 h at room temperature. The larvae were washed with PBS-DTx and stored at 4 °C until imaging.