Animals

Gli1-CreER (JAX# 007913),36 tdTomato (JAK# 007905), Fgfr1fl/fl (from Dr. Philippe Soriano),37K14rtTA (JAX# 007678),38Teto-Cre (JAX# 006234),39 and β-cateninfl/fl (JAX# 004152)40 mouse lines were used in this study. The primers for genotyping were designed according to protocols from Jackson Labs using Integrated DNA Technologies products. All mice were housed in pathogen-free conditions. All animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Southern California (USC).

Tamoxifen and doxycycline administration

Tamoxifen (Sigma, T5648) was dissolved in corn oil (Sigma, C8267) at 20 mg/mL. Fgfr1fl/fl, Gli1-CreER;Fgfr1fl/fl and Gli1-CreER;Fgfr1fl/fl;β-cateninfl/+ mice were injected once intraperitoneally at a dosage of 1.5 mg/10 g body weight at PN3.5. Doxycycline rodent diet was fed to dams of K14rtTA;tetO-Cre;Fgfr1fl/fl mice (Envigo, TD.08541) every day beginning when the suckling pups were at PN3.5. A dosage of 50 mg/mL doxycycline (Sigma-Aldrich; D9891) was injected into the pups intraperitoneally at PN3.5 and PN5.5.

MicroCT analysis

We collected mandibles from mice 30 days, 50 days, 60 days, and 9 months of age and then fixed them with 4% paraformaldehyde, and mandibles from more than three mice were collected for each group. MicroCT analysis was performed using a Skyscan 1174v1.2 (Bruker Corporation, USA) at 50 kVp, 800 μA and a resolution of 16.7 mm, followed by visualization and three-dimensional reconstruction performed using Avizo/Amira 9.5.0 (Visualization Sciences Group, France).

Histological analysis

Mouse mandibles were fixed in 4% paraformaldehyde (PFA) overnight after dissection. After 10% EDTA treatment for 2–4 weeks, the samples were decalcified and then dehydrated in an ethanol and xylene series. The samples were embedded in paraffin and cut into 6-7 μm sections using a microtome (Leica). H&E staining was performed according to standard protocols. We performed serial sectioning to make sure different levels were captured and checked every section of both control, Fgfr1 mutant, and Gli1-CreER;Fgfr1fl/fl;β-cateninfl/+ rescue samples. All staining protocols in this study used this method.

In situ hybridization

For cryosections, sections were stained according to the manufacturer’s instructions using an RNAscope Multiplex Fluorescent v2 kit (Advanced Cell Diagnostics, 323100). For cells, staining was performed after fixation with 10% Neutral Buffered Formalin (NBF) for 30 min. All probes used in this study were synthesized by Advanced Cell Diagnostics: Probe-Mm-Fgfr1 (454941), Probe-Mm-Dspp (448301), Probe-Mm-Fzd6 (404921), Probe-Mm-Piezo2 (400191), Probe-Mm-Piezo1 (500511), Probe-Mm-Axin2 (400331), Probe-Mm-Ibsp (415501), Probe-Mm-Pthlh (456521), and Probe-Mm-Ctnnb1 (311741).

Immunofluorescence

The decalcified samples were dehydrated in serial sucrose solutions and then embedded in an optimal cutting temperature compound (Tissue-Tek). The samples were cut into 8 μm cryosections using a cryostat (Leica CM1850). The cryosections were treated with a blocking solution (PerkinElmer) for 1 h. The primary antibodies used were the following: Sp7 (Abcam; ab209484, 1:100), Periostin (1:100, Abcam, ab14041), Ki67 (1:100, Abcam, ab15580), Col1a1 (1:100, CST, 72026), and Cleaved Caspase3 (9661, Cell signaling, 1:200 with TSA). After being incubated with primary antibodies at 4 °C overnight, signals were detected with Alexa-conjugated secondary antibody (1:200, Invitrogen), and nuclei were stained with DAPI (Invitrogen, 62248). Images were captured with a Keyence microscope (Carl Zeiss).

RNA sequencing

First mandibular molars from the control and Gli1-CreER;Fgfr1fl/fl mice were dissected at PN7.5 after tamoxifen induction. The apical region of the molar was collected following RNA extraction with an RNeasy Micro Kit (Qiagen, 74004). For RNA-sequencing analysis, cDNA library preparation and sequencing were performed on NextSeq500 High Output equipment for three pairs at the Technology Center for Genomics & Bioinformatics at the University of California, Los Angeles (UCLA). Raw reads were trimmed, aligned with the mm10 genome, and normalized in Partek Flow. Differential analysis was performed by selecting transcripts with a significance of P < 0.05.

Plasmid transfection and qPCR

Plasmids were from OriGene, including pCMV6-AC-GMP (PS100010) and Piezo2 (NM_001039485 Mouse Tagged ORF Clone, MR226955). Plasmid transfection was performed following the manufacturers’ protocols (QIAGEN, 301704, and OriGene, TF81001). Briefly, with 1 µg/µL stock solution, the plasmid was transfected into cells in 24-well plates for 2 days followed by real-time qPCR.

The total RNA was isolated using RNeasy Plus Micro Kit (QIAGEN, 74034) after cells were collected. cDNA transcription was performed using iScript™ cDNA Synthesis Kit (Bio-495 Rad, 1708891). qPCR quantification was performed using SsoFast™ EvaGreen® Supermix (Bio-Rad, 1725202) on a Bio-Rad CFX96 Real-Time System. The primer sequences used in this study were as follows: Gapdh (forward primer 5′-AGGTCGGTGTGAACGGATTTG-3′, reverse primer 5′-TGTAGACCATGTAGTTGAGGTCA-3′), Piezo2 (forward primer 5′- TCAACTGCTCCTTGCCCAAT-3′, reverse primer 5′-ATGGCGGTAAACGGTGACTT-3′), Etv5 (forward primer 5′- TCAGTCTGATAACTTGGTGCTTC-3′, reverse primer 5′-GGCTTCCTATCGTAGGCACAA-3′).

Cell culture and osteogenic differentiation

The apical mesenchymal tissues of the first mandibular molars from control and Fgfr1 mutant mice were collected at PN3.5, then cut into small pieces and cultured in α-MEM (Thermo Fisher, 12571071) with 10% FBS (Thermo Fisher, 12662029) at 37 °C in a 5% CO2 incubator.

StemPro osteogenesis differentiation kit (Thermo Fisher, A1007201) was used for the osteogenic differentiation following the manufacturer’s protocol.

Alizarin Red S staining and quantification

Cells were washed twice with PBS after removing the medium, then fixed with 4% paraformaldehyde for 30–40 min. After washing with PBS twice, cells were stained with 0.2% Alizarin Red S solution for 15 min and then washed with PBS to remove unspecific staining. Calcified nodules stained with red were photographed with a microscope. To quantify the calcium deposition, the stain was solubilized with 10% cetylpyridinium chloride monohydrate (CPC, Sigma-Aldrich) in 0.1 mol/L PBS (pH 7.0) for 15 min. The absorbance was read at 570 nm.

siRNA transfection

Cells were passaged for siRNA transfection when they reached sub-confluence. AllStars Negative Control siRNA (QIAGEN,1027280), Etv5 siRNA (QIAGEN, 1027416, Mm_Etv5_1 FlexiTube siRNA SI00996744; Mm_Etv5_2 FlexiTube siRNA SI00996737; Mm_Etv5_3 FlexiTube siRNA SI00996730; Mm_Etv5_4 FlexiTube siRNA SI00996723), Piezo2 siRNA (QIAGEN, 1027416, Mm_Piezo2_1 FlexiTube siRNA SI04723404; Mm_Piezo2_2 FlexiTube siRNA SI04723397; Mm_Piezo2_3 FlexiTube siRNA SI04723390; Mm_Piezo2_4 FlexiTube siRNA SI04723383), Fzd6 siRNA (QIAGEN, 1027416, Mm_Fzd6_1 FlexiTube siRNA SI02708510; Mm_Fzd6_2 FlexiTube siRNA SI02686684; Mm_Fzd6_3 FlexiTube siRNA SI02666979; Mm_Fzd6_4 FlexiTube siRNA SI00171451), Opti-MEM I Reduced Serum Medium (Thermo Fisher, 31985062) and lipofectamine™ RNAiMAX (Thermo Fisher, 13778075) were used in this study. SiRNA was transfected into cells at a final concentration of 10 nmol/L.

ChIP-qPCR

The mandibular first molars were dissected from wild-type mice at PN3.5. 60–80 mg tissue from multiple animals was combined for each replicate. Samples were prepared for ChIP following the manufacturer’s protocol (Chromatrap, 500191). Briefly, tissue was cut into small pieces, fixed with 1% formaldehyde at room temperature for 15 min, and incubated with 0.65 mol/L glycine solution. After washing with PBS twice, the tissue was resuspended in Hypotonic Buffer and incubated at 4°C for 10 min to obtain nuclei. The pellet was resuspended using Digestion Buffer. Chromatin was sheared into 100–500 bp fragments with Shearing Cocktail. 10 µg chromatin with ETV5 antibody (Proteintech, 13011-1-AP) or immunoglobulin G-negative control was added to the Column Conditioning Buffer. Immunoprecipitation (IP) slurry was mixed thoroughly and incubated on a rotor for 1 h at 4 °C. An equivalent amount of chromatin was set as an input. A chromatrap spin column was used to purify the IP slurry at room temperature, and chromatin was eluted using the ChIP-seq elution buffer. The chromatin sample and input were further incubated at 65°C overnight to reverse cross-linking. DNA was treated with proteinase, then purified with the Chromatrap DNA purification column. Primers were designed using the promoter region of Piezo2. Input, negative control, and ChIP eluates were assayed using real-time qPCR. Primers were designed using the promoter region of Piezo2. Forward: 5’- CGCTCCCAGGAAATGTTCTCTG-3’; Reverse: 5’-GCTATGTCTCCACGTAGGCATCT-3’.

Western blot

First mandibular molars from Fgfr1fl/fl control, Gli1-CreER;Fgfr1fl/fl mutant and Gli1-CreER;Fgfr1fl/fl;β-cateninfl/+ rescue mice were dissected at PN8.5 after tamoxifen induction. The apical region of the molar was collected and incubated in RIPA buffer (Cell Signaling, 9806) for 30 min, then centrifuged at 14 000 × g at 4 °C. Total protein was loaded in 4%–15% precast polyacrylamide gel and transferred to PVDF membranes. After blocking for 1 h, samples were incubated with primary antibody against beta Catenin (1:100, Abcam, ab6302) at 4 °C overnight, and detected with secondary antibodies on an Azure 300 (Azure Biosystems).

Statistical analysis

Statistical analysis was performed with GraphPad Prism. All statistical data are presented as individual points and mean ± SD. Unpaired Student’s t-test or one-way ANOVA analysis was used for comparisons, with P < 0.05 considered statistically significant. n ≥ 3 for all experiments.