Animals
Pregnant miniature pigs were obtained from the Animal Science Institute of Chinese Agriculture University. The gestation age was calculated from the day of insemination. Pregnancy was verified by B-type ultrasonography. All procedures were approved by the Animal Care Use Committee of Capital Medical University (Beijing, China) (Permit Number: AEEI-T-212). Pregnant miniature pigs were anesthetized and sacrificed as previously described [33]. The DM3 and mandible were harvested at embryonic days 40 (E40), E50, and E60, which corresponded to the cap, early, and late bell (secretory) stages.
Tissue preparation for HE and immunofluorescence
Mandibular samples were fixed with paraformaldehyde, 4% in phosphate-buffered saline (PBS), at 4°C overnight. The samples were rinsed with PBS twice before being decalcified with 10% EDTA-PBS for 3–14 days according to the degree of calcification. The samples were dehydrated in an ethanol series and embedded in paraffin. Paraffin-embedded samples were sectioned at 7μm thickness for later staining. Sections were stained with hematoxylin and eosin and observed by whole slide image (Pannoramic scan, 3DHistech, Budapest, Hungary). The images were processed with Case Viewer software (3DHistech, Budapest, Hungary). Immunofluorescence (IF) was performed as described previously [34]. Briefly, deparaffinized sections were microwaved for 3 min in citrate buffer (pH 6.0). Then the slides were reheated with adding additional buffer, which processes were repeated for three times for antigen retrieval. When the slides were cooled down to room temperature, non-specific antigens were blocked with 10% normal goat serum (C-0005, Bioss, Beijing, China) for 1 h. The sections were incubated with primary antibodies at 4°C overnight. The primary antibodies used in this research were as follows: rabbit anti-FGF3 antibody (bs-1255R, Bioss, Beijing, China), rabbit anti-FGF4 antibody (bs-1256R, Bioss, Beijing, China), rabbit anti-KGF antibody (bs-0734R, Bioss, Beijing, China), rabbit anti-FGF8 antibody (bs-0735R, Bioss, Beijing, China), and rabbit anti-FGF9 antibody (bs-5906R, Bioss, Beijing, China). Donkey anti-Rabbit IgG (H+L), Alexa Fluor 594 (A-21207; Thermo, CA, USA) as secondary antibody was subsequently applied at 37°C for 1 h. The nuclei were stained with DAPI (F6057, Sigma, St. Louis, MO, USA). Finally, the sections were observed using confocal microscopy (TCS SP8, Leica, Wetzlar, Germany). Objective lenses were used 20× and 40×, N.A. 0.75. The images were processed with LAS AF software.
Fluorescence in situ hybridization
Fluorescence in situ hybridization (FISH) staining was performed using tooth tissues fixed in 4% DEPC-treated paraformaldehyde/PBS, embedded in paraffin, and sectioned at 7μm. The procedure for non-radioactive in situ hybridization was described previously [35]. Briefly, total RNA was extracted from DM3 tooth germs at E40–E60. Sequences of the degenerate primers for Fgf3, 4, 7, 9, Fgfr1, 2, 3, and Spry2, 4 were listed in Supplementary Table 1. After reverse transcriptase-polymerase chain reaction, the correct-sized bands were extracted from agarose gels and their DNA sequences were determined. Digoxigenin-(DIG)-labeled RNA probes were synthesized with DIG-UTP and T7 RNA polymerase (10881767001; Roche, Basel, Switzerland) and DIG RNA labeling mix solution (11277073910; Roche, Basel, Switzerland). Sections were prepared using standard procedures and then incubated in horseradish peroxidase-conjugated, polyclonal sheep anti-digoxigenin antibody (90420, Merck Millipore) at 37°C for 4 h. Immunoreactive cells were visualized with TSA Plus Fluorescence Kits (NEL741001KT, PerkinElmer, Massachusetts, USA). Paraffin sections were processed for mucin 2 staining with rabbit anti-Cytokeratin-14 (ab119695, Abcam, Cambridge, England). Donkey anti-Rat IgG (H+L), Alexa Fluor 488 (A-21208; Thermo, CA, USA) as secondary antibody was subsequently applied at 37°C for 1 h. Meanwhile. Slides were counterstained with DAPI, mounted and imaged on a Leica TCS SP8 confocal microscope. Objective lenses were used 20× and 40×, N.A. 0.75. The images were processed with LAS AF software.
Real-time RT-PCR
DM3 tooth germs at E40, E50, and E60 were harvested and the dental epithelium was separated from the mesenchyme by dispase Ⅱ from miniature pigs described previously [22]. Total RNA was extracted with RNeasy Mini Kit (74106, Qiagen, Hilden, Germany) according to its supplied method. Reverse transcription was performed using the SuperScript III First-Strand Synthesis System (18080051; Thermo Fisher Scientific). Real-time RT-PCR was conducted in triplicate using SYBR Green PCR Master Mix (A25742; Applied Biosystems, Foster City, CA, USA) on a CFX96 Touch Real-time RT-PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA), after which melting curve analysis was performed. Expression level of each gene was normalized to that of Gapdh. The relative expression of each gene was determined using the 2-ΔΔCT method. Forward and reverse primers for Fgf3, 4, 7, 9, Fgfr1, 2, 3, and Spry2, 4 and Gapdh were listed in Supplementary Table 2.
Statistical analysis
Statistical analysis was performed using GraphPad Prism 8.3 software. One-way analysis of variance and Newman-Keuls multiple comparison test were used to calculate statistical significance. Differences were considered to be statistically significant if p<0.05 (*), p<0.01 (**), or p<0.001 (***).