Role of PTH1R signaling in Prx1+ mesenchymal progenitors during eruption

Chen Cui, Ruiye Bi, Weiqing Liu, Shuyuan Guan, Peiran Li, Dongzhe Song, Ruoshi

Xu, Liwei Zheng, Quan Yuan, Xuedong Zhou, Yi Fan

Appendix Material and Methods

Micro-computed tomographic (μCT) analyses

Mandibular bones from postnatal day (P) 10，P21 and P42 animals were used for

μCT with Scanco Medical μCT 35 system (Scanco), with an isotropic voxel size of 7

mm, according to guidelines (Dempster et al. 2013). The images were reconstructed

and perform three dimensional. Regions of interest were defined from alveolar bone

tissue nearby first molar. The selected regions were analyzed to determine the

trabecular bone volume fraction (BV/TV), trabecular thickness (Tb. Th), and trabecular

separation (Tb. Sp) at P10. Alveolar bone volume under first molar as well as the

corresponding enamel and dentin volume of the incisor was analyzed at P21.

Histology and Immunostaining

Tissues were dissected and fixed in 4% paraformaldehyde overnight. After

decalcification, samples were embedded in paraffin. 5μm sections were cut using

HM360 microtome (Microm). The sections were stained with hematoxylin (VWR) and

eosin (Sigma-Aldrich). Tartrate-resistant acid phosphatase (TRAP) (Sigma-Aldrich)

and Masson's trichrome (Solarbio) staining were performed according to the

manufacturers’ protocols, respectively. For immunostaining, slides were stained with

anti-Runx2 (1:200, Abcam, ab23981), anti-Osx (1:200, Abcam, ab22552), anti-ColαI

(1:200, Abcam, ab21286), anti-Periostin (1:200, Abcam, ab14041), anti-Red

Fluorescent Protein (1:100, Santa Cruz, sc-390909), anti-Sox9 (1:200, Abcam,

ab3697), anti-ColX (1:200, Abcam, ab58632), anti-OPN (1:400, R&D systems, AF808),

anti-PTH1R (1:200, Assay biotech, G220), anti-CD200 (1:200, Abcam, ab33734)

overnight at 4°C and stained with Alexa Fluor 488 (Invitrogen, 1:1000), Alexa Fluor

568 (Invitrogen, 1:1000) or a biotinylated secondary antibody, respectively. DAPI

(Vector) or hematoxylin (VWR) was used as counterstaining. Immunostaining images

were performed with Olympus confocal microscope FV3000 (Olympus). Quantitative

analysis including the numbers of Runx2+/tdTomato+ cells, Osx+/tdTomato+ cells and

tdTomato+ osteoblasts in ColαI-labeled alveolar bone region, were conducted by a

blinded observer with Image J software. At least three different sections were used

from each sample and three or more different samples were analyzed for each group.

Histomorphometry

Calcein mineral labels was performed to evaluate dynamic mineral apposition and

bone formation in mandibles of 3-week-old mice. Mice were injected with 20 mg of

calcein (Sigma-Aldrich) per kg of body weight in a 2% sodium bicarbonate solution at

5 and 2 days prior to sacrifice. Then, mandibles were collected and processed for

histologic sectioning and histomorphometry. 5μm frozen sections from undecalcified

samples were processed using Multipurpose Cryosection Preparation kit (Section-

LAB Co. Ltd, Japan) and cutting by a Cryomicrotome at -35°C (Leica Microsystems

KK, Germany). To determine the label, sections were directly viewed under fluorescent

light. Mineral surface/bone surface (MS/BS, %), mineral apposition rate (MAR, μm/day)

and bone formation rate/bone surface (BFR/BS, μm3/μm2/day) were measured.

Mouse Orofacial Bone-marrow-derived mesenchymal stem cells (OMSCs)

Culture

Isolation of OMSCs was as described previously (Yamaza et al. 2011). Briefly, after

removing attached soft tissues and teeth (incisors and molars), mice mandible was

digested with 3 mg/mL collagenase type I (Worthington Biochem) and 4 mg/mL

dispase II (Roche Diagnostic) for 60 min at 37°C. All nucleated cells were obtained

through 70-μm cell strainers (Corning) and seeded at 1-1.5 x 106 on a 100-mm dish

(Corning). Cells were cultured in α-MEM (Gibco) containing 1% penicillin/streptomycin

(HyClone) and 10% fetal bovine serum (FBS) (Gibco). Non-adherent cells were

removed by replacing the medium after 24 hours. The attached OMSCs were used for

experiments at passage 3 and plated at 3 x 104 cells/cm2. Osteogenic differentiation

was induced with osteogenic differentiation medium (α-MEM supplemented with 1%

PS, 10% FBS, 50 μg/ml ascorbic acid (Sigma-Aldrich), 10 mM β-glycerophosphate

(Sigma-Aldrich)). The medium was changed every other day. Alkaline phosphatase

(ALP) staining was performed using ALP assay kit (Beyotime) at 7 days and Alizarin

red S staining (Sigma-Aldrich) was performed at 14 days according to the

manufacturer’s instructions. For adipogenic differentiation, cells were induced with

adipocyte differentiation medium (α-MEM supplemented with 1% PS, 20% FBS, 500

μM IBMX, 1 μM Dexamethasone, 5 μg/ml Insulin and 1 μM Rosiglitazone) for first 2

days. Then the medium was changed to adipocyte differentiation base medium (α-

MEM supplemented with 1% PS, 20% FBS, 5 μg/ml Insulin and 1 μM Rosiglitazone)

for next 4 days, which was changed every other day. After 6 days, cells were washed

in PBS and collected for qPCR analysis or fixed with 10% formalin for oil red O staining.

For chondrogenic introduction, OMSCs were plated at 1.5mL microcentrifuge tube

with 3 × 106 cells/tube to form three-dimensional cartilage balls. Cells were cultured in

chondrogenic differentiation media (Cyagen) and were changed every other day. After

14 days, cartilage balls were fixed with 10% formalin and embedded in paraffin for

alcian blue staining and immunofluorescence staining.

Transfection

Recombinant adenoviruses CRE expression (Ad-CRE) were gotten from Dr. Dongzhe

Song which also express GFP as the marker for monitoring infection efficiency.

Adenovirus expressing only GFP (Ad-GFP) was used as a control. OMSCs were

transfected with Ad-CRE or Ad-GFP for 12 hours and then exchanged normal medium.

In order to enhance transgene transduction efficiency, polybrene (5 μg/ml) was added

to the culture medium for all adenovirus infections (Song et al. 2017; Wu et al. 2014).

Flow Cytometry

For preparation of flow cytometry analysis, OMSCs were digested with trypsin

(HyClone), and then suspended in αMEM and washed with 5% BSA. Cells were

incubated with PE anti-CD29 (BioLegend), PE anti-CD90 (BioLegend), FITC anti-

CD45 (BioLegend) and FITC anti-CD11b (BioLegend). Flow cytometry was carried out

using the FACSCalibur (FC500, Beckman Coulter). Each data set was analyzed by

FlowJo software (FLOWJO, LLC, OR, USA).

RNA Extraction and Quantitative Real-time PCR (qRT-PCR)

Total RNA from mice mandible at P0, P10 and P14 and in vitro cultured OMSCs were

extracted using Trizol (Invitrogen) according to the manufacturer’s protocol. RNA

concentration was determined with NanoDrop ND-1000 (Thermo Fisher Scientific).

cDNA was generated using PrimeScript RT reagent Kit (Takara) and qRT-PCR was

performed with SybrGreen Supermix (Bio-Rad Laboratories). Relative gene

expression levels were normalized by GAPDH (glyceraldehyde-3-phosphate

dehydrogenase) using the 2ΔΔCT method.

Protein Extraction and Western Blot

Protein from OMSCs was extracted by whole cell lysis assay (KeyGEN) and the

concentrations were evaluated by enhanced BCA protein assay kit (Beyotime). Equal

amount of protein samples was separated by NuPAGE 4-12% Bis-Tris SDS/PAGE

using precast gels (Invitrogen) and transferred onto polyvinylidene fluoride membrane

(Millipore). Following incubation in primary antibodies, we probed proteins by

enhanced chemiluminescence kit (Bio-Rad Laboratories, USA). Bands were

quantified by Image J software. Primary antibodies included anti-Osterix (abcam,

1:1000, ab22552), anti-Runx2 (abcam, 1:1000, ab23981), anti-Col αI (abcam, 1:1000,

ab21286), anti-Tubulin α (Signalway Antibody, 1:500, 41517). Horseradish

peroxidase-conjugated anti-rat or rabbit IgG was used as the secondary antibody

(Jackson ImmunoResearch Laboratories). Signal was detected by Amersham ECL

Prime Western Blotting Detection Reagent (GE Healthcare).

Statistics

For statistical analysis, GraphPad Prism 8.0 (GraphPad Software Inc.) was used.

Unpaired two-tailed Student’s t test was used between two groups. All values are

expressed as mean ± SEM. P values

Appendix Figure 1

Appendix Figure 1: Incisor eruption phenotype and distribution pattern of Prx1+

progenitors. (A) Gross phenotype of the incisors of PTH1Rfl/fl (upper) and

Prx1Cre;PTH1Rfl/lfl (lower) mice at postnatal days P7, P10, P14, P21 and P180 shows

arrested mandibular incisor eruption in mutants. Scale bars: 2mm. (B, C)

Quantification of mandibular incisor and molar eruption length or height measured in

mm at P10 and P21. n=4-7. *p

Appendix Figure 2

Appendix Figure 2: Characterization of incisor cervical loop and molar root. (A) H&E

staining of sagittal section of mandible at P0, P4, P10 and P14 indicate comparable

cervical loop histology in mutant when compared to age-matched control. n=4-6. Scale

bars: 200μm. (B) qRT-PCR analysis of gene expressions related to amelogenesis,

including Amelogenin (Amelx), Ameloblastin (Ambn) and Enamelin (Enam) in

mandibles of mutant and control littermates at P10 and P14. n=4-6. (C) 3D μCT

images of control and mutant at P42. Scale bars: 1mm. Individual slides of μCT images

at an interval of 6μm indicate the molar root are not dilacerated in mutants. (D) CD200

staining of P21 mice showed unchanged expression in root cementum. n=3. Scale

bars: 50μm.

Appendix Figure 3

Appendix Figure 3: Multilineage differentiation capacity of OMSCs and decreased

osteogenesis in PTH1R-deficient OMSCs after Ad-Cre transfection. (A,B) Alizarin red

staining (ARS) and quantitative analysis of osteogenic gene expressions of OMSCs

after 14-day osteogenic induction. (C,D) Oil red O staining and adipogenic-related

gene expressions of OMSCs after 7-day adipogenic induction. (E,F) Alcian blue and

immunofluorescence (Col X and Sox 9) staining of cartilage balls and gene expression

after chondrogenic induction of OMSCs. n=3. Scale bars: 100μm in C, E. *p

Appendix Figure 4

Appendix Figure 4: Alveolar bone resorption remained unchanged in

Prx1Cre;PTH1Rfl/fl mice (A) qRT-PCR analysis of osteoblast/osteoclast-relative gene

expressions (Rankl, Opg, Rank) in mandibles of mutant and control littermates at P10

and P14. n=4-6. *p

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