Neural Crest-Specific TSC1 Deletion in Mice Leads to Sclerotic Craniofacial Bone Lesion

Abstract

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder caused by mutations in either TSC1 or TSC2. TSC has high frequency of osseous manifestations such as sclerotic lesions in the craniofacial region. However, an animal model that replicates TSC craniofacial bone lesions has not yet been described. The roles of Tsc1 and the sequelae of Tsc1 dysfunction in bone are unknown. In this study, we generated a mouse model of TSC with a deletion of Tsc1 in neural crest-derived (NCD) cells that recapitulated the sclerotic craniofacial bone lesions in TSC. Analysis of this mouse model demonstrated that TSC1 deletion led to enhanced mTORC1 signaling in NCD bones and the increase in bone formation is responsible for the aberrantly increased bone mass. Lineage mapping revealed that TSC1 deficient NCD cells overpopulated the NCD bones. Mechanistically, hyperproliferation of osteoprogenitors at an early postnatal stage accounts for the increased osteoblast pool. Intriguingly, early postnatal treatment with rapamycin, an mTORC1 inhibitor, can completely rescue the aberrant bone mass, but late treatment cannot. Our data suggest that enhanced mTOR signaling in NCD cells can increase bone mass through enlargement of the osteoprogenitor pool, which likely explains the sclerotic bone lesion observed in TSC patients.

Keywords: CRANIOFACIAL; NEURAL CREST; OSTEOBLASTS; OSTEOPROGENITOR; RAPAMYCIN; SCLEROTIC; TUBEROUS SCLEROSIS; mTORC1.

Figure 1. TSC1 deletion in osteoblasts with P0-Cre leads to increased mTORC1 activity in neural crest-derived osteoblasts

(A) X-gal staining of skulls of one-week-old P0-Cre⁻;Rosa26 and P0-Cre⁺;Rosa26 mice. P=parietal bone; F=frontal bone; N=nasal bone. (B) Immunoblotting analysis of TSC1, p-S6, S6 and Vinculin protein level in frontal and nasal bones of one-week-old CTR and CKO mice. (C, D) Quantification of TSC1 (C) and p-S6 (D) protein level, normalized to Vinculin and S6 respectively. ^#p<0.001, n=5 per group. (E) Immuno-staining of p-S6 in frontal bone of one-month-old CTR and CKO mice. Arrows point to p-S6⁺ cells. Scale bar=40µm. (F) Quantification of p-S6 positive cell number per bone perimeter at the frontal bone extracranial periosteum of one-month-old CTR and CKO mice. *p<0.05, n=5 per group. For C, D, and F, the data were presented as mean ± SD.

Figure 2. TSC1 deletion by P0-Cre leads to increased neural crest-derived bone mass

(A) MicroCT images of skulls of 1-year-old CTR and CKO mice. P=parietal bone; F=frontal bone; Z=zygomatic bone; N=nasal bone. (B) Coronal section view of the frontal and parietal bone of skulls shown in (A). (C) Thickness of frontal and parietal bones of CTR and CKO mice at 1-, 2-, 3-month and 1-year-old. *p<0.05, n=15–19 for 1-month-old samples; n=19–23 for 2-month-old samples; n=10–19 for 3-month-old samples; n=5–7 for 1-year-old samples. Data were presented as mean ± SD.

Figure 3. Loss of TSC1 increases bone formation

(A) Representative calcein double labeling images of the frontal bone (5 days apart between two labelings). Scale bar=50µm. (B–G) Dynamic histomorphometry for the extracranial surface (B–D) and intracranial surface (E–G) of frontal bone of one-month-old CTR and CKO mice: (B, E) mineralizing surface; (C, F) mineral deposition rate (MAR); (D, G) bone formation rate (BFR). (H) Osteoclast number per bone perimeter of frontal bone of one-month-old CTR and CKO mice. Region A: extracranial surface; Region B: Intracranial surface; Region C: bone marrow cavity surface. All region: region A, B, and C. (I, J) Quantitative-PCR analysis of the Rankl (I) and Opg (J) mRNA expression in frontal bone of one-month-old mice. *p<0.05, ^#p<0.001, n=5–6 per group for B-H, n=10 per group for (I) and (J). Data were presented as mean ± SD.

Figure 4. TSC1 deletion by P0-Cre leads to increased neural crest derived osteoblasts

(A–D) X-gal staining of skulls of one-week-old Tsc1^flox/+;P0-Cre;Rosa/+ (cHet-LacZ) and Tsc1^flox/flox;P0A-Cre;Rosa/+ (CKO-LacZ) mice. (A) Representative whole skull image after staining. (B) Coronal section of frontal bones, counter-stained with neutral red. Scale bar=40µm. Arrows point to LacZ positive cells. (C–D) LacZ positive cell number per bone perimeter (C) and percentage of LacZ positive cells (D) in the periosteum areas shown in B. n=4 per group. (E, F) X-gal staining of primary osteoblasts isolated from frontal bones of newborn cHet-LacZ and CKO-LacZ mice. (E) Representative X-gal staining images. Red (converted from Dapi staining) indicates nucleus and blue shows LacZ positive signal. Scale bar=200µm. (F) Percentage of LacZ positive cells. Data were the average of three independent experiments. (G-H) Immuno-staining with anti-Osterix antibody and quantification of Osterix positive cell per bone perimeter in frontal bone of one-month-old CTR and CKO mice. n=5 per group. Scale bar=50µm. (I) H&E staining images of frontal bones of one-month-old CTR and CKO mice. Arrows point to osteocytes. Scale bar=20 µm (J) Quantitative analysis of osteocyte number per bone area. *p<0.05, ^#p<0.001, n=5–6 per group. Data were presented as mean ± SD.

Figure 5. TSC1 deletion leads to increased osteoprogenitor cell proliferation at early postnatal stage

(A, B) Immunofluorescence staining with anti-Ki67 antibody was performed in the frontal bone of one-week-old CTR and CKO mice. (A) Representative fluorescent images in the extracranial periosteum area. Scale bar=50µm. (B) Ki67 positive cell number per bone perimeter. *p<0.05, n=5 per group. (C–E) Quantitative-PCR analysis of the mRNA expression of cell cycle G1/S transition gene Cyclin D1 (C), Wnt signaling target genes: Axin2, Gja143, Col6a1, Mmp16 (D), and osteoblast differentiation markers: Col1a1, Alpl, Runx2, Osx, Atf4, Bsp, Ocn (E) of frontal bone of one-week-old CTR and CKO mice. ^#p<0.001, n=11–12 per group. Data were presented as mean ± SD.

Figure 6. Early but not late rapamycin treatment can rescue the increased frontal bone thickness in CKO mice

(A) Frontal bone thickness of CTR and CKO mice at two-month-old after 1mg/kg rapamycin/vehicle i.p injection every other day starting from one-week-old. *p<0.05, n=8–18 per group. (B) Representative pictures of X-gal stained skulls of one-week-old cHet-LacZ and CKO-LacZ mice after 1mg/kg rapamycin injection every other day starting at one-day-old. (C) Immunoblotting analysis of p-S6, S6 and Vinculin protein level in frontal and nasal bones of one-week-old CTR and CKO mice after 1mg/kg rapamycin/vehicle injection every other day starting at one-day-old. Short/long means short/long time film exposure. (D) Frontal bone thickness of CTR and CKO mice before rapamycin treatment at one-month-old and after two months treatment (1mg/kg i.p injection every other day starting at one-month-old) at three-month-old. *p<0.05, n=6–12 per group. (E) Schematic diagram of the mechanism by which neural crest-specific TSC1 deletion leads to sclerotic craniofacial bone lesion: Up-regulated mTORC1 signaling (indicated by upward pointing arrow) resulting from TSC1 deletion in neural crest-derived cells promotes osteoprogenitor expansion (indicated by plus sign) at early postnatal stage. Enlarged osteoprogenitor pool generates more osteoblasts to form thicker bone in CKO mice.