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. Author manuscript; available in PMC: 2022 Dec 1.
Published in final edited form as: Bone. 2021 Aug 5;153:116142. doi: 10.1016/j.bone.2021.116142

Chondrocyte Tsc1 controls cranial base bone development by restraining the premature differentiation of synchondroses

Yuan-Lynn Hsieh 1,#,&, Xiaoxi Wei 1,2,&, Yating Wang 1,3, Honghao Zhang 1, Shuqun Qi 1, Di Xie 1, Yuji Mishina 1, Daniela Mendonça 1, Nan Hatch 4, Fei Liu 1,*
PMCID: PMC8543925  NIHMSID: NIHMS1747917  PMID: 34365025

Abstract

Cranial base bones are formed through endochondral ossification. Synchondroses are growth plates located between cranial base bones that facilitate anterior-posterior growth of the skull. Coordinated proliferation and differentiation of chondrocytes in cranial base synchondroses is essential for cranial base bone growth. Herein, we report that constitutive activation of the mechanistic target of rapamycin complex 1 (mTORC1) signaling via Tsc1 (Tuberous sclerosis 1) deletion in chondrocytes causes abnormal skull development with decreased size and rounded shape. In contrast to decreased anterior-posterior growth of the cranial base, mutant mice also exhibited significant expansion of cranial base synchondroses including the intersphenoid synchondrosis (ISS) and the spheno-occipital synchondrosis (SOS). Cranial base synchondrosis expansion in TSC1-deficient mice was accounted for by expansion in the resting zone due to increased cell number and size without alteration in cell proliferation. Furthermore, our data showed that mTORC1 activity is inhibited in the resting and proliferating zone chondrocytes of wild type mice, and Tsc1 deletion activated mTORC1 signaling of the chondrocytes in the resting zone area. Consequently, the chondrocytes in the resting zone of TSC1-deficient mice acquired characteristics generally attributed to pre-hypertrophic chondrocytes including high mTORC1 activity, increased cell size, and increased expression level of PTH1R (Parathyroid hormone 1 receptor) and IHH (Indian hedgehog). Lastly, treatment with rapamycin, an inhibitor of mTORC1, rescued the abnormality in synchondroses. Our results established an important role for TSC1-mTORC1 signaling in regulating cranial base bone development and showed that chondrocytes in the resting zone of synchondroses are maintained in an mTORC1-inhibitory environment.

Keywords: Tsc1, mTORC1, chondrocyte, cranial synchondrosis, skull, cranial base, ISS, SOS, endochondral

Introduction

The cranial skeleton is distinct from the rest of the skeleton in its complexity in organization, embryonic origin, and molecular mechanisms inducing skeletogenesis [1–3]. Most of our knowledge about skeletal development and repair comes from analyses of long bones and vertebrae. However, the cellular and molecular mechanisms regulating both physiological and pathological cranial skeletal development are much less understood [4, 5].

The cranial skeleton is composed of the neurocranium that surrounds the brain and the viscerocranium that comprises of the facial bones. The neurocranium includes the cranial vault and the cranial base, which develop through intramembranous and endochondral ossification, respectively. The cartilaginous segments persisting between the ossification centers of the cranial base bones are synchondroses, such as the intersphenoid synchondrosis (ISS) and the spheno-occipital synchondrosis (SOS) according to their anatomical location [1, 3]. The synchondroses are growth sites that drive cranial base growth.

The synchondrosis is comprised of mirror-image growth plates with a central resting zone flanked by proliferative and hypertrophic zones on both sides. Similar to the growth plate in long bone, the proliferation and maturation of chondrocytes contribute to the elongation of the cranial base. In contrast to the unidirectional growth in the growth plate of long bones, cranial synchondrosis drives bidirectional growth and has unique molecular mechanisms regulating its development [1, 2]. Because of its structural similarities to growth plate, it is generally assumed that the chondrocytes in synchondroses undergo the same path of proliferation, differentiation towards hypertrophy, and subsequent apoptosis [6]. However, the regulatory mechanisms in proliferation and differentiation at synchondroses are not identical to those at growth plate [1, 6].

The mammalian/mechanistic target of rapamycin complex 1 (mTORC1) signaling is essential to mouse embryonic skeletal development [7], yet its roles in cranial skeletal development and disease are poorly defined. We reported that mTORC1 hyperactivation by deleting Tsc1 gene, encoding an upstream inhibitor of mTORC1, in neural crest-derived cells results in increased craniofacial bone mass partly through enhancing proliferation of osteoprogenitor cells [8]. We also showed that Tsc1 regulates the balance between osteoblast and adipocyte differentiation of bone marrow stromal cells, and mTORC1 hyperactivation by Tsc1 deletion in osterix-expressing cells results in osteopenia in mice [9]. In this report, we used Col2a1-Cre transgenic mice to delete Tsc1 in chondrocytes and discovered that Tsc1 deletion disrupted skull development due to premature differentiation of chondrocytes in the resting zone of cranial base synchondroses. To the best of our knowledge, this is the first report to suggest that TSC1-mTORC1 signaling plays an important role in regulating cranial base bone development and our data showed that the chondrocytes in the resting zone of synchondroses need to be maintained in an mTORC1-inhibitory environment.

Materials and Methods

Mice

The floxed Tsc1 (Tsc1flox/flox), Col2a1-Cre, ROSA26-LacZ Cre reporter (R26R) transgenic mice were described previously [10–12]. Tsc1flox/flox and R26R mice had been backcrossed to C57BL/6 background for at least 8 generations; Col2a1-Cre mice had been backcrossed to C57BL/6 background for 4 generations. The Col2a1-Cre transgenic mice were bred with Tsc1flox/flox mice to generate Tsc1flox/+;Col2a1-Cre mice, which were then bred with Tsc1flox/flox mice to generate Tsc1flox/flox;Col2a1-Cre (hereafter CKO), Tsc1flox/+;Col2a1-Cre (hereafter CHet), Tsc1flox/flox and Tsc1flox/+ (hereafter CTR). Litter-matched CTR, CHet, and CKO mice were used in the study. The genotyping of the mice was determined by the previously described PCR strategy using tail DNA [10, 13]. All mice were kept in a controlled environment (standard temperature and humidity) at the University of Michigan School of Dentistry and all animal procedures followed protocols approved by Institutional Animal Care and Use Committee at the University of Michigan.

X-gal staining

The X-gal staining was performed similar to our previous reports [8, 13]. Col2a1-Cre+;R26R mice and Col2a1-Cre−;R26R were euthanized at postnatal day 0 and day 7 and fixed with 2.5% glutaraldehyde. Ten μm sagittal-cut cryosections of cranial base were obtained for X-gal staining. The X-gal staining solution containing 1 mg/ml X-gal, 5 mM potassium ferrocyanide, 5mM potassium ferricyanide, 2% NP-40 (IGEPAL), 2 mM magnesium chloride, 0.1% sodium deoxycholate in phosphate buffered saline (PBS) was freshly made before use. The slides were incubated overnight at 37°C room in humidified box. The next day, after rinsing with PBS, the slides were shortly counter-stained by eosin red solution. The slides were mounted and photos were taken via bright-field microscope (Olympus, Tokyo, Japan). The cell/tissue with β-galactosidase activity was stained blue.

μCT scanning and analysis

Skull scanning and analysis were performed similar to our previous report [14]. A high-resolution cone beam, micro-computed tomography (μCT, eXplore Locus SP, GE Healthcare Pre-Clinic Imaging, London, ON, Canada) was applied to assess postnatal craniofacial bone structure and development. Skull specimens of 30-day-old CKO, CHet, and CTR groups were collected, fixed by 4% paraformaldehyde and stored in 70% ethanol. Mouse skulls were scanned by μCT. Using defined landmarks (Fig S1, Table S1), 27 parameters including 11 in A-P length, 3 in transversal width, 4 in vertical height, and 9 in angle, were measured via software MicroView (version ABA 2.2), Image J (version 1.50i), and ITK-SNAP (version 3.6). Additionally, bone parameters including BV/TV, BMD, and thickness were calculated using MicroView for cranial vault bones (frontal and parietal bone) and/or cranial base bones (presphenoid, basisphenoid and basioccipital bones). Five regions of interest (ROI) were selected similar to our previous study [14]. For the presphenoid bone, a coronal plane that is 0.09 mm anterior to the anterior border of ISS and perpendicular to the long axis of the bone was identified; and the entire bone structure 0.5 mm anterior to this plane was selected. For the basisphenoid bone, a coronal plane that is 0.09 mm anterior to the anterior border of SOS and perpendicular to the long axis of the bone was identified; and the entire bone structure 0.5 mm anterior to this plane was selected. For the basioccipital bone, a coronal plane that is 0.09 mm posterior to the posterior border of SOS and perpendicular to the long axis of the bone was identified; and the entire bone structure 0.5 mm posterior to this plane was selected. For the frontal bone, a 0.5 mm × 0.5 mm area was located 1.5 mm anterior to the bregma and 1.5 mm lateral to the posterior frontal suture. For the parietal bone, a 0.5 mm × 0.5 mm area was located 1.5 mm posterior to the bregma and 1.5 mm lateral to the sagittal suture.

Histomorphometry analysis

The cranial base of CTR and CKO mice at postnatal 7, 14, and 30 days were dissected after euthanization. After fixation in 4% paraformaldehyde and decalcification in 14% EDTA, the samples were horizontally embedded in Tissue-Plus O.C.T. compound (Fisher Healthcare, USA). Ten μm frozen tissue specimens were sectioned at the sagittal midline of the cranial base and H&E staining of synchondroses (ISS and SOS) were performed. Zone area, zone fraction, cell number, and cell size in synchondroses were analyzed using Image J software (version 1.50i).

Immunofluorescence analysis

Immunofluorescence staining was performed similar to our previous studies [9, 15]. Ten μm cryosections were used for immunofluorescence staining. The process included permeabilization (0.1% PBST), blocking (5% BSA), incubation with primary antibodies (4°C, overnight) and secondary antibodies (room temperature, 1h), mounting with DAPI, and photo taking using a confocal microscope (Nikon Eclipse Ti, Tokyo, Japan). The primary antibodies used in this study include TSC1 (Santa Cruz, sc-377386, 1:100), P-S6 (CST, #2215, 1:600), Ki67 (CST, #9129, 1:400), Ihh (Santa Cruz, sc-271101, 1:100), PTH1R (PTH/PTHrP-R, Santa Cruz, sc-12722, 1:100), and Runx2 (Abcam, ab23981, 1:300). The secondary antibodies used in this study include goat anti-rabbit IgG-TR (Santa Cruz, sc-2780, 1:400), donkey anti-mouse IgG (H+L), and Alexa Fluor 555 (ThermoFisher, 1:400).

In situ hybridization

RNA in situ hybridization was carried out according to standard procedure. Briefly, cranial base tissues freshly dissected were immediately fixed in 4% PFA overnight, decalcified overnight, before cryo-protected in 30% sucrose in PBS. Then, 14 μm sections were treated with proteinase K, post-fixed with 4% PFA, before treated with acetic anhydride solution (Sigma, St. Louis, MO, USA). Sections were hybridized with an RNA probe for collagen X in a hybridization solution containing 5X SSC, 50% formamide, 1mg/mg tRNA (Sigma, St. Louis, MO, USA), 0.1mg/ml Heparin (Sigma, St. Louis, MO, USA) at 65 °C. Sections were then incubated with RNase A (Roche) and washed in post-hybridization solution containing 0.2X SSC, before incubation with alkaline phosphatase conjugated mouse anti-digoxigenin overnight. Purple color for positive signal was developed through incubating sections with BM Purple for alkaline phosphatase substrate (Roche).

Rapamycin treatment

Vehicle (5% PEG400, 5% Tween80 in PBS) or rapamycin (LC laboratory) was administrated to mice via intraperitoneal injection daily from birth to postnatal day 6 at a dose of 5mg/kg/day. At postnatal day 7, one day after the last treatment, the cranial base samples were collected for analysis.

Statistics

Student’s t-test and one-way or two-way ANOVA analysis with Turkey’s post hoc test was performed for statistical analysis using SAS (SAS Institute, Gary, North Carolina, US). P value < 0.05 was considered statistically significant.

Results

Targeted deletion of Tsc1 in chondrocytes during skull development

Col2a1-Cre transgenic mice were used to generate chondrocyte-targeted Tsc1 conditional knockout mice. Col2a1-Cre mice were crossed with ROSA26-LacZ Cre reporter (R26R) mice to examine the Cre recombinase activity, shown by X-gal staining, in the skull. Col2a1-Cre-mediated recombination was examined in the entire cranial base structure including the sphenooccipital synchondrosis (SOS), the intersphenoid synchondrosis (ISS), and cranial base bones. Our data showed that Col2a1-Cre targeted the perichondrium and chondrocytes in the resting, proliferative, and hypertrophic zones in the ISS and SOS as well as the cranial base bones (Fig 1A and Fig S2).

Figure 1. Chondrocyte-targeted Tsc1 deletion leads to skull developmental defects in mice.

Figure 1.

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(A) X-gal staining of the cranial base of newborn Col2a1-Cre−;R26R and Col2a1-Cre+;R26R mice. SOS represents spheno-occipital synchondrosis; and ISS represents intersphenoid synchondrosis. (B-C) Body weight of Tsc1flox/flox and Tsc1flox/+ mice (CTR), Tsc1flox/+;Col2a1-Cre mice (CHet), and Tsc1flox/flox;Col2a1-Cre mice (CKO) at birth (B) and one month (1M) of age (C). (D) Representative images showing body size of CTR, CHet, and CKO mice at one month of age. (E) Representative microCT images of the sagittal plane of skulls of one-month-old CTR and CKO mice. The dotted box and its magnified image indicate the cranial base. The scale bar = 1 mm. (F) Skull length of indicated mice and the percent change of skull length compared to controls at one month of age. (G) Cranial base length of indicated mice and the percent change of cranial base length compared to controls at one month of age. (H) SOS length of indicated mice and the percent change of SOS length compared to controls at one month of age. (I) ISS length of indicated mice and the percent change of ISS length compared to controls at one month of age. * p<0.05, ** p<0.01, *** p<0.001, and **** p<0.0001. Values are presented as mean + SD for Fig 1C (n=6, 4, 8 for the male CTR, CHet, and CKO, respectively; n=4, 6, 8 for the female CTR, CHet, and CKO, respectively) and as median and interquartile range with individual data points for other panels.

Col2a1-Cre transgenic mice were bred with Tsc1flox/flox mice, and we subsequently obtained Tsc1flox/flox (hereafter CTR), Tsc1flox/+;Col2a1-Cre (hereafter CHet), and Tsc1flox/flox;Col2a1-Cre (hereafter CKO) mice. CKO mice were born at the expected Mendelian ratios with similar body weight to CTR and CHet mice (Fig 1B). However, CKO mice had significantly less body weight and smaller body size than CTR and CHet mice at one month of age (Fig 1C, D). Similar to previous reports [16, 17], CKO mice started to die around one month of age, thus subsequent phenotypic analyses were done on or before one month of age.

Tsc1 deletion by Col2a1-Cre leads to decreased skull size, altered skull shape, expanded synchondroses, and compromised cranial base bone acquisition

Besides being smaller in size, some CKO mice exhibited visible changes in skull shape. Thus, we characterized the effect of Tsc1 deletion on skull structure by analyzing μCT images. The mid-sagittal section view showed that CKO mice had a more rounded skull compared to control mice (Fig 1E). Using the mouse skull landmarks (Fig S1) to define the parameters used in linear and angle measurements (Table S1), the morphology of the skulls of CKO and control mice was quantitatively characterized by length, width, height, and angle parameters in a similar method to our previous study [14]. Our data showed that CKO mice had skull development defects (Fig 1F–I, S3). Compared to control mice, CKO skulls were smaller in nearly all parameters of length, width, and height. However, the dimensional changes were not proportionate in all directions, which explained the change in skull shape aside from size decrease. The rounded shape of the skull was consistent with the significant angular changes including a 9% increase (p<0.0001) in rostral angle of cranial cavity, a 4% decrease (p<0.0001) in anterior-middle cranial vault angle, a 4% decrease (p<0.0001) in posterior cranial vault angle, a 9% increase (p<0.0001) in cranial vault-cranial base angle, a 4% decrease (p<0.0001) in cranial base angle, a 2% decrease (p<0.001) in snout angle, and a 3% decrease (p<0.0001) in cranial-maxilla angle (Fig S4). Intriguingly, despite a small but significant 7% decrease (p<0.0001) in skull length (Fig 1F) and cranial base length (Fig 1G), CKO mice had a significant 217% and 140% increase (P<0.0001) in the length of cranial base synchondroses, SOS (Fig 1H) and ISS (Fig 1I), respectively. In contrast to CKO mice, CHet mice had normal body weight, skull size, shape, and comparable values in all measured parameters compared to CTR (Fig 1C–D, F-I, and Fig S4). Thus, Tsc1 deletion by Col2a1-Cre led to reduction in skull size, rounded skull, and abnormal increase in the length of SOS and ISS.

SOS and ISS are the growth centers located in the cranial base and play important roles in the cranial base bone development. The abnormality in SOS and ISS in CKO mice prompted us to determine the effect of Tsc1 deletion by Col2a1-Cre on cranial base bone acquisition. Using our previously established method [14], μCT analyses were performed in the cranial base bones (Fig 2A–F). Our data showed 19% decrease (p<0.0001) in presphenoid bone length (Fig 2B), 13% decrease (p<0.0001) in basisphenoid bone length (Fig 2C), and no change in basioccipital bone length (Fig 2D) in CKO mice. In addition, the bone volume fraction (Fig 2E) and bone mineral density (Fig 2F) of presphenoid, basisphenoid, and basioccipital bones were significantly decreased in CKO mice. In contrast to the cranial base bones that were formed through endochondral ossification, our data showed that Tsc1 deletion by Col2a1-Cre did not affect the development of cranial vault frontal and parietal bones (Fig 2G–I), which were formed through intramembranous ossification.

Figure 2: Bone Analysis of the skull.

Figure 2:

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MicroCT bone analyses were performed on the skull samples as in Fig 1F–I in both cranial base (A-F) and vault (E-I). (A) Illustration showing the measured areas in cranial base: PS represents presphenoid; BS represents basisphenoid bone; BO represents basioccipital bone; SOS represents spheno-occipital synchondrosis; and ISS represents intersphenoid synchondrosis. (B-D) MicroCT analysis of bone length and the percentage change compared to the mean of control group of presphenoid bone (B), basishphenoid bone (C), and basioccipital bone (D). (E) Bone volume fraction of presphenoid, basishphenoid, and basioccipital bones. (F) Bone mineral density of presphenoid, basishphenoid, and basioccipital bones. (G) Illustration showing the measured areas in cranial vault. (H-I) MicroCT analysis on thickness (H) and bone volume fraction (I) of cranial vault bones as illustrated in (G). * p<0.05, ** p<0.01, and **** p<0.0001. Values are presented as mean + SD for Fig 2H and 2I (n=7 per group) and as median and interquartile range with individual data points for other panels.

Altogether, our data revealed that SOS and ISS were enlarged but dysfunctional in CKO mice as indicated by the compromised cranial base bone development.

Tsc1 deletion by Col2a1-Cre results in an expansion of the resting zone area with increased resting zone cell number in cranial synchondroses

As a first step to determine the nature of the expansion of synchondroses in CKO mice, histological analysis was performed. The histological view showed a longer but irregular SOS and ISS in CKO compared to CTR (Fig 3A). In SOS, quantitative analysis showed that the CKO had a 33% (p<0.0001), 47% (p<0.0001) and 128% (p<0.0001) increase in total synchondrosis zone area at postnatal day 7, day 14, and day 30, respectively (Fig 3B). Further analysis revealed that SOS area increase in CKO was mainly contributed by the increase in resting zone area, in which CKO had a 65% (p<0.0001), 107% (p<0.0001), and 275% (p<0.0001) increase at postnatal day 7, day 14, and day 30 respectively (Fig 3B). Zone fraction analysis showed that CKO had a 23% (p<0.0001), 40% (p<0.0001), and 54% (p<0.0001) increase in the resting zone fraction, a significant 20% (p<0.0001), 28% (p<0.0001), and 43% (p<0.001) decrease in hypertrophic zone fraction at postnatal day 7, day 14, and day 30 respectively without significant changes in the proliferative zone fraction (Fig 3C), indicating that resting zone expansion was the major contributor to SOS expansion. In ISS, CKO had a 28% (p<0.01), 82% (p<0.01) and 90% (p<0.0001) increase in total synchondrosis zone area at postnatal day 7, day 14 and day 30, respectively. Similar to SOS, ISS area increase in CKO was mainly contributed by the increase in resting zone area, in which CKO had a significant 88% (p<0.001), 183% (p<0.01), and 207% (p<0.0001) increase at postnatal day 7, day 14, and day 30 respectively (Fig 3D). Zone fraction analysis showed that CKO had a 45% (p<0.0001), 57% (p<0.0001), and 58% (p<0.0001) increase in the resting zone fraction, and a 29% (p<0.0001), 37% (p<0.0001), and 41% (p<0.0001) decrease in hypertrophic zone fraction at postnatal day 7, day 14, and day 30 respectively, without significant changes in the proliferative zone fraction (Fig 3E).

Figure 3. Chondrocyte-targeted Tsc1 deletion leads to the expansion of the resting zone in cranial base synchondroses.

Figure 3.

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Histological analysis of cranial base synchondroses in Tsc1flox/flox and Tsc1flox/+ mice (CTR) and Tsc1flox/flox;Col2a1-Cre mice (CKO) at indicated postnatal ages. H: hypertrophic zone; P: proliferative zone; R: resting zone. 7D: postnatal day 7; 14D: postnatal day 14; and 30D: postnatal day 30. (A) H & E staining showing the histological structure of spheno-occipital synchondroses (SOS) and intersphenoid synchondroses (ISS) at indicated ages. (B-C) Zone area (B) and zone fraction (C) analysis of SOS at indicated ages (n= 6, 11, 7 for 7D, 14D, and 30D of CTR group respectively, and n=8, 6, 8 for 7D, 14D, and 30D of CKO group respectively). (D-E) Zone area (D) and zone fraction (E) analysis of ISS at indicated ages (n= 6, 11, 7 for 7D, 14D, and 30D of CTR group respectively, and n= 8, 6, 8 for 7D, 14D, and 30D of CKO group respectively). (F-G) Cell number (F) and size (G) in the resting zone of SOS and ISS at postnatal day 7 (n=9 for CTR and n=9 for CKO). * p<0.05, ** p<0.01, *** p<0.001, and **** p<0.0001, comparing to the control at the same time point or the same zone or the same synchondrosis. Values are presented as mean + SD in bar graphs.

Histological analysis suggested that synchondroses enlargement in CKO mice was mainly due to the expansion of the resting zone. To further determine the underlying mechanism behind resting zone expansion, we analyzed the cell number and size in the resting zone. Our data showed that there was a 30% (p<0.01) and 35% (p<0.001) increase in cell number (Fig 3F) and 42% (p<0.0001) and 59% (p<0.0001) increase in cell size (Fig 3G) in the SOS and ISS of CKO mice, respectively. Altogether, our data indicated that the increase in resting zone cell number and size is mainly responsible for the expansion of synchondroses in CKO mice.

Tsc1 deletion leads to increased mTORC1 activity without affecting proliferation

To determine the underlying mechanism of resting zone expansion in CKO mice, we first examined the deletion of TSC1 by Col2a1-Cre in the SOS and ISS. In control mice, TSC1 was expressed mainly in the resting zone and proliferating zone chondrocytes of ISS and SOS; in CKO mice, TSC1 was effectively deleted in most of the cells in these two zones (Fig 4A). The major known function of TSC1 is to inhibit mTORC1 signaling, thus, we examined the effect of TSC1 on the expression level of phospho-S6, a well-established downstream effector of mTORC1 signaling. Our data showed that in control mice, phospho-S6 signals were mainly detected in the pre-hypertrophic chondrocytes but mostly absent in the resting and proliferating zones; however, in CKO mice, strong phospho-S6 signal was detected in resting zone chondrocytes of both ISS and SOS (Fig 4B). To determine whether increased mTORC1 activity in CKO synchondroses altered proliferation, we examined the ratio of cells that were positive for Ki67, a proliferation marker, in ISS and SOS. Our data showed a similar Ki67-positive cell ratio in both the proliferating and resting zones of the CKO and CTR mice (Fig 4C–D). Thus, the increase in cell number in the resting zone of CKO synchondroses correlated with the abnormally upregulated mTORC1 signaling in this zone, but it was unlikely caused by an alteration in proliferation.

Figure 4. Tsc1 deletion leads to upregulated mTORC1 activity in the resting zone of cranial base synchondroses without alteration in proliferation.

Figure 4.

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Immunofluorescence staining was performed in the intersphenoid synchondroses (ISS) and spheno-occipital synchondroses (SOS) of one-week-old Tsc1flox/flox and Tsc1flox/+ mice (CTR) and Tsc1flox/flox;Col2a1-Cre mice (CKO), using anti-TSC1 (A), anti-phospho-S6 (B), and anti-Ki67 (C) antibodies. Scale bar=100 μm. Panel D shows the percentage of Ki67 positive cells in the resting zone (R) and proliferative zone (P) of ISS and SOS of CTR and CKO mice as shown in (C) (n=4 for CTR and n=5 for CKO).

Tsc1 deletion leads to premature differentiation and aberrant PTH1R and IHH expression in resting zone chondrocytes

Because the resting zone chondrocytes in CKO mice had increased cell size and high mTORC1 activity, two characteristics of pre-hypertrophic chondrocytes seen in CTR mice, we determined the effect of Tsc1 deletion on the expression of some chondrocyte differentiation regulators/markers in the synchondroses. Although there was no difference in the expression pattern of FGFR3, RUNX2, and OPN between CKO and CTR mice (data not shown), CKO had abnormally high expression of PTH1R in the resting zone, which is mainly expressed in the pre-hypertrophic zone in control mice (Fig 5A). Similarly, CKO had abnormally high expression of IHH in the resting zone, which is mainly expressed in the pre-hypertrophic zone in control mice (Fig 5B). Notably, there was no upregulated expression of PTH1R and IHH in the chondrocytes of proliferating zone of CKO mice, which was consistent with the unaltered mTORC1 activity in this zone (Fig 4B). Next, we performed in situ hybridization to examine the mRNA expression of collagen X, a well-established marker of hypertrophic chondrocytes, in synchondroses. As expected, our data showed positive signals at hypertrophic zones of synchondroses, which were absent in other zones in CTR mice (Fig 5C, upper left). However, in addition to in the hypertrophic zone, there were collagen X-positive cells in the resting zone of CKO ISS (Fig 5C, lower left), suggesting that resting zone chondrocytes in CKO ISS had already undergone premature hypertrophic differentiation to the stage of collagen X expression. Interestingly, there were no collagen X-positive cells in the resting zone of CKO SOS (Fig 5C, lower right). Because collagen X is a late differentiation marker of chondrocytes, the absence of collagen X in the CKO SOS indicated that the premature differentiation in CKO SOS was less advanced compared to CKO ISS, Altogether, Tsc1 deletion by Col2a1-Cre led to a premature differentiation-like phenotype of the resting zone cells of CKO synchondroses evidenced by increased cell size, increased mTORC1 activity, increased expression of PTH1R and IHH in ISS and SOS as well as ectopic expression of collagen X in ISS.

Figure 5. Tsc1 deletion leads to premature differentiation of chondrocytes in the resting zone of cranial base synchondroses.

Figure 5.

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(A-B) Immunofluorescence staining was performed in the ISS and SOS of one-week-old CTR and CKO, using anti-PTH1R (A) and anti-IHH (B) antibodies. Scale bar=100 μm. (C) In situ hybridization using anti-sense probe for collagen X mRNA was performed in the intersphenoid synchondroses (ISS) and spheno-occipital synchondroses (SOS) of one-week-old Tsc1flox/flox and Tsc1flox/+ mice (CTR) and Tsc1flox/flox;Col2a1-Cre mice (CKO). Asterisks indicate the hypertrophic zone expressing collagen X, arrows point to the center of the resting zone, and the dotted area highlights the ectopic expression of collagen X in the ISS resting zone of CKO.

Rapamycin treatment rescued the resting zone expansion and cell size enlargement

While the major role of TSC1 is to function as a negative regulator of mTORC1 activity, it also has mTORC1-independent function [18]. Our data showed that the abnormality in synchondroses of CKO mice was associated with an upregulated mTORC1 signaling in the resting zone. To determine whether the increased mTORC1 activity was responsible for resting zone expansion in CKO mice, we determined whether mTORC1 inhibition could rescue the abnormality in synchondroses of CKO mice by administering rapamycin, a widely used mTORC1 inhibitor (Fig 6A). Our data showed that daily rapamycin treatment starting from birth effectively rescued the total zone area (Fig 6B, 6F), resting zone area (Fig 6C, 6G), resting zone cell number (Fig 6D, 6H), and resting zone cell size (Fig 6E, 6I) in both SOS (Fig 6B–E) and ISS (Fig 6F–I) of CKO mice, supporting the notion that synchondrosis abnormalities in CKO mice were due to the abnormally activated mTORC1 signaling and the chondrocytes in the resting zone of synchondroses need to be maintained in an mTORC1-inhibitory environment. At the molecular level, our data showed that rapamycin treatment effectively decreased PTH1R and IHH expression in the resting zone of CKO mice (Fig 6J–K).

Figure 6. Rapamycin rescues the expansion of cranial base synchondroses in CKO mice.

Figure 6.

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(A) Cartoon showing the design of rescue experiment: rapamycin or vehicle was administered to newborn mice daily at the first 6 days and the synchondroses were analyzed at day 7. N= 10, 9, 6, 9 for vehicle treated control, rapamycin treated control, vehicle treated CKO, rapamycin treated CKO, respectively. (B-E) The histological measurements conducted at SOS on total zone area (B), resting zone area (C), resting zone cell number (D) and cell size (E). (F-I) The histological measurements conducted at ISS on total zone area (F), resting zone area (G), resting zone cell number (H) and cell size (I). (J-K) Immunofluorescence staining was performed in the ISS and SOS of one-week-old vehicle-treated or rapamycin-treated CKO mice, using anti-PTH1R (A) and anti-IHH (B) antibodies. Scale bar=100 μm. * p<0.05, ** p<0.01, *** p<0.001, and **** p<0.0001. Values are presented as mean + SD.

Discussion

In this study, we focused on elucidating the role of TSC1, an upstream inhibitor of mTORC1 signaling [19], in skull development. Our data showed that Tsc1 deletion by Col2a1-Cre limited skull growth by disrupting the development of cranial synchondroses. Besides the cartilage, collagen 2 gene is expressed in the heart, eye, fetal brain, liver, tongue, and salivary gland during embryonic development [20, 21]. It has been demonstrated that Col2a1-Cre targets many non-cartilaginous tissues including head mesoderm and notochord, cranial mesenchyme, submandibular glands, epithelial cells of the kidney, pancreas, lungs, intestine, ovaries, central nervous system [11, 17, 22–24]. Our mutant mice have a smaller body size, thus the decrease in the skull size could be contributed by overall growth retardation resulting from Tsc1 deletion in non-cartilaginous tissues by Col2a1-Cre. However, besides the decrease in skull size, our data showed significant change in skull shape, which could not be simply explained by the overall body/skull size decrease. The skull shape change could be contributed by several potential factors such as altered brain development, premature suture fusion, and impaired cranial base development. Analysis of the μCT images did not reveal any premature fusion of cranial sutures in CKO mice, including frontonasal suture, coronal suture, lambdoid suture, sagittal suture, and metopic suture (Fig S4). Synchondroses are important growth centers that mainly facilitate anterior-posterior growth of the mouse skull and our data revealed abnormal expansion of the cranial synchondroses in association with a premature differentiation phenotype in the resting zone, thus, we concluded that the defective cranial base development, at least partially, contributed to the observed skull abnormality. Importantly, although CKO mice had expanded synchondroses, they had a significant decrease in cranial base length and bone volume fraction of cranial base bones. In addition, the cranial vault bone development was not affected in CKO mice. Thus, our data suggest that the defective synchondrosis development is a primary phenotype in this CKO mouse model.

mTORC1 signaling plays a crucial role in the development of long bone growth plate [7, 16]. Disruption of mTORC1 signaling greatly delays chondrocyte hypertrophy and bone formation in limbs [7]. The effect of mTORC1 hyperactivation on the development of long bone growth plate is debatable. Yan et al. reported that hyperactivation of mTORC1 by deleting Tsc1 in chondrocytes leads to increased proliferation and blockage of differentiation in the growth plate of long bone [16]. On the contrary, Newton et al. used the same mouse model but concluded that hyperactivation of mTORC1 does not affect proliferation or differentiation in the growth plate of long bone [17]. In this report, we used the same Col2a1-Cre transgenic mice to delete Tsc1 in chondrocytes focusing on the role of mTORC1 in skull development. We showed that constitutive hyperactivation of mTORC1 compromised the development of cranial base synchondroses, which was due to the premature differentiation of the resting zone chondrocytes without significant alteration in proliferation.

Our data showed a very high level of mTORC1 activity specifically in the pre-hypertrophic chondrocytes in both the ISS and SOS in control mice; while in long bone growth plate, high mTORC1 activity is reported in both proliferating and pre-hypertrophic chondrocytes [16]. Upon Tsc1 deletion, mTORC1 activity was significantly enhanced in resting zone chondrocytes but not in proliferating zone chondrocytes of cranial synchondroses. In contrast, Tsc1 deletion by Col2a1-Cre leads to increased mTORC1 activity in chondrocytes of hypertrophic zone but not resting zone in long bone growth plate [16, 17]. These differences between cranial synchondroses and long bone growth plate clearly indicate different underlying mechanisms regulating mTORC1 signaling in these two distinct anatomic locations despite being similar in structure. Importantly, the hyperactivated mTORC1 signaling in the resting zone chondrocytes of synchondroses correlates well with the phenotypic abnormalities in this zone; thus, our data suggest that the activation of mTORC1 signaling in resting zone chondrocytes of synchondroses contributed to resting zone expansion. Indeed, the phenotypic rescue by rapamycin, an mTORC1 inhibitor, strongly supported this conclusion. However, the underlying molecular mechanisms remain to be elucidated. In the rescue experiment, the mice were treated with rapamycin starting at birth and analyzed at one week old. Although our data showed that this treatment regimen could effectively rescue the major histological and molecular abnormalities in CKO mice, the effect of mTORC1 inhibition in rescuing the phenotypes in later development stages such as at one month of age is unknown.

Yan et al. and Newton et al. showed disorganization of the resting zone in the long bone growth plate of Tsc1 CKO mice [16, 17], which is congruent with our analysis of the cranial synchondroses showing an irregular shape of resting zone. Further analysis showed that the irregularly expanded synchondrosis resting zone was due to cell number/size increase. Interestingly, our data showed abnormal expression of PTH1R and IHH in the resting zone chondrocytes of both ISS and SOS of CKO mice. Both PTH1R and IHH are mainly expressed in the pre-hypertrophic zone of long bone growth plate and cranial synchondroses [1, 2]. Our data on the expression of PTH1R and IHH in the synchondroses of control mice were consistent with literature [4, 5]. In addition, our data showed that mTORC1 signaling is mainly active in the pre-hypertrophic chondrocytes in the control synchondroses, but it was abnormally activated in the resting zone chondrocytes of both ISS and SOS in CKO mice. Altogether, our data showed that Tsc1 deletion by Col2a1-Cre led to a premature differentiation phenotype in the resting zone chondrocytes of synchondroses resembling pre-hypertrophic chondrocytes, characterized by increased cell size, high mTORC1 activity, and high expression of PTH1R and IHH. We speculate that this premature differentiation prevents resting zone chondrocytes from achieving a timely transition to the proliferating zone resulting in the accumulation of cells, expansion and disorganization of resting zone, and leading to compromised cranial base bone development and elongation (Fig 7). Of note, in this model, the nature of the cells in the resting zone of CKO mice is not completely clear. Our data showed that they have comparable proliferation activity despite more advanced differentiation characteristics. It is possible that the cells in the resting zone of CKO mice represent a mixture of cells differentiating towards pre-hypertrophic stage.

Figure 7. Diagram depicting the major changes caused by Tsc1 deletion in cranial synchondroses:

Figure 7.

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Tsc1 deletion in the chondrocytes of the resting zone leads to high mTORC1 activity as shown by the increased phosphor-S6 level. The ectopic high mTORC1 activity drives the premature differentiation of chondrocytes in the resting zone in association with the abnormal upregulation of PTH1R and IHH. The premature differentiation diminishes the progression of chondrocytes in resting zone towards downstream lineage and leads to the accumulation of cells with increased size.

In our CKO mice, although the cells in the resting zone area appear to have acquired some characteristics generally attributed to pre-hypertrophic chondrocytes, their characteristics do not support a late hypertrophic chondrocyte phenotype. For example, they have normal expression levels of the SPP1 and RUNX2, two markers reported to be mainly expressed in late hypertrophic chondrocytes [2]. In addition, the resting zone cells in the SOS of CKO mice did not express Collagen X. Thus, our data suggest that CKO resting zone cells have a premature differentiation status with some features associated with pre-hypertrophy but have not reached a more terminal differentiation stage.

Although the cells in the resting zone of both SOS and ISS of CKO mice acquired some similar characteristics resembling pre-hypertrophic chondrocytes, our data revealed that only the cells in the center of ISS but not SOS of CKO mice had abnormally expressed collagen X, a marker of hypertrophic chondrocyte. These results suggest that constitutive activation of mTORC1 could lead to the premature differentiation of resting zone chondrocytes in both synchondroses but to different extents: in SOS, the chondrocytes in resting zone were prematurely differentiated enough to cause expansion but not to the stage of collagen X expression. This difference at two locations is not surprising considering the many significant differences between ISS and SOS. In humans, ISS and SOS have very different timing in completion of ossification: between 2 and 3 years of age in ISS and between 16 and 18 years of age in SOS [25], indicating independent regulatory mechanisms during development. In mice, our previous study showed that both ISS and SOS start to form bony bridges around 2 months of age and complete fusion occurs between 6 and 13 months [1]. Histologically, mouse ISS and SOS have some significant structural differences. A recent report showed a mineralized central zone, called “tether”, in the SOS of male C57bl/6j mice at postnatal day 28. Similar structures appear much later at postnatal day 78 in the ISS [26]. Similarly, our previous study showed that “tether” structure starts to appear at 3 weeks in SOS and at 2 months in ISS of female C57bl/6NCrl mice [1]. In many reported genetically modified mice, it is not uncommon to have some phenotypical differences between ISS and SOS [6]. One significant biological difference between ISS and SOS is their distinct embryonic origin. ISS originates completely from neural crest, while SOS has a dual origin: the rostral part is from the neural crest and the caudal part from the mesoderm [27]. This significant difference in embryonic origin may be one factor affecting their developmental regulation and response to genetic manipulation including the constitutively active mTORC1 signaling in this study. However, despite of the difference in the extent of premature differentiation at these two sites, our data demonstrated a common importance of restraining mTORC1 activity in both ISS and SOS.

Our previous study showed that Tsc1 deletion in Osterix-expressing cells results in decreased femoral trabecular bone mass, which is partly attributed by an increase in osteoclast number [9]. Mechanistically, our data suggested that increased Mcsf expression in Tsc1-deficient osteoblasts contributes to enhanced osteoclastogenesis [9]. In this report, our data showed that Tsc1 deletion in Col2a1-expressing cells results in decreased cranial base bone mass. Consistent with a recent report [28], X-gal staining showed the positive signals in the majority of the osteoblasts in cranial base of Col2a1-Cre+;R26R mice. Thus, the previously identified Tsc1/Mcsf/osteoclastogenesis regulatory mechanism may also play a role in the decrease of cranial base bone mass in the Col2a1-Cre model.

Our data showed that the basioccipital bone in CKO had a decrease in bone volume fraction but had no change in length. This differs from other cranial base bones including basisphenoid bone and presphenoid bone, which had a decrease in both bone volume fraction and length. There are two likely contributors to this difference. First, basioccipital bone is located at the posterior end of the cranial base and lies anterior to the foramen magnum, through which the spinal cord, an extension of the medulla oblongata, passes as it exits the cranial cavity. Because of the need to maintain the shape of the foramen magnum, it is possible that there is a unique regulator mechanism that controls the growth of the basioccipital bone and the loss of Tsc1 could be compensated by unknown mechanism. Second, the basioccipital bone is completely derived from the mesoderm, while presphenoid bone is completely derived from the neural crest and basisphenoid bone is derived from both neural crest and mesoderm. Our data showed that in CKO mice, the neural crest-derived presphenoid bone had a greater decrease in length compared to the basisphenoid bone, which has mixed origins; and the mesoderm-derived basioccipital bone had no change in length. which may suggest a differential effect of Tsc1 deletion on bone length in neural crest-derived bone compared to mesoderm-derived bone.

Supplementary Material

1

  • Tsc1 regulates cranial base bone development

  • Deletion of Tsc1 in chondrocytes leads to the expansion of synchondroses

  • Resting zone chondrocytes are maintained in an mTORC1-inhibitory environment

  • Tsc1 deletion leads to aberrant expression of PTH1R and IHH

  • mTORC1 activation results in the premature differentiation of synchondroses

Acknowledgments

We thank Ms. Andrea Clark for microCT scanning and reconstruction. MicroCT work was partly supported by P30 Core Center award to University of Michigan from NIH/NIAMS (AR 69620). FL was supported by the MCubed Award, University of Michigan. YH was supported by Research Initiative Award from Department of Biologic and Materials Sciences & Prosthodontics, University of Michigan School of Dentistry, and Rackham Graduate Student Research Grant, University of Michigan. The authors declare no potential conflicts of interest with respect to the authorship and/or publication of this article.

Footnotes

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Supplementary Materials

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