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. Author manuscript; available in PMC: 2015 Nov 1.
Published in final edited form as: J Bone Miner Res. 2014 Nov;29(11):2357–2368. doi: 10.1002/jbmr.2267

Constitutive expression of GsαR201C in mice produces a heritable, direct replica of human fibrous dysplasia bone pathology and demonstrates its natural history

Isabella Saggio 1, Cristina Remoli 2, Emanuela Spica 2, Stefania Cersosimo 2, Benedetto Sacchetti 2, Pamela G Robey 3, Kenn Holmbeck 3, Ana Cumano 4, Alan Boyde 5, Paolo Bianco 2, Mara Riminucci 2
PMCID: PMC4205271  NIHMSID: NIHMS597175  PMID: 24764158

Abstract

Fibrous dysplasia of bone (FD) is a crippling skeletal disease associated with post zygotic mutations (R201C, R201H) of the gene encoding the α subunit of the stimulatory G protein, Gs. By causing a characteristic structural subversion of bone and bone marrow, the disease results in deformity, hypomineralization, and fracture of the affected bones, with severe morbidity arising in childhood or adolescence. Lack of inheritance of the disease in humans is thought to reflect embryonic lethality of germline-transmitted activating Gsα mutations, which would only survive through somatic mosaicism. We have generated multiple lines of mice that express GsαR201C constitutively and develop an inherited, histopathologically exact replica of human FD. Robust transgene expression in neonatal and embryonic tissues, and embryonic stem (ES) cells was associated with normal development of skeletal tissues and differentiation of skeletal cells. As in humans, FD lesions in mice developed only in the postnatal life; a defined spatial and temporal pattern characterized the onset and progression of lesions across the skeleton. In individual bones, lesions developed through a sequence of three distinct histopathological stages: a primary modeling phase defined by endosteal/medullary excess bone formation, and normal resorption; a secondary phase, with excess, inappropriate remodeling; and a tertiary fibrous dysplastic phase, which reproduced a full-blown replica of the human bone pathology in mice of age ≥1 year. Gsα mutations are sufficient to cause FD, and are per se compatible with germline transmission and normal embryonic development in mice. Our novel murine lines constitute the first model of FD.

Introduction

Fibrous dysplasia of bone (FD, OMIM#174800) is a crippling skeletal disease associated with activating mutations (R201C and R201H) of the α subunit of the stimulatory G protein, Gs (1–3). The two mutations are the consequence of methylation-deamination of cytosine in the CpG dinucleotide at codon 201, in either DNA strand (4,5), and convey a significant reduction of the intrinsic GTPase activity of Gsα (6). This results in prolonged overstimulation of adenylyl cyclase, and excess cAMP production (7). Gsα signaling is thus enhanced, leading to pleiotropic cell responses that vary in different cell types (8). As many endocrine cells are regulated by Gs protein-coupled receptors, activating Gsα mutations in endocrine cells cause the autonomous overproduction of specific hormones, resulting in hyperfunctional endocrinopathies (8,9). The net effect in bone is the production of a peculiar pattern of bone pathology in which abnormal bone and abnormal marrow replace the normal layout of the bone-bone marrow organ (10,11). Bone mass is high (12), the bone matrix is woven and undermineralized (12,13) and the trabecular architecture is abnormal [“Chinese writing” pattern (14)] The bone marrow is locally depleted of hematopoietic tissue and adipocytes, and is replaced by a connective tissue made of abnormal osteogenic cells (10,11,13). This complex constellation of changes results in bone that is mechanically incompetent, brittle and overly compliant, leading to fracture, pain and deformity, and more severe consequences such as wheelchair confinement or loss of sensory functions (4,15). The skeletal disease, by far the most serious and the least understood of the multiple organ lesions associated with activating Gsα mutations, does not have a cure. At the same time, the disease and its largely unknown mechanisms provide a unique window on the physiological role of Gsα signaling in skeletal physiology at large. Located downstream of major endocrine regulators affecting both bone and bone marrow (parathyroid hormone, β-adrenergic signaling), and upstream of pleiotropic cell responses ranging from cytoskeletal organization to cell differentiation, Gsα can play crucial roles in bone development and postnatal remodeling, as well as in hematopoietic physiology.

The human disease is thought to result from postzygotic Gsα mutations, giving rise to a somatic mosaic state (2,3,9,16,17). Involvement of tissues and organs derived from the three germ layers indicates that the embryonic cell undergoing mutation is pluripotent, and mutation must occur between fertilization and gastrulation (4). To account for the invariable lack of inheritance of the human disease, Happle postulated that the disease genotype would be lethal if germline-transmitted, and only able to survive through mosaicism (18). Following the identification of Gsα activating mutations in monostotic and polyostotic FD and in the McCune-Albright syndrome [FD, endocrinopathies and skin pigmentation (4)], direct demonstration of mosaicism in patients seems to support this notion (2,3,9,16,17,19). Nonetheless, direct demonstration of embryonic lethality of germline-transmitted mutations of Gsα cannot be obtained in humans, and lack of inheritance of the disease could also be attributed, in principle, to alternative mechanisms (such as, gene lethality in gametes). Clinical reports suggesting that human FD can occasionally be inherited should be mentioned (20), although evidence of inheritance of the FD disease gene has never been provided.

Animal models of the disease have been missing. To date, the only direct in vivo models of the disease are based on xenotransplantation of human Gsα-mutated (natural or Gsα mutation-transduced) skeletal stem cells into immunocompromised mice (13,16,21). This approach highlighted a specific role of cells of the osteogenic/stromal lineage in FD pathogenesis, and also contributed to elucidate some key aspects of the disease (16,17,22). However, this approach is insufficient to model developmental aspects of the bone lesions and their organ dimension. Murine models of overactivity of different players in the cAMP signaling pathway (23–26), upstream or downstream of Gsα, have resulted in phenotypes interpreted as reminiscent of FD. However none of these models represent either a direct replica of human FD lesions or a direct in vivo model of the effects of the specific, activating Gsα mutations. The development of mouse models is an indispensable tool to elucidate the natural history of the disease and to design and test novel treatments. Many aspects of the biology of the disease have remained poorly understood. For example, while Gsα mutations must arise in early embryonic development (4,5), bone lesions develop postnatally (4,27,28). Furthermore, the histological, clinical and radiographic diversity of bone lesions (15) is not easily traced to current understanding of the disease biology, but has a major direct impact on diagnosis, management and prognosis of the disease. Therapeutically, FD is an orphan disease: medical treatment available is only palliative (29,30); surgical approaches are complex and often with high risk (15) and disseminated multifocal disease defies any plausible approach for cure. Development of therapeutic strategies may rely either on identification of specific drug targets or on more innovative approaches such as cell and gene therapy (31,32). Of note, specific gene correction in skeletal progenitors has been shown to be technically feasible ex vivo (21), but significant experimentation lies ahead on this avenue.

We show here that constitutive expression of GsαR201C in mice results in a direct replica of human FD, establishing a direct causative role of Gsα mutations in FD. Defining pathological features of FD are faithfully reproduced in this model and the timed onset and progression of individual skeletal lesions can be monitored. Surprisingly, these models demonstrate that the FD phenotype is indeed heritable in mice, indicating that mutated Gsα per se is not embryonic lethal.

Materials and Methods

Generation of transgenic mice

EF1α-GsαR201C and PGK-GsαR201C recombinant viruses were produced as previously described [(21) and Fig. 1A]. Viral titers were determined by p24 antigen quantification (Alliance HIV-1 p24 ELISA KIT, Perkin Elmer, Boston, MA) following the manufacturer’s instructions. EF1α-GsαR201C and PGK-GsαR201C mice were produced by collecting one-cell stage embryos and microinjecting 10–100pl of 108TU/ml viral solution into the perivitelline space (Telethon Core Facility for Conditional Mutagenesis, San Raffaele Research Institute, Milan, Italy) as previously described (33). All manipulated embryos were implanted into pseudo-pregnant CD-1 mice (Harlan). Two founders from EF1α-GsαR201C (#184 and #33A) and PGK-GsαR201C (#60 and #61) mice were serially backcrossed with WT animals (FVB and C57Bl6, respectively, Harlan). Animals were maintained in cabin-type isolators at standard environmental conditions (temperature 22–25°C, humidity 40–70%) with 12:12 dark/light photoperiod. Food and water were provided ad libitum. Experiments involving animals were performed in compliance with the relevant Italian laws and Institutional guidelines and all procedures were IACUC approved. Transgenic offspring were weaned at 3 weeks of age and genotyped. Transgene expression was routinely assessed in tail biopsies taken at two months in all mice. Transgene expression was also assessed in whole embryos between E10 and E16 and at multiple sites across the skeleton of newborn and 4-months old mice. Mice were anesthetized by intramuscular injection of Zoletil 20 (Virbac S.A., France). When required, animals were euthanized by asphyxiation with carbon dioxide. Additional methods (Genotyping, Trasngene expression, ES cell culture, infection and transplantation, osteoblastic cultures, cAMP assay, X-ray studies, histology and statistics are reported in supplemental online material.

Fig. 1.

Fig. 1

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(A) Vectors used to generate FD transgenic mice. (B) Expression of GsαR201C mRNA in non-skeletal tissues and in skeletal segments (including affected tail and femur and unaffected cervical vertebrae, foot, humerus and calvariae) in adult transgenic mice. (C) Increased intracellular levels of cAMP in osteogenic cells isolated from adult non-affected vertebrae from EF1α- GsαR201C (left panel) and PGK- GsαR201C (right panel) mice. Points correspond to measurements performed on single mice in triplicate. (D) Expression of GsαR201C mRNA in embryos (E10–E16, left panel) and different skeletal segments of newborn mice (right panel). (E) Alcian blue/alizarin red stain of whole mount E16 GsαR201C embryos showing normal skeletal patterning. (F) Normal microscopic appearance of the femur in E16 GsαR201C embryos as shown by Sirius red stain and transmitted (upper panel) and polarized (lower panel) light microscopy.

Results

Generation of EF1α-GsαR201C and PGK-GsαR201C transgenic lines

Lentiviral constructs, containing the mutant GsαR201C under control of constitutive promoters were used to generate mice with constitutive expression of GsαR201C, one of the two mutations associated with human FD. Two different backgrounds (FVB and C57/Bl6) and two different constitutive promoters [the human elongation factor 1α (EF1α) and the human phosphoglycerate kinase (PGK) (34,35)] (Fig. 1A), were used to control for the potential influence of each variable on the potential resulting phenotype. Multiple founders developed a radiographically detectable skeletal phenotype, and were backcrossed to generate transgenic lines. Transmission of a skeletal phenotype was obvious in all lines. For each genotype, two lines were selected for further analysis. In both PGK-GsαR201C transgenic lines, and one EF1α-GsαR201C line, high frequencies of genotype inheritance in F1–F4 (>70%) indicated the presence of multiple integrations. In one EF1α-GsαR201C line, in contrast, a Mendelian pattern of inheritance was observed in F1–F2. Determination of copy numbers by qPCR revealed 0.33 proviral copies/cell in the founder, consistent with chimerism, and one proviral copy/cell in F1 and F2 progenies, which continued to transmit the transgene with a Mendelian pattern, and the skeletal phenotype, at least until F9 (data not shown). In all lines, transgene expression was ubiquitous as expected (Fig. 1B, left). Between birth and 6 months, expression of the transgene was demonstrated throughout the skeleton, in radiographically affected or unaffected bones (Fig. 1B, right). Cyclic AMP production was significantly increased in bone cells from transgenic animals compared to WT (Fig. 1C) demonstrating expression and functional activity of the transgenic mutated Gsα. Excess cAMP production was documented in bone cells isolated in culture both from lesion-free bone (Fig. 1C) and skeletal lesions (data not shown).

GsαR201C expression does not impair bone development or embryonic bone cell differentiation

As inheritance of the transgene and the skeletal phenotype suggested embryonic tolerance of activating Gsα mutations in mice, we confirmed that transgene was expressed in embryos (Fig. 1D). Whole mount staining of whole embryos and newborns with alcian blue/alizarin red demonstrated the absence of changes in skeletal patterning (Fig. 1E) and histological analysis of developing bones failed to reveal changes in bone formation or modeling in embryos and newborns (Fig. 1F), suggesting that expression of GsαR201C in embryonic, differentiating skeletal cells did not bring about abnormal bone formation and modeling. To confirm this, we transduced murine ES cells (36) with the LV-PGK-GsαR201C vector, also used to generate the transgenic mouse strains, and then induced their differentiation into skeletal cells in vitro and in vivo. In undifferentiated WT ES cells, only trace amounts of Gsα were expressed (Fig. 2B). Consistent with published data indicating that Gsα expression is robust in differentiating ES cells [embryoid bodies (37)], we found that Gsα expression was in fact markedly upregulated upon differentiation with retinoic acid (RA), along with that of other GNAS transcripts (NESP, Xlαs, data not shown). In contrast, clonal undifferentiated GsαR201C ES cells robustly expressed GsαR201C in vitro (Fig. 2A, B) and produced excess cAMP (Fig. 2C). Consistent with differentiation-related upregulation of WT Gsα, cyclic AMP production was enhanced by RA-induced differentiation, both in WT and GsαR201C ES cells (data not shown). When transplanted into immunocompromised mice, GsαR201C–expressing ES cells formed teratomas that were indistinguishable from those formed by WT ES cells (Fig. 2D). Teratomas retained robust transgene expression in vivo (Fig. 2E), and included multiple areas of cartilage and bone differentiation (Fig. 2F).

Fig. 2.

Fig. 2

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(A–B) Expression of transgene mRNA (A) and protein (B) in murine, undifferentiated GsαR201C ES cells. (C) Increased intracellular cAMP in transgenic ES cells compared to WT ES. (D) Histology of teratomas formed by WT and GsαR201C ES cells. (E) Expression of the transgene in tumors generated by GsαR201C ES cells. (F) Cartilage and bone tissue in GsαR201C teratomas (HE stain). (G) GsαR201C ES cells generated normal cartilage in vitro (toluidine blue stain) that underwent normal endochondral ossification in vivo (Sirius red stain). (H) During differentiation, markers of pluripotency were down-regulated, markers of chondrogenesis and osteogenesis were up-regulated.

To analyze specifically the ability of GsαR201C ES cells to give rise to skeletal cells, we used an established model (38) in which ES cells are induced to ex vivo differentiation to cartilage on hydroxyapatite-tricalcium phosphate (HA/TCP) scaffolds, and then transplanted heterotopically into immunocompromised mice to obtain endochondral ossification (Fig. 2G). In vitro, GsαR201C-expressing cells upregulated cartilage genes and formed histology-proven cartilage, as did WT cells (Fig. 2H). In vivo, the ex vivo formed cartilage underwent endochondral ossification, with no appreciable difference between WT and GsαR201C transplants (Fig. 2G). Taken together, these data indicated that expression of mutated Gsα does not impair either embryonic bone development, or differentiation of skeletal cells.

Temporal and spatial patterns of skeletal lesions

To monitor the onset and the evolution of the postnatal skeletal lesions, 89 EF1α-GsαR201C and 105 PGK-GsαR201C mice were subjected to monthly radiography up to 24 months of age. By 6 months of age, a radiographically detectable skeletal phenotype was obvious in all EF1α-GsαR201C, and in 50–80% of PGK-GsαR201C mice. Both in EF1α-GsαR201C and PGK-GsαR201C lines, lesions in the tail vertebrae were the first to appear (at 2 and 3 months, respectively), and the only obligate, site of skeletal lesions. Seventy percent of the EF1α-GsαR201C mice also developed multiple additional lesions at other skeletal sites, resulting in a polyostotic pattern of skeletal disease. The femur was the second earliest and the second most common site of involvement, followed by tibia, spine, humerus, acropodial short bones, cranium, ribs and pelvis. A closely similar pattern of lesion distribution was observed in PGK-GsαR201C transgenic mice, in which, however, the cranium was only rarely affected. In all lines, involvement of the spine was progressive over time and followed a regular caudal-cranial pattern (Fig. 3A, B).

Fig. 3.

Fig. 3

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(A) Progression of skeletal changes in a caudal-cranial direction along the spine. Serial radiograms of two EF1α-GsαR201C mice. Lesions appear first in the tail, and later progress cranially. Arrows point to the same individual vertebra at indicated time points. (B) Detail of the fifth lumbar vertebra of one mouse at 4 and 17 months of age. (C) Progression of femoral lesions. The same mice are shown in successive radiograms. Note that femoral lesions begin as subtle thickening of the cortex at mid-shaft. As the lesions extend distally, mid-shaft thickening becomes more prominent and combines with newly arising cortical lytic changes, “ground glass” areas, before culminating in complex lytic-sclerotic patterns. (D) Macroscopic appearance of tail lesions in EF1α-GsαR201C mice and PGK-GsαR201C mice. (E) Serial radiographic analysis of EF1α-GsαR201C mice. Progression of tail lesions between 4 and 18 months of age. The same mice are shown in the successive radiograms. Note the progressive deformity, sclerosis, and lytic changes in individual vertebrae, leading in some cases to spontaneous fracture (two examples are shown in the bottom panel magnifications).

A regular pattern of lesion progression was likewise apparent in individual bones. In tail vertebrae, early radiographic lesions (2 months) consisted in subtle, irregular endosteal thickening at the metaphyses, with or without associated small lytic intracortical lesions. The latter became progressively more obvious between 3 and 9 months, along with increasing hyperostosis and deformity of individual vertebrae, which continued to progress steadily after 1 year of age (Fig. 3D, E). Radiographically obvious, spontaneous fractures of tail vertebrae occurred in 9.1% of the EF1α-GsαR201C mice and 7.3% of PGK-GsαR201C mice between 12 and 18 months. In the femur (Fig. 3C), early lesions consisted in localized endosteal thickening at the midshaft or the meta-diaphyseal junction. The thickening extended proximally and distally progressively, while lytic changes developed within the thickened bone. The distal metaphyses ultimately became globally hyperostotic and deformed, after 1 year of age. Spontaneous partial fractures of the distal femur were detected radiographically in 2/89 of the EF1α-GsαR201C mice.

FD develops stepwise in the post-natal skeleton

Histological studies guided by serial radiographic analysis were then focused on lesions that were classified as early (<2 months from radiographic appearance), intermediate (2–6 months from appearance) and late (>10 months from appearance).

As seen in tail vertebrae of 6–8 week old mice, the earliest histological changes consisted of a distinct pattern of endosteal thickening, associated with an excess of abnormal bone trabeculae. The marrow cavities were narrowed and distorted as a result, while the outer contour of the vertebra was unchanged (Fig. 4A–D). Endosteal/medullary bone trabeculae were irregular in shape and made of woven bone, did not contain a cartilage core (were not endochondrally formed), and were separated from each other by a richly cellular tissue, which did not include Sirius-red stainable, birefringent collagen (fibrosis) (Fig. 4E–L). Histomorphometric analysis reflected the excess bone formation and abnormal trabecular density and architecture revealed by histology, but (along with TRAP cytochemistry, Fig. 4O) revealed no excess of osteoclast activity relative to bone surfaces, qualifying early changes as dominated by abnormal and excessive bone formation (Fig. 4M). qBSE analysis of early lesions demonstrated a mineralization deficit in the lesional bone (Fig. 4N). Alkaline phosphatase was uniformly expressed in the intertrabecular cellular tissue, indicating its osteogenic nature (Fig. 4P).

Fig. 4.

Fig. 4

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Early stage of development of FD lesions in tail vertebrae of EF1α-GsαR201C mice. (A–D) H&E staining demonstrating the formation of new bone inside the bone marrow cavity (perimeter outlined by black lines) in transgenics (B–D) compared to WT mice (A). (E–H) Sirius red staining and Nomarski optics images, demonstrating de novo formed bone inside the marrow cavity in transgenic mice (F–H, green lines), occupied by yellow (adipose) marrow in WT mice (E). (I–J) Transmitted (I) and polarized light (J), views of the same field stained with Sirius red from transgenic mice. (K–L) Nomarski (K) and polarized light (L) images of the same field stained with Sirius red. Note that the bone is woven, but there is no fibrosis in the adjacent soft tissue at this stage. (M) Histomorphometric analysis of bone formation and resorption, demonstrating increased bone mass, reduced intertrabecular space, and increased bone formation (Obs/Bs), with normal osteoclastic bone resorption (OcS/Bs). (N) qBSE analysis of mineralization demonstrating reduced mineral content in the new “medullary” bone in transgenic mice. (O) TRAP-positive osteoclasts in tail vertebrae of WT and transgenic mice. (P) ALP immunostaining demonstrating intense immunoreactivity in the soft, cellular tissue separating newly formed medullary bone in transgenic mice.

In intermediate lesions, the marrow cavity was obliterated or markedly narrowed and distorted; large, focal structural gaps within an abnormally expanded cortical bone could be recognized as the basis of the lytic changes seen radiographically (Fig. 5A–D). Sirius red staining and polarized light microscopy again demonstrated, however, the absence of fibrosis (Fig. 5E–H). The excess bone was significantly and uniformly undermineralized as assessed by BSE and qBSE analysis (Fig. 5I–Q). At variance with early lesions, intermediate lesions were noted for prominent osteoclastic resorption, as documented by histomorphometry (Fig. 5R). Confocal light microscopy and iodine-contrasted BSE SEM images of undecalcified, PMMA embedded whole vertebrae readily identified intracortical gaps as sites of ectopic intracortical remodeling, mimicking the tunneling resorption pattern seen in human FD and hyperparathyroidism (Fig. 6A–H). Intense osteoclastic activity extended up to the outer regions of the cortex causing microfractures (Fig. 6E, G). In addition, a complex system of cement lines documented the occurrence of multiple, recurrent cycles of (ectopic) intracortical remodeling (Fig. 6F, H). ALP and TRAP cytochemistry demonstrated spatially segregated resorptive and formative phases within the ectopic remodeling sites (Fig S1). Florid osteoclastogenesis was obvious in resorptive lacunae (Fig. 6I, J, L), where ALP+ elongated processes of loosely distributed perivascular stromal cells established contact with TRAP+ mononuclear osteoclast precursors (Fig. 6K). Formative-type lacunae, in contrast, were filled with densely packed ALP+ stromal cells, did not contain osteoclasts or their precursors and were bordered by osteoblasts depositing osteoid (Fig. 6M–P).

Fig. 5.

Fig. 5

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Histology, SEM and qBSE analysis of tail vertebrae from 7 months old WT and transgenic mice. Transmitted light (A–D) and polarized light (E–H) views of Sirius red stained tail vertebrae; SEM views of triiodide stained PMMA block surfaces (I–K). Note the narrowing of marrow cavities, and the opening of sites of intracortical osteolysis. (L–Q) qBSE analysis, demonstrating overall reduced mineral content in tail vertebrae from transgenic mice. (R) Histomorphometric analysis demonstrating enhanced osteoclastic resorption (remodeling) in transgenic mice.

Fig. 6.

Fig. 6

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Details of intracortical remodeling in 7 months-old EF1α-GsαR201C mice. (A–B) Tandem scanning reflected light confocal microscopy. (C–H) SEM of triodide stained PMMA blocks. Note osteoclasts in E (arrows), microcracks in the severely thinned cortex in G (arrows) and a mosaic of cement lines demonstrating multiple repeated cycles of intracortical remodeling in F and H (arrows). (I–L) Sites of intracortical remodeling as seen in undemineralized GMA sections reacted for TRAP (red) and ALP (blue) activities. Note the wealth of mononuclear TRAP-positive cells in the remodeling “sites” (I, J, L) and the thin elongated cell processes of ALP-positive stromal cells contacting mononuclear cells (K). (M–P). Sites of intracortical remodeling as seen in undemineralized GMA sections reacted for ALP activities. Note that the entire stromal tissue filling the remodeling cavities (stroma) is intensely ALP-positive, and merges with recognizable mature osteoblasts (obs) at bone surfaces.

It was only in late lesions that a full-blown FD-like picture became obvious. In mice older than 1 year (Fig. 7), individual bones were grossly deformed and/or fractured; the internal bone architecture was effaced (cortical bone and marrow cavity could no longer be discerned), a characteristic “Chinese writing” pattern of abnormal bone trabeculae was readily recognizable, Sirius red-stainable birefringent collagen was abundant in intertrabecular regions and filled broad areas of the lesions, Sharpey fibers adjoining woven bone and adjacent fibrosis were prominent. In brief, the classical, diagnostic histopathological hallmarks of human FD (4,39,40) had developed. Accordingly, three histopathologists and 2 orthopedic surgeons with specific experience in bone pathology/FD invariably identified (“mock-diagnosed”) 15 different digital histological images from 15 different mice as demonstrating typical FD when blinded to their actual source.

Fig. 7.

Fig. 7

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Ultimate histopathological pattern in old (17 months) transgenic mice. (A–B) Overview of the distal femur of EF1α-GsαR201C mice, Sirius red staining, transmitted (A) and polarized (B) light view. In the polarized light image, fibrotic areas are green, bone is red. (C–N) Details of Sirius red (C–F), Nomarski-sirius red (G–J), and polarized light-sirius red (K–N) images of the femur, demonstrating “Chinese writing” trabecular patterns and fibrosis. Higher magnification images (F, J, N) highlight Sharpey fibers. (O–R) FD lesions in the skull of EF1α-GsαR201C mice. Fibrosis (asterisk) is better shown by polarized light (R). (S–V) Four tail vertebrae of two different PGK-GsαR201C mice stained with Sirius red demonstrating combinations of lytic (S, T, U), sclerotic changes, fracture (T) and “Chinese writing” (U, V) patterns.

Taken together, these data defined three successive temporal phases in the development of FD-like lesions in the mouse: an initial stage in which excess primary bone formation occurs at endosteal surfaces and within medullary cavities, an intermediate stage during which enhanced remodeling occurs and a final stage during which marrow fibrosis and overall architectural subversion are established, generating a murine replica of human FD.

Discussion

We have shown here that constitutive expression of GsαR201C, one of the two activating mutations associated with human FD, causes an inherited replica of human FD in mice, independent of genetic background and type of constitutive promoter used. Our data conclusively demonstrate that Gsα activating mutations are sufficient to establish FD in mice, bringing the detection of the mutations in humans out of the realm of association and into that of determinism. Our data also show that contrary to a widely held assumption, expression of mutated Gsα per se is not embryonic lethal if germline transmitted, at least in mice. Transgenic lines described in this report have now been in existence for 5 years, with no significant change in phenotype or inheritance thereof. Thus, expression of an activating mutation of Gsα, as a randomly integrated transgene, is not necessarily lethal, and the transgene survives independent of genetic mosaicism, at variance with Happle’s classical postulate regarding the human disease gene (18). FD-associated human mutations at codon 201 in the Gsα gene also involve other transcripts of the GNAS locus that share with Gsα the mutation-bearing exon 8 [such as XLαs (41,42), which remains WT in our model]. Therefore, our data cannot formally exclude that the same mutation could be lethal (as assumed) and non-inheritable, if occurring in the natural GNAS locus. We are currently addressing this theoretical possibility through different murine models.

Our data also suggest that downstream of robust expression of GsαR201C, development of the disease phenotype, are linked through a complex, and yet to be elucidated, array of involves regulatory and developmental events independent of genetic mosaicism. Transgene expression in embryos, newborns, and postnatal murine bones does not necessarily, or immediately, result in generation of FD-like bone. Expression of the transgene in clonal pluripotent cells does not impair their survival, pluripotency and differentiation in vitro and in vivo, including their ability to form bone and cartilage. Development of FD lesions is entirely postnatal, while transgene expression is not, and FD lesions remains focal, asymmetric, and metachronous in mice, in the absence of genetic mosaicism. Thus, expression of functional, mutated Gsα is per se not necessarily incompatible with normal, osteoblastic function and bone formation, which proceed unscathed during development. Likewise, large portions of the skeleton remain unaffected throughout life independent of genetic mosaicism. As in humans, a regular temporal and spatial pattern of multifocal skeletal involvement can be discerned in mice, which do not represent, at variance with humans, somatic mosaics. Such patterns may reflect specific anatomic and physiological determinants of skeletal growth and remodeling, interplaying with the function of mutated Gsα and its cellular regulation. For example, remodeling of cAMP signaling, occurring through site and time-specific patterns of PDE expression, contributes to disease development in other organ systems, and might have a role in the development and evolution of FD (21,43–45) (46).

Our data show that the diagnostic pathological picture of human FD is in fact the endpoint of a timed sequence of events. Primary, secondary and tertiary phases of FD lesion development, are each dominated by distinct histological changes: excess endosteal/medullary bone formation, excess remodeling, and ultimately architectural subversion and true fibrosis, respectively. Likely, similar stages occur in humans. They may form the basis for clinical-pathological diversity, but also for its clinical interpretation. The diversity of cellular events underlying tissue changes in each stage (e.g., excess bone formation in early stages, osteoclastic remodeling in later stages) predicts that lesions at different stage of development would respond differently to different treatments.

For testing existing or potential treatments, modeling human disease in the mouse must aim at the closest possible approximation to the specific histopathology from which morbidity arises. In human FD, morbidity factors include the fragility, deformity and abnormal compliance of FD bone (4). Several transgenic models portraying the effects of enhanced Gsα signaling, but obtained through transgenes other than mutated Gsα (acting upstream or downstream of Gsα), have been considered as indirect mimics of human FD. Compared to each of these models (23) ((24,25) (26) our model seems to be the only one (and the first) truly reproducing the FD bone pathology and the dependent morbidity factors. On the other hand, specific differences in anatomy and physiology between humans and mice must be taken into account when modeling human disease for translational purposes. Paradoxically, we efficiently modeled a human pediatric disease in aging mice. However, the absolute time required for the development of a histology-proven FD lesion in the mouse reflects the organ-specific remodeling events needed to establish the specific pathological pattern. Indeed, the biological time of FD development (>1 year) is more directly similar in humans and mice, than its relationship to the organism lifespan in each species.

In conclusion, we have generated the first direct model(s) of human FD in mice, which provide(s) suitable and long-missed resource(s) for applicative and fundamental studies on the pathogenesis and treatment of the human disease.

Supplementary Material

Suppl. Table 1
Suppl. Text

Acknowledgments

Supported by grants from Telethon (GGP09227) to PB, from Fondazione Roma to PB and MR, from Istituto Pasteur-Fondazione Cenci-Bolognetti to PB, from MIUR and EU (PLURIMES) to PB. Funding for IS was supported by EU FP7 Brainvectors n. 286071. Funding for PGR and KH was supported by the Division of Intramural Research, NIDCR, a part of the Intramural Research Program of the NIH, DHHS.

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Suppl. Table 1
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