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, 165 (4), 1107-15

Transgenic Mice Overexpressing BMP4 Develop a Fibrodysplasia Ossificans Progressiva (FOP)-like Phenotype

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Transgenic Mice Overexpressing BMP4 Develop a Fibrodysplasia Ossificans Progressiva (FOP)-like Phenotype

Lixin Kan et al. Am J Pathol.

Abstract

Fibrodysplasia ossificans progressiva (FOP) is a rare hereditary connective tissue disease characterized by progressive postnatal heterotopic bone formation. Although the genetic defects of FOP are not known, several lines of evidence have suggested that bone morphogenetic protein-4 (BMP4) may be involved in the pathophysiology. Nevertheless BMP4-transgenic mice have previously failed to develop the disorder and there has been no good animal model of the disease. Here, we report that a unique transgenic mouse line that overexpresses BMP4 under control of the neuron-specific enolase (NSE) promoter develops a FOP-like phenotype. Mating of these animals with transgenic animals that overexpress the BMP inhibitor noggin prevents the disorder, confirming the role of BMP4 in the pathogenesis of the disease. Heterotopic bone formation in these animals appears to follow the classic endochondral ossification pathway. Sex-mismatched cell transplantation experiments indicate that multiple cell sources contribute to the heterotopic ossification. This remarkable animal model provides a unique opportunity to further study the role of the BMP signaling pathway in heterotopic ossification and to improve our understanding of the clinical aspects of FOP.

Figures

Figure 1-4266
Figure 1-4266
Early skeletal phenotypes of NSE-BMP4 transgenic mice. A: The first noticeable phenotype of NSE-BMP4 transgenic mice was stiffness of the hind limbs. a: When picked up by the tail, hind limbs of affected mouse showed sign of stiffness and cannot fully extend and move freely, (b) while WT littermate can fully extend their hind legs and move freely. B: The first gross phenotype of NSE-BMP4 transgenic mice was enlargement of the hind limb circumference. Lane 1, WT littermates; Lane 2, NSE-BMP4 transgenic mice before abnormality; and Lane 3, NSE-BMP4 transgenic mice after abnormality. C: The first histological phenotypes were infiltration of CD45+ mononuclear cells and muscle degeneration. FITC was used to stain CD45, DAPI was used to stain nuclei, and autoflorescence in Cy3 channel was intentionally collected to show the basic structure of muscle fibers. a: A large amount of CD45+ mononuclear cells was detected. Brace indicated the region where muscle fibers degenerated. b: WT littermates showed no sign of CD45+ mononuclear cells infiltration or muscle degeneration.
Figure 2-4266
Figure 2-4266
Sequential events of heterotopic endochondral ossification in NSE-transgenic mice. A: Phase I of heterotopic endochondral ossification. a: The early histological finding is the local proliferation of fibroblast-like cells, usually located subcutaneously (shown) or between muscles (not shown). Note the fibroblast-like morphology (H&E staining). b: The early cells mass were positive for ALP (blue staining). c: Most of these cells bear MSCs markers Sca-1+ (FITC, shown) CD34+ (not shown). d: These cells expressed BMP4 (Cy3): the surrounding region had higher level of BMP4, while the inner region had lower level and (e) Lin (FITC) was negative. B: Phase II of heterotopic endochondral ossification. a: Inner cells lose CD34 (FITC) staining (shown) and Sca-1 staining (not shown) b: Early cell mass continually proliferated and expressed BMP4 (Cy3) (especially the surrounding region). c: Obvious de novo angiogenesis initiated in the connected tissue between the dense fibrotic region and skin. Arrows indicated blood vessels. d: H&E stain of dense fibrotic and its surrounding region. Note most of the cells in fibrotic region still kept fibroblast-like morphology at this stage. Arrows indicated blood vessels. e: Dense fibrotic region still kept ALP activities (surrounding region had stronger activities). f: Alcian blue staining was negative. C: Phase III of heterotopic endochondral ossification. a: At this stage, inner cell region already condensed and differentiated, and show typical chondrocyte morphology. b: ALP staining showed the inner cells lost ALP activities. c: Alcian blue staining showed the inner cells were positive. D: Phase IV of heterotopic endochondral ossification. Multi-focal central hypertrophic chondrocytes were surrounded by profound proliferative fibroblast-like cells. Around the marginal hypertrophic chondrocytes, nascent trabeculae-like calcium deposits appeared.
Figure 3-4266
Figure 3-4266
A: Heterotopic bone showed characteristic structure of cancellous or woven bone. a: ALP staining of typical heterotopic bone. ALP activities (blue stain) were detected in trabeculae, especially on surfaces contacting with marrow space. b: Alizarin red staining of typical heterotopic bone. Note that trabeculae were strongly positive for the stain. c: TRAP staining of typical heterotopic bone. Arrow indicated typical multinucleated TRAP-positive cells and untypical mononucleus TRAP-positive osteoclasts cells located in marrow space, may or may not contact with trabeculae. d: Typical heterotopic bone under polar light. Lighter regions in trabeculae indicated regular arrangement of collagen fibers and darker regions indicated irregular arranged collagen fibers. B: Expression patterns of key transcription factors and markers implicated in osteogenesis; DAPI was used to stain nuclei in all case. a: Msx2 was detected in proliferative fibroblast-like mesenchymal cells. b: col II was detected mostly in typical chondrocytes or hypertrophic chondrocytes. c: col I was detected in presumably pre-osteoblasts or osteoblasts in newly formed trabeculae. (d) Double-staining of N-cad(Cy3) and CD45(FITC). Typical N-Cad+ CD45 cells were detected in newly formed trabeculae. e: Runx-2 was detected in newly formed trabeculae. f: BSP was detected in newly formed trabeculae, matrix, or cytoplasmic.
Figure 4-4266
Figure 4-4266
X-ray images of NSE-BMP4 transgenic mice and littermates. A: X-ray image at different time points of a typical NSE-BMP4 transgenic mouse and its littermate indicated the heterotopic ossification is progressive. a: Three-month-old NSE-BMP4 transgenic mouse and its littermate. Arrows indicated the defused radiopaque regions. b: Four-month-old NSE-BMP4 transgenic mouse and its littermate. Arrows indicated the radiopaque regions. Note that the radiopaque regions were expended. c: Five-month-old NSE-BMP4 transgenic mouse and its littermate. Arrows indicated the radiopaque regions. Note, not only the radiopaque regions were expended but also the typical kyphoscoliosis. d: Comparison of 8-month-old NSE-BMP4, NSE-Noggin, and NSE-BMP4, NSE-Noggin double-transgenic mouse. Note, only the NSE-BMP4 transgenic mouse had severe heterotopic ossification and kyphoscoliosis. e: One example of NSE-BMP4 mouse with fully developed heterotopic ossification. B: Spectrum of skeletal phenotypes in transgenic mice. Phenotypical comparison of six individual 5-month-old NSE-BMP4 transgenic mice. Arrows indicated the radiopaque regions. Note, some mice had severe and some mice had milder heterotopic ossification and kyphoscoliosis.
Figure 5-4266
Figure 5-4266
A: Summary of tumorogenesis activities tests. The heterotopic bones were surgically removed from NSE-BMP4 transgenic mice and implanted subcutaneously in the back of NES-BMP transgenic mice or WT littermates. Cells extracted from heterotopic bones also subjected to soft agar colony analysis. There was no positive colony in soft agar colony analysis, and no growth of implants was observed in recipient mice either. B: Summary of sex-mismatched cell transplantation. Peritoneal cells and mononuclear cells were isolated from 2-month-old male donor NES-BMP transgenic mice (red lines) or male WT littermates (black lines), and were injected subcutaneously to the inner side of female left hind limb of 2-month-old NES-BMP4 transgenic mice or WT littermates. “no” indicated there was no heterotopic ossification within 2 weeks. “H.O.” indicated there was heterotopic ossification within 2 weeks. Note that only female NES-BMP4 transgenic mice receiving the male NES-BMP4 donor cells developed heterotopic ossification. C: Dual-color FISH experiment to follow the donor cells in recipient mice. a: Y-chromosome (Cy3), (b) X-chromosome (FITC), (c) DAPI staining, (d) merge of a–c frozen section of tissue with heterotopic ossification from recipient mice. Note the typical chondrocytes or hypertrophic chondrocytes morphology and nuclei staining, and more importantly, many of typical chondrocytes or hypertrophic chondrocytes had both X and Y chromosome staining.

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