Heterotopic Ossification in Mouse Models of Fibrodysplasia Ossificans Progressiva

Abstract

Fibrodysplasia ossificans progressiva (FOP), a rare genetic disorder of progressive extra-skeletal ossification, is the most disabling form of heterotopic ossification (HO) in humans. Most people with FOP carry an activating mutation in a BMP type I receptor gene, ACVR1^R206H, that promotes ectopic chondrogenesis and osteogenesis and in turn HO. Advances in elucidating the cellular and molecular events and mechanisms that lead to the ectopic bone formation are being made through the use of genetically-engineered mouse models that recapitulate the human disease. We describe methods for inducing heterotopic ossification in a mouse model that conditionally expresses the Acvr1^R206H allele.

1. Introduction

The BMP/TGFβ family of ligands and their receptors regulate many diverse and biologically critical cellular functions including cell differentiation, apoptosis, proliferation, migration, and stem cell reprogramming and are highly evolutionarily conserved in order to maintain precise control of these processes[1–6].

A human genetic disease, fibrodysplasia ossificans progressive (FOP; MIM #135100, http://omim.org/entry/135100), is caused by rare mutations in the BMP type I receptor ACVR1 (also known as ALK2), with most cases caused by a recurrent heterozygous gain-of-function mutation c.617G>A; R206H [7–9]. People with FOP are born with no clinical manifestations of the disease except for a characteristic malformation of the great toe. However, during childhood and throughout adult life, extra-skeletal bone episodically forms in soft connective tissues such as skeletal muscle, often in response to injury. Non-genetic forms of such ectopic bone formation, known as heterotopic ossification (HO), are associated with a number of common conditions in adults that involve severe trauma such as spinal cord and head injuries, hip replacement surgery, and war-related blast injuries [10,11].

In 2012, we published a detailed characterization of the first genetically-engineered FOP mouse model with a knock-in allele of the Acvr1 R206H mutation [12]. This mouse model closely mimics the clinical features of FOP in humans and was the first demonstration that the ACVR1 R206H mutation is solely responsible for the FOP clinical phenotype and that FOP could be modeled in the mouse with high fidelity. However, genetic transmission of the Acvr1^R206H allele in mice caused perinatal lethality, therefore development of mouse models that conditionally activate the Acvr1 R206H mutation was necessary. Studies using a floxed conditional-on Acvr1^R206H knock-in mouse were recently reported [13,14].

Heterotopic ossification in FOP can occur in response to tissue trauma/injury or spontaneously in the absence of overt trauma. In our hands, post-natal expression of the mutant allele is not sufficient to allow robust and predictable HO formation in the absence of injury. However, we have found that global post-natal Cre recombinase activation and induction of Acvr1^R206H expression in the conditional mouse model is sufficient to form HO in response to skeletal muscle injury [13]. Here, we describe our approach to activating and characterizing HO lesion formation in a conditional Acvr1^R206H knock-in mouse by muscle injury.

2. Materials

2.1. Mouse Models

1. Conditional-on knock-in mouse model: Acvr1^[R206H]FlEx was described in Hatsell et al., 2015. The floxed Acvr1^[R206H]FlEx allele is expressed in response to Cre recombinase expression.

2. Mice double transgenic for R26-rtTA and tetO-Cre (heterozygous Gt(ROSA)26Sor^{tm1(rtTA*M2)Jae} and hemizygous Tg(tetO-Cre)1Jaw; Jackson Laboratories) (see Note 1).

2.2. Doxycycline chow

1. Sterilized doxycycline embedded chow containing 625mg/kg doxycycline.

2. Store doxycycline chow in sealed packets at 4°C (see Note 1).

2.3. Cardiotoxin

• 1.1X Phosphate buffered saline (PBS): 137 mM NaCl, 2.7 mM KCl, 110 mM Na₂HPO₄, 1.8 mM KH₂PO₄, pH 7.2. Weigh 8 g of NaCl, 0.2 g of KCl, 1.44 g of Na₂HPO₄, and 0.24 g KH₂PO₄; dissolve in 800 mL of deionized water. Adjust the pH to 7.2 with HCl, and add water to 1 L. Sterilize by autoclaving for 20 min at 15 psi.
• 2.100 μM Cardiotoxin stock solution: Cardiotoxin (from Naja mossambica mossambica) is obtained as lyophilized powder. Add 1 mg cardiotoxin to 1426 ul of sterile 1X PBS with gentle shaking. Store at −20°C in 50 μl aliquots.
• 2.10 μM Cardiotoxin working solution: Just prior to use, dilute 100 μM cardiotoxin stock to 10 μM with sterile 1X PBS (10 μl of stock diluted with 90 μl of PBS). Keep on ice.
• 3.28 gauge 1/2 U-100 insulin syringes
• 4.alcohol pads
• 5.Gas vaporizer chamber for isoflurane-induced anesthesia

2.4. Genotyping

1. Mouse genomic DNA.

2. PCR primers to detect the knock-in allele post-Cre recombination:.

   Forward: 5’-TGTATTGCAGGACGCTGAAG-3’

   Reverse: 5’-CCCCTGAAGTGGAATAACCA-3’

3. 2X PCR master mix: 2.5 U of Taq DNA polymerase, 20 mM Tris-HCl (pH 9.0), 3 mM MgCl₂, 20 mM KCl, 16 mM (NH₄)₂SO₄, 0.1% NP-40, 1.6 mM dNTPs.

4. 10X Tris acetate EDTA (TAE): 400 mM Tris acetate, 10 mM EDTA, pH 8.3. Dilute to 1X with deionized water for preparing agarose gels and gel electrophoresis running buffer

5. Agarose

6. Ethidium bromide at 10 mg/ml

2.5. Histology (see Note 8)

1. 4% Paraformaldehyde (PFA)

2. Decalcifying solution containing 5% formic acid

3. Harris Haematoxylin

4. 0.5% (w/v) eosin Y in acidified 90% (w/v) ethanol (can be obtained commercially)

5. Acid alcohol: 5 ml of 12 N hydrochloric acid added to 500 ml 70% ethanol.

6. Ammonium water: Add 2.5 ml of 28.0–30.0% ammonium hydroxide stock solution to 500 ml deionized water.

7. Parafilm

8. Scotch tape (or other similar tape).

3. Methods

Injury-induced heterotopic ossification in Acvr1^R206H/+;R26-rtTA;tetO-Cre (Acvr1^R206H/+) mice

3.1. Post-natal global induction of the Acvr1 R206H mutation

1. Cross Acvr1^{[R206H]FlEx/+} mice with mice double transgenic for R26-rtTA and tetO-Cre (heterozygous Gt(ROSA)26Sor^{tm1(rtTA*M2)Jae} and hemizygous Tg(tetO-Cre)1Jaw; Jackson Laboratories) to generate Acvr1^{[R206H]FlEx/+};Gt(ROSA)26Sor^{tm1(rtTA*M2)Jae}; Tg(tetO-Cre)1Jaw mice (referred to as Acvr1^cR206H/+;rt-tetO-Cre below). (see Note 2)

2. Induce Cre recombinase expression in Acvr1^cR206H/+;rt-tetO-Cre mice at 4–6 weeks old (see Note 3) by replacing normal food chow with doxycycline chow for 3 days (see Note 4).

3. PCR amplification to detect Cre recombination: Add 40 ng mouse genomic DNA (in 2–3 μl) to 10 μl of 2X PCR master mix and 0.5 μl each of forward and reverse primers (50 μM); add water to a total volume of 20 μl. PCR amplification: initial denaturation 94°C (3 min); followed by 35 cycles of denaturation 94°C (30 s), annealing 55°C (45 s), and extension 72°C (30 s); then final elongation at 72°C for 10 min.

4. Agarose gel electrophoresis: Electrophorese 10 μl of each PCR amplified sample through 4% (w/v) agarose gels (4 g of agarose added to 50 ml of 1× TAE with 0.5 μl of ethidium bromide stock solution) (Fig 1).

Fig 1. Agarose gel electrophoresis of PCR products to verify Cre recombination of the Acvr1^R206H allele.

Genomic DNA samples were isolated from a Acvr1^cR206H/+;rt-tetO-Cre mouse and a control Acvr1^+/+ mouse after 3 days on doxycycline chow. Following PCR amplification to detect Acvr1, samples were electrophoresed through a 4% agarose/TAE/ethidium gel. PCR products from wild-type Acvr1 (336 bp) and recombined Acvr1^cR206H (370 bp) alleles are indicated.

3.2. Hind limb injury by cardiotoxin to induce heterotopic ossification

1. Restrain mice by isoflurane-induced anesthesia (3–5% isoflurane) using a gas vaporizer chamber (see Note 5).

2. Load sterile syringes aseptically with 50 μl of 10 μM cardiotoxin working solution; place on ice. Clean hind limbs of mice with alcohol pad. Inject cardiotoxin aseptically into the quadriceps muscle (see Notes 6 and 7). Inject contralateral limbs with 1X PBS as controls.

3. Place mice into cages and confirm recovery from anesthesia by noting that mice are awake with normal movement.

3.3. Tissue harvest and fixation

1. Euthanize mice at the desired assay time point for analysis (see Note 7). Hind limbs are evaluated by standard radiography for presence of HO formation (Fig 2A). HO volume can be quantified by microCT analyses (Fig 2B). Progression and stages of HO can be examined by histology (Fig 2C).

2. Dissect hind limbs from euthanized mice. Place in 50 ml tubes and fix by filling tubes with 4% paraformaldehyde (4% PFA) to fully immerse sample. Incubate at room temperature with gentle shaking/rocking overnight (12–16 hr).

3. Pour off PFA. Wash the PFA-fixed samples by filling tube with 1X PBS and rock gently for 5 minutes. After 3 washes, remove hind limb from the tube and process for imaging and/or histologic analyses.

Fig 2. Cardiotoxin-injured skeletal muscle in Acvr1^cR206H/+ mice induces heterotopic ossification.

Acvr1^R206H/+ mice (at 4 weeks old) were provided doxycycline chow for 3 days to induce global expression of the Acvr1^cR206H allele, followed by cardiotoxin injury to quadriceps muscles. A. X-ray images of the hind limbs at 6, 8 10, 12 days post-cardiotoxin injection are shown; circled areas indicate HO formation. B. MicroCT images of a control and a mutant leg showing extensive HO formation at the site of cardiotoxin injection in the mutant (circle). C. Decalcified tissue section from a hind limb of a cardiotoxin-injected mouse was stained with haematoxylin and eosin. Evidence of heterotopic lesion formation is shown by the presence of cartilage (C) and bone (B); fibroproliferative cells are also present (F).

3.4. Imaging analyses

1. Following PFA and washes, carefully blot excess PBS from fixed hind limb with paper towels. Place tissue on a square of Parafilm that has been taped to scanning surface to prevent movement. Image using a standard small animal X-ray instrument to detect HO.

2. For more detailed analysis of HO, acquire micro-computed tomography (μCT) volumetric data at the following parameters: 80 kVp and 80 μA X-ray tube voltage and current, 250 μm aluminum filter, 1.7 s integration time, 400 views at 0.5° increments, 2×2 detector bin mode, 4 averages. Reconstruct image data at a resolution of 40.5 μm isotropic voxels using a Feldkamp cone beam algorithm. Analyze and determine volume from the reconstructed 3D data using software such as OsiriX (www.osirix-viewer.com).

3.5. Histological evaluation of heterotopic ossification

1. Following PFA and washes (see Note 8), decalcify samples by filling 50 ml tube containing the sample with decalcifying solution containing 5% formic acid; shake gently at room temperature for 3 days, replacing with fresh decalcifying solution each day. Pour off decalcifying solution. Wash the decalcified samples by filling tube with 1X PBS and rock gently for 5 minutes. After 3 washes, remove hind limb from the tube and process for paraffin embedding and section at 7 microns. Place sections on slides for histological staining.

2. Follow standard protocols to remove paraffin using three changes of xylene, 5 min each.

3. Re-hydrate sections by immersing slides in 3 changes each of 100%, 95%, 70% ethanol for 3 minutes each followed by immersion in deionized water twice.

4. Immerse hydrated sections on slides in Harris haematoxylin for 5 minutes. Rinse gently in running deionized water until excess stain no longer leaches from tissue. Dip slides three times in acid alcohol then rinse by immersing in three changes of deionized water. Place slides in ammonium water for 30 seconds for developing the blue stain (bluing).

5. Dip slides in 3 changes of deionized water, then place in 95% ethanol for 1 minute. Immerse in 1% Eosin Y solution for 5 minutes. Dehydrate in 3 changes of 95% ethanol and 3 changes of 100% ethanol, then cleare by immersing in xylene, 3 changes of 5 minutes each.

6. Cover slip and mount tissue sections and dry overnight. Observe microscopically for HO formation.

4. Notes

1. The rtTA-tetO ‘on’ system [15] is responsive to doxycycline to activate Cre expression. Use fresh doxycycline chow for induction of Cre recombinase. Moisture and increased temperature can cause hydrolysis and inactivation of doxycycline.

2. We (and others [14]) have also induced global expression of the floxed Acvr1^R206H allele using tamoxifen and ERT2-Cre (B6.129-Gt(ROSA)26Sor^{tm1(cre/ERT2)Tyj}/J). Since tamoxifen, an estrogen analog, could plausibly affect bone formation and growth, we preferred to use the rt-tetO system in our initial studies [13]. To induce global expression, tamoxifen is administered by intraperitoneal injection. Dissolve tamoxifen in corn oil to 20 mg/ml by gentle rocking overnight at 37°C. For adult mice, 100 μl (75 mg tamoxifen/kg body weight) is administered via intraperitoneal injection. Repeat for 3 consecutive days (https://www.jax.org/research-and-faculty/tools/cre-repository/tamoxifen).

   Alternatively, to allow the expression of the Acvr1^R206H allele during embryonic development but avoid the lethality that is associated with global expression of Acvr1^R206H during embryogenesis, we have induced Acvr1^R206H expression prenatally in a limited population of skeletal progenitor cells (Prrx1⁺). During embryonic development, Prrx1 is expressed in a population of skeletal progenitor cells that gives rise to lateral plate mesoderm-derived limb mesenchymal cells. Acvr1^R206H;Prrx1-Cre mice are born at the expected Mendelian frequency. Acvr1^R206H;Prrx1-Cre mice form extensive spontaneous extra-skeletal bone by one month of age [13].

3. For cardiotoxin-induced heterotopic ossification, mice are used within 6 weeks of age. We have found that the amount of HO formation by injury decreases as the mouse ages.

4. This diet delivers a daily dose of 2–3 mg of doxycycline based on an average 4–5 g/day of food consumption by a mouse.

5. When injecting mice, immobilization using isoflurane anesthesia is recommended. Avoid injecting into blood vessels, as this can be fatal to mice. Care should be taken during injections to avoid needle contact with skeletal bone; injury to periosteum can stimulate orthotopic ossification even in wild-type mice.

6. Vigorous shaking/vortexing of cardiotoxin can cause denaturation. Prepare diluted working solutions of cardiotoxin in pre-loaded syringes and keep on ice.

7. Progression to heterotopic bone occurs through endochondral ossification and forms mineralized bone by ~14 days post-cardiotoxin muscle injury. Cartilage formation is detected after 5–7 days. Impaired hind limb movement from day 7 post-injury is typically observed. Note that mice must be handled gently; pinching or squeezing appears to result in sufficient tissue trauma to stimulate HO.

8. Histological analysis can use PFA-fixed samples that have been processed specifically for histology or can use the same fixed sample used for imaging analysis without further processing.

All animal procedures were reviewed and approved by the Institutional Animal Care and Use Committee at University of Pennsylvania.