Histological examination of the skin reveals bone formation.

<p>Histological examination of the skin from the trunk (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021755#pone-0021755-g005" target="_blank">Fig. 5B,5D,5F,5H</a>) and feet (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021755#pone-0021755-g005" target="_blank">Fig. 5A,5C,5E,5G</a>) of male and female −m/+ and −p/+ mice reveal mineralized bone formation often associated with a dense eosinophilic osteoid-like matrix in the dermis and perifollicular areas. No abnormalities were observed in WT mice. Occasional osteoid-like material was present without bone, immediately adjacent to hair follicles, especially in female mice which had milder lesions (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021755#pone-0021755-g005" target="_blank">Fig. 5F, 5H</a>). Subcutaneous bone frequently contained central bone marrow elements making it histologically indistinguishable from mature skeletal bone (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021755#pone-0021755-g005" target="_blank">Fig. 5B, 5D</a>). Scale bars = 50 µm.</p

Radiographic analyses of mice reveal multiple subcutaneous ossifications.

<p>X-rays of 12 month +/−p and WT mice. A) 12 month +/−p female with no ossifications visualized; B) 12 month +/−p male, inset and arrows demonstrate areas consistent with ossifications; C) 12 month WT without areas of ossifications.</p

The dermis and subcutis of 3 month old male mice with heterozygous targeted disruption of exon 1 of the <i>Gnas</i> gene.

<p>The −m/+ and +/−p male and female mice had no heterotopic bone formation. However, there were subtle lesions in the dermis of both −m/+ and +/−p male mice that comprised widely scattered plaque-like areas with pale collagen and increased cellularity in periadnexal areas of the reticular dermis. A) −m/+ male; B) +/−p male; Scale bar = 50 µm.</p

The ossifications appear to originate in the perifollicular areas.

<p>Ossifications are emanating near the hair follicle both with hematoxylin and eosin staining (A) as well as Alizarin Red staining (B). Scale bar = 50 µm.</p

Analysis of human subcutaneous ossification.

<p>A) Surgically removed area of ossification from a 20 year old male AHO (−p/+) patient with a documented <i>GNAS</i> mutation. The site contained multiple irregular firm masses 0.5–4 cm in length with branching extensions from a central mass. B) Ossified lesion in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021755#pone-0021755-g001" target="_blank">Figure 1A</a> stained with hematoxylin and eosin. The lesion is composed of mature bone with frequent central bone marrow elements. Scale bar = 50 µm.</p

Analyses of subcutaneous ossifications in mice.

<p>A) feet of a <i>Gnas</i> −m/+ male compared to WT (wild type) male at 12 months of age; B) ear of a <i>Gnas</i> −m/+ male at 12 months of age with nodular subcutaneous ossifications; C) X-ray of <i>Gnas</i> −m/+ male at 3 months of age showing occasional subcutaneous ossifications; D) X-ray of 12 month −m/+ obese male with extensive subcutaneous ossifications.</p

Whole body computerized tomographic (CT) images reveal subcutaneous ossifications.

<p>CT scans of 12 month (A–D) and 3 month (E, F) male and female mice with heterozygous targeted disruption of exon 1 in the <i>Gnas</i> gene. Arrows indicate a subset of the subcutaneous ossifications. A) male −m/+,. B) female −m/+. C) male +/−p. D) female +/−p. E) male −m/+. F) female −m/+.</p

<i>In situ</i> hybridization analysis of dermal heterotopic bone using probes to markers of osteogenesis.

<p>Nonisotopically labelled osteonectin-specific and osteopontin-specific probes hybridized with cells located in and directly adjacent to foci of dermal heterotopic bone in the mice (no counterstain). Scale bars A, B, C = 50 µm; D, E, F = 10 µm.</p

Radiographic Imaging of Subcutaneous Ossifications Over Time.

<p>*1+ = 1–3 foci.</p><p>2+ = 4–10 foci.</p><p>3+ = 11–20 foci.</p><p>4+ = >20 foci.</p