Sam68 Is Expressed in the Developing Skeleton of Embryonic Mice
The Sam68 mRNA is known to be widely expressed [38], whereas its pattern of protein expression in vivo remains to be defined. Sections of paraffin-embedded wild-type E14.5 and E16.5 embryonic mice were immunostained with the well-characterized AD1 anti-Sam68 antibody, raised in rabbits against an immunizing peptide that corresponds to amino acids 330 to 348 of the mouse Sam68 protein [46]. Sam68 immunoreactivity was observed in the skeleton and soft tissues of developing wild-type mice at E14.5 and E16.5 (Figure 1). Intense staining was seen in the nucleus of cells in the developing brain, heart, and small intestine (Figure 1B), as well as in chondrocytes in the nasal septum and the glandular tissue adjacent to the nasal cartilage (Figure 1C, panels A–D), in the vertebra and intervertebral discs (panels E–H) and in the epiphysis (panels I–K) and metaphysis (panel L) of long bones. Nuclear staining was observed in proliferating chondrocytes and hypertrophic chondrocytes (Figure 1C, panel H), in osteoblasts (panel L; Dataset S1), and in osteoclasts (Dataset S1). No staining was observed with preimmune serum or when the antiserum was preadsorbed with the immunizing peptide (Figure 1C, panels B, F, and J). Taken together, these data demonstrate that Sam68 protein is selectively expressed in the developing mouse embryo, with particularly elevated expression in cartilage and bone.
Figure 1  Immunohistochemical Localization of Sam68 in Embryonic Mice
(A) Embryonic mice were removed from pregnant dams at E14.5 and E16.5, fixed in 4% paraformaldehyde, and embedded in paraffin. The entire embryo was immunostained with the AD1 anti-Sam68 antibody and counterstained with methyl green, and the image was captured at ×1.2 magnification.
(B) Embryonic soft tissues from the brain, heart and gut were stained with hematoxylin (left) and immunostained with anti-Sam68 antibody (right), and images were captured at ×20 magnification.
(C) Intense anti-Sam68 immunoreactivity was seen in chondrocytes in the nasal septum (panels A–D), in developing vertebra (panels E–H), and in the femoral epiphysis (panels I–K), as well as in diaphyseal osteoblasts (panel L). Adjacent sections were stained with hematoxylin and eosin (panels A, E, and I) or with antibody preadsorbed with the immunizing peptide (panels B, F, and J). Sam68 was localized primarily in the nucleus of cells in a variety of tissues but was also found occasionally in the cytoplasm. Magnification at source ×20, except for panels D, H, and L, which were ×40. Staining patterns are representative of three to five embryos.