First bone formation and the dissection of an osteogenic lineage in the embryonic chick tibia is revealed by monoclonal antibodies against osteoblasts

Abstract

Detailed studies of the origin and differentiation of osteogenic cells can be facilitated by cell-specific markers. To this end, we immunized mice with a heterogeneous population of chick embryonic bone cells and subsequently generated and selected for monoclonal antibodies against cell surface determinants. Supernatants from growing hybridoma colonies were screened immunohistochemically against frozen sections of embryonic stage 35 (day 9.5) chick tibiae. Three cell lines, SB-1, SB-2, and SB-3, which each secrete a different antibody against osteogenic cells, have been successfully cloned, stabilized, and immortalized. Antibody SB-1 reacts with a family of cells in embryonic bone, liver, kidney, and intestine, which are identically stained by the histochemical stain for alkaline phosphatase. The SB-2 antigen is present only on osteoblasts, while the SB-3 antigen is expressed on the surface of osteoblasts, ependymal cells and ventricular myoblasts. Studies on the developmental progression of osteoblasts in the embryonic tibia indicate that the determinants recognized by SB-1, SB-2, and SB-3 are temporally coupled to the appearance of the pre-osteoblast marker alkaline phosphatase. Detailed morphologic analyses reveal that SB-1 reacts with a large family of osteogenic cells residing between the surface of newly formed bone and the overlying periosteal osteoprogenitor cells. By contrast, SB-2 and SB-3 appear to react with those mature osteoblasts involved in the secretion and mineralization of osteoid. Cells which are imprisoned within bone matrix (osteocytes) of the developing tibia are not recognized by these antibodies, but are immunostained by an osteocyte-specific monoclonal antibody which does not react with SB-1, SB-2 or SB-3 positive cells. The results reported here suggest the existence of an osteogenic cell lineage which is characterized by a series of discrete cell states prior to the overt expression of the Secretory Osteoblastic phenotype. We propose that the emergence and abatement of phenotypically distinct osteogenic cell surface antigens follows a precise spatial and temporal sequence which reflects the position of cells within the osteogenic lineage.