Different pattern of alkaline phosphatase, osteopontin, and osteocalcin expression in developing rat bone visualized by in situ hybridization

TLDR

Alkaline phosphatase, osteopontin, and osteocalcin are expressed during osteoblastic differentiation, but prior studies indicate differences in their timing and spatial expression. This study employed in situ hybridization to map the distribution of these osteoblastic markers during rat bone development. Frozen sections of neonatal rat long bones and calvariae were hybridized with 35S‑labeled antisense RNA probes for AP, OP, and OC, with sense probes and RNase pretreatment as controls. High osteopontin and osteocalcin expression was confined to osteoblasts adjacent to bone, whereas alkaline phosphatase was expressed at lower levels in many cells, including preosteoblasts distant from the bone surface, indicating that AP is an earlier differentiation marker than OP and OC during endochondral and membranous bone formation.

Abstract

Abstract Alkaline phosphatase (AP), osteopontin (OP), and osteocalcin (OC) are expressed during osteoblastic differentiation. However, previous studies suggested differences in the timing and possibly the site of their expression. In this study we used in situ hybridization to follow the distribution of these osteoblastic markers during bone development. Frozen sections of neonatal rat long bones and calvariae were hybridized with 35S-labeled RNA probes complementary to the AP, OP, and OC mRNAs. Controls included sections hybridized with the sense (mRNA) probes or pretreated with RNase. Positive cells were identified in all areas of bone formation of the long bones and calvariae. Based on quantitative silver grain distribution and density, high levels of OP expression were present only in osteoblasts in close proximity to bone (one to two cell layers). OC expression, apparently at lower levels than OP, was also localized to osteoblasts in contact with bone. In contrast AP, which was expressed at lower levels than OP, was present in a large number of cells, including preosteoblasts that were many layers removed from the bone-forming surface. These findings are consistent with the asynchronous expression of phenotypically related genes and suggest that AP is an earlier differentiation marker than OP and OC during the formation of endochondral and membranous bone.