Developmental restriction of embryonic calvarial cell populations as characterized by their in vitro potential for chondrogenic differentiation.

Abstract

The mechanism(s) by which the cells within the calvaria tissue are restricted into the osteogenic versus the chondrogenic lineage during intramembranous bone formation were examined. Cells were obtained from 12-day chicken embryo calvariae after tissue condensation, but before extensive osteogenic differentiation, and from 17-day embryo calvariae when osteogenesis is well progressed. Only cell populations from the younger embryos showed chondrogenic differentiation as characterized by the expression of collagen type II. The chondrocytes underwent a temporal progression of maturation and endochondral development, demonstrated by the expression of collagen type II B transcript and expression of collagen type X mRNA. Cell populations from both ages of embryos showed progressive osteogenic differentiation, based on the expression of osteopontin, bone sialoprotein, and osteocalcin mRNAs. Analysis using lineage markers for either chondrocytes or osteoblasts demonstrated that when the younger embryonic cultures were grown in conditions that were permissive for chondrogenesis, the number of chondrogenic cells increased from approximately 15 to approximately 50% of the population, while the number of osteogenic cells remained almost constant at approximately 35-40%. Pulse labeling of the cultures with BrdU showed selective labeling of the chondrogenic cells in comparison with the osteogenic cells. These data indicate that the developmental restriction of skeletal cells of the calvaria is not a result of positive selection for osteogenic differentiation but a negative selection against the progressive growth of chondrogenic cells in the absence of a permissive or inductive environment. These results further demonstrate that while extrinsic environmental factors can modulate the lineage progression of skeletal cells within the calvariae, there is a progressive restriction during embryogenesis in the number of cells within the calvaria with a chondrogenic potential. Finally, these data suggest that the loss of cells with chondrogenic potential from the calvaria may be related to the progressive limitation of the reparative capacity of the cranial bones.