HB-GAM/pleiotrophin: localization of mRNA and protein in the chicken developing leg.

The heparin-binding growth-associated molecule HB-GAM (also named pleiotrophin) is a developmentally-regulated protein that belongs to a new family of heparin-binding molecules with putative functions during cell growth and differentiation. In order to study the localization of HB-GAM during chicken embryogenesis, we produced specific monoclonal antibodies to this factor. HB-GAM protein is first observed at stage 23 in the developing nervous system and later in the forming cartilage. We present an investigation of the HB-GAM mRNA expression and HB-GAM protein distribution in the developing leg by in situ hybridization and immunocytochemical studies. We focused our attention on the development of the tibia, where the HB-GAM protein appears at stage 27-28, i.e., just after the condensation of the mesodermal precursor cells of the chondrocytes. The protein then progressively accumulates in the central part of the embryonic cartilage (diaphysis). It persists until stage 42-44 in the regions where hypertrophic cartilage is being replaced by bone marrow. In contrast to the protein, the transcript is first detected at stage 26-27 and later expressed essentially in the epiphysis until stage 37. Therefore the localization of the mRNA does not parallel that of the protein and our data suggest a long half-life of the protein in the hypertrophic cartilage. In addition, the layer of stacked cells surrounding the cartilage core (usually considered as the osteoprogenitor cells) clearly expresses the HB-GAM message between stages 30-37 whereas differentiated osteoblasts do not. Furthermore, the distribution of HB-GAM protein in the osteoblast/osteoid layer suggests an involvement of this protein in early steps of osteogenesis. HB-GAM is absent from the newly formed bone.