CHONDROGENESIS IN THE REGENERATING ANTLER TIP IN RED DEER - EXPRESSION OF COLLAGEN TYPE-I, TYPE-IIA, TYPE-IIB, AND TYPE-X DEMONSTRATED BY IN-SITU NUCLEIC-ACID HYBRIDIZATION AND IMMUNOCYTOCHEMISTRY

The annual regrowth of antlers in male deer is a unique example of com plete bone regeneration occurring in an adult animal. Growth is initia ted at the distal antler tip, which is similar to the epiphyseal growt h plate in some respects. However, there is some debate as to whether this process represents ''true'' endochondral ossification. As part of the characterization of the developmental process in pre-osseus antle r tissue, we have studied, by in situ hybridization, the spatial expre ssion of mRNAs for types I, II, and X collagen. Viewed in a coronal pl ane, type I procollagen mRNA was observed in skin, the fibrous pericho ndrium, and the densely cellular area immediately adjacent to the peri chondrium. Below this area, as cells began to assume a columnar arrang ement and coincident with the appearance of a vasculature and synthesi s of a cartilaginous matrix, transcripts for types I, IIA, IIB procoll agen and X collagen were detected. Further down in the cartilage zone, the pattern of type I procollagen mRNA expression was altered. Here, the signal was detected only in a morphologically distinct subpopulati on of small, flattened cells within the intercellular matrix at the pe riphery of the columns of chondrocytes. The alternative splice form of type II procollagen mRNA (IIA), characteristic of chondroprogenitor c ells (Sandell et al. [1998] J. Cell Biol. 114:1307-1319), was expresse d by a subset of cells in the upper region of the columns, indicating that this zone contains a population of prechondrocytic cells. Positiv e hybridization to type IIA was most abundant in these cells. In contr ast, transcripts for the other procollagen splice form (IIB) and type X collagen were expressed by chondrocytes throughout the whole of the cartilage region studied. The translation and export of type II collag en and type X collagen were confirmed by detecting specific immunoreac tivity for each. The spatial distribution of immunoreactivity for coll agen types II and X was consistent with that of corresponding mRNAs. T hese data demonstrate for the first time the distinct pattern of expre ssion of genes for major cartilage matrix macromolecules, the expressi on of the differentially spliced form of type II procollagen mRNA (IIA ), and specifically the colocalization of types II and X collagen in t he developing antler tip. Taken together, they strongly indicate that antler growth involves an endochondral process. (C) 1996 Wiley-Liss, I nc.