EVIDENCE FOR SEQUENTIAL APPEARANCE OF CARTILAGE MATRIX PROTEINS IN DEVELOPING MOUSE LIMBS AND IN CULTURES OF MOUSE MESENCHYMAL CELLS

The initiation of synthesis and the accumulation of four cartilage matrix proteins (type II collagen and three noncollagenous proteins, one of Mr 148, one of Mr 59, and an oligometric protein of Mr above 500 with 100-kDa subunits, respectively) were studied in developing mouse limbs and in cultures of limb bud mesenchyme by means of immunolocalization. On day 13 of gestation, type II collagen was observed throughtout the entire humerus, whereas the 148-kDa protein was localized only in the central portion. Neither the 100-kDa-subunit protein nor the 59-kDa protein could be demonstrated in the humerus at that stage. On day 14 1/2, type II collagen and the 148-kDa protein were codistributed throughout the humerus. The 100-kDa-subunit protein was detectable in the periphery of the humerus, whereas little 59-kDa protein could yet be demonstrated. On day 18, all four proteins being studied could by detected immunologically in the developing mouse humerus. They differed in immunolocalization. Type II collagen, the 148-kDa protein, and the 100-kDa-subunit protein were codistributed throughout the distal and proximal parts of the cartilage. However, the 148-kDa protein could no longer be detected immunochemically in the outermost part of the cartilage in the proximal shoulder joint. The 148-kDa protein codistributed with type II collagen and the 100-kDa-subunit protein in the distal cartilaginous region, where joint development was less advanced. On the other hand, the 59-kDa protein was not demonstrated directly within the hyaline cartilaginous structures, but surrounded the entire structure. This protein was also present in the same part of the proximal joint region as that in which the 148-kDa protein was not detected. To develop an in vitro model for studies of skeletogenesis, mesenchymal cells prepared from mouse limb buds were cultured as micromass cultures at high initial cell density to favor chondrogenesis. On day 3 of culture, type II collagen was the only protein that could be detected immunochemically in the cultures, whereas on day 6 the 148-kDa protein was demonstrated, and a few chondrocytes in the central portion of each cartilaginous nodule were associated with the 100-kDa-subunit protein. The 59-kDa protein could not yet be immunochemically detected. Only day 14, type II collagen, the 148-kDa protein, and the 100-kDa-subunit protein could be detected throughout the cartilaginous nodules, whereas the 59-kDa protein was distributed in tissues surrounding each nodule. On day 55, the distribution of the four cartilage matrix proteins was the same on day 14, except that the 59-kDa protein was also demonstrated near some of the chondrocytes. In conclusion, the four cartilage matrix proteins being studied appeared in sequence. The order of appearance in the developing mouse humerus was identical to that in cultures of mesenchymal cells from the early mouse limb bud. These results indicate that the mesenchymal cell culture system can be used as an in vitro model for studies for skeletogenesis.