Association of enhanced expression of gap junctions with in vitro chondrogenic differentiation of rat nasal septal cartilage-released cells following their dedifferentiation and redifferentiation

The nasal septum is an important centre of endochondral ossification during the development of the facial region. Previous studies have shown that it is possible to recapitulate the differentiation programme of 21-day-old rat nasal chondrocytes in vitro. The purpose now was to investigate, in vitro, the cell condensation phase that represents the earliest morphological event associated with cartilage differentiation in skeletal development. The study focuses on the ability of the cells to form condensations before overt differentiation, with special emphasis on gap-junction expression. The gap-junction protein connexin 43 was localized by indirect immunofluorescence as primarily intracellular and, on day 5, at the condensation stage, as spot-like contacts between cells. Intracellular injection of the permeable dye Lucifer yellow led to the staining of up to 20 neighbouring cells, indicating functional gap junctions and coupling. In contrast, treatment of cultures with the gap-junction blocker glycyrrhetinic acid inhibited dye coupling and reduced cartilage differentiation. Northern blotting of connexin 43 mRNA showed a faint band during the first days of culture, with a striking increase after day 4. In addition, the mRNA of the homeodomain-containing gene Cart-1 began to be expressed in prechondrogenic condensations and corresponded to the expression of type II collagen mRNA. These data indicate that the early stage of in vitro chondrocyte differentiation is the formation of cell condensations and the ability to establish cell-to-cell communication. Connexin 43, together with other molecular mechanisms, mediates the condensation phase of chondrogenesis and sets up the optimal environment in which nasal septal cells may terminally differentiate into chondrocytes.