DIVIDE, ACCUMULATE, DIFFERENTIATE - CELL CONDENSATION IN SKELETAL DEVELOPMENT REVISITED

Cell condensation is a pivotal stage in skeletal development. Although prechondrogenic condensations normally exist for some 12 h, duration can vary. Variation is seen both between condensations for different c artilages (Meckel's vs. elastic ear cartilage) and within a single con densation from which more than one skeletal element will form, as in t he three components of the single first arch chondrogenic condensation . Understanding how duration of the condensation phase is established - how the condensation phase is entered and exited during cell differe ntiation - remains a major area for future study. During chondrogenesi s, cell-specific products such as collagen types II and IX and cartila ge proteoglycan appear concomitant with condensation. Therefore, durin g chondrogenesis, condensation precedes commitment of cells as prechon droblasts. During osteogenesis, however, differentiation of preosteobl asts precedes condensation. Therefore, during osteogenesis, condensati on amplifies the number of committed osteogenic cells. Further compara tive analysis of skeletogenesis should provide us with a more rigorous understanding of cell commitment, when differentiation is initiated, how commitment and differentiation are measured and the relationship o f condensation to onset of differentiation. Current knowledge of molec ules characteristic of condensations focused attention on extracellula r matrix and cell surface components on the one hand, and on growth fa ctors homeobox genes and transcription factors on the other. We have d rawn together the molecular data for pre-chondrogenic condensations in diagrammatic form in Figure 2. Three major phases of chondrogenesis a re identified: (a) epithelial-mesenchymal interactions that precede co ndensation, (b) Condensation itself, and (c) cell differentiation. Alt hough we label the third phase differentiation, it is important to rec ognize that phases a and b also constitute aspects of chondroblast cel l differentiation (see Dunlop and Hall, 1995 for a discussion of this point. The pre-condensation phase is characterized by expression of Ho x genes, growth factors (TGF-beta and BMP-2) and the cell surface prot eoglycan receptor, syndecan-1. Expression of Msx-1 and Msx-2, growth f actors and syndecan continues into the condensation phase. Other molec ules, such as versican, syndecan-3 and tenascin, present in low concen trations before condensation, are up-regulated during condensation. Ye t other molecules - Hox genes, transcription factors, growth factors ( activin, BMP-4 and -5, GDF-5), cell adhesion molecules and proteoglyca ns - are only expressed during the condensation phase, while the trans cription factor Pax-1, fibronectin, hyaluronan and hyaladherin are exp ressed both during and after condensation. During condensation mRNAs f or collagen types II and IX and for the core protein of cartilage prot eoglycan are up-regulated. Late in condensation and increasingly there after, the protein products of these genes accumulate as chondroblasts differentiate (see Fig. 2 for details). Not all the molecules present before, during of after condensation can be placed into causal sequen ces. Some however can. In Figure 3 we summarize the causal sequences d iscussed in this paper as they relate to initiation of condensation an d to transit from condensation to overt differentiation during chondro genesis. Condensations form following activation of at least three pat hways: (1) Initiation of epithelial-mesenchymal interactions by tenasc in, BMP-2, TGF beta-1 and Msx-1 and -2. (2) Up-regulation of N-CAM by activin. (3) Up-regulation of fibronectin by TGF-beta, further enhanci ng N-CAM accumulation (Fig. 3). It is by these three pathways that con densations are initiated and grow. Transition from condensation to ove rt cell differentiation is under both positive and negative control (F ig. 3). Syndecan blocks fibronectin and so blocks N-CAM accumulation, preventing accumulation of additional cells to the condensation. By bl ocking condensation, syndecan enhances differentiation. BMP-2, -4 and -5, Hox genes and Msx-1 and -2 act directly on condensed cells to init iate differentiation. Clearly, many steps still have to be elucidated. These include further elaboration of relationships between the molecu les summarized in Figure 3 and already known to play roles in condensa tion or differentiation. How other molecules shown in Figure 2 (Pax-1, Barx-1, Ck-erg, Cart-1) fit into Figure 3 has to be determined. And d oubtless, there are other molecules/pathways to be identified. Neverth eless, our knowledge of the importance and regulation of condensations and of the molecules that are involved when condensation formation is perturbed has advanced enormously over the last three years. Talpid, Brachypod and Short Ear mutations are three cases in point. Over-expre ssion of N-CAM, a frameshift mutation in GDF-5 and a mutation in BMP-5 respectively, have been shown to perturb skeletal development by acti ng at the condensation phase. We look forward with eager anticipation to the next triennium.