Temporal and spatial analysis of hyaluronidase activity during development of the embryonic chick limb bud

The glycosaminoglycan hyaluronate (HA) appears to play an important role in limb cartilage differentiation. The large amount of extracellular HA accumulated by prechondrogenic mesenchymal cells may prevent the cell-cell and/or cell-matrix interactions necessary to trigger chondrogenesis, and the removal of extracellular HA may be essential to initiate the crucial cellular condensation process that triggers cartilage differentiation. It has generally been assumed that HA turnover during chondrogenesis is controlled by the activity of the enzyme hyaluronidase (HAase). In the present study we have performed a temporal and spatial analysis of HAase activity during the progression of limb development and cartilage differentiation in vivo. We have separated embryonic chick wing buds at several stages of development into well-defined regions along the proximodistal axis in which cells are in different phases of differentiation, and we have examined HAase activity in each region. We have found that HAase activity is clearly detectable in undifferentiated wing buds at stage 18/19, which is shortly following the formation of a morphologically distinct limb bud rudiment, and remains relatively constant throughout subsequent stages of development through stage 27/28, at which time well-differentiated cartilage rudiments are present. Moreover, HAase activity in the prechondrogenic distal subridge regions of the limb at stages 22/23 and 25 is just as high as, or even slightly higher than, it is in proximal central core regions where condensation and cartilage differentiation are progressing. We have also found that limb bud HAase is active between pH 2.2 and 4.5 and is inactive above pH 5.0. This suggests that limb HAase is a lysosomal enzyme and that extracellular HA would have to be internalized to be degraded. These results indicate that the onset of chondrogenesis is not associated with the appearance or increase in activity of HAase. We suggest that possibility that HA turnover may be regulated by the binding and endocytosis of extracellular HA in preparation for its intracellular degradation by lysosomal HAase. Finally, we have found that the apical ectodermal ridge (AER)-containing distal limb bud ectoderm possesses a relatively high HAase activity. We suggest the possibility that a high HAase activity in the AER may ensure a rapid turnover and remodeling of the disorganized HA-rich basal lamina of the AER that might be essential for limb outgrowth.