A gradation of hyaluronate accumulation along the proximodistal axis of the embryonic chick limb bud

We are currently investigating the mechanism by which the apical ectodermal ridge (AER) of the embryonic chick limb bud exerts its negative effect on the cytodifferentiation of limb mesenchymal cells directly subjacent to it, and the mechanism by which cytodifferentiation is triggered when the cells leave the influence of the AER. Since there is a gradation of differentiation along the proximodistal axis of the limb bud, we have dissected limb buds into discrete segments along the proximodistal axis, and biochemically examined the accumulation of hyaluronate (HA) and other glycosaminoglycans (GAG) in each segment. The unspeciajized subridge region of stage-25 limb buds was separated into distal (segment 1) and proximal (segment 2) regions, and the remaining proximal portion of the limb was separated into four segments (segments 3, 4, 5 and 6) along the proximodistal axis. Stage-24 limb buds were separated into corresponding regions. Since in the proximal regions of the limb (segment 3 through 6), only the cells comprising the central cores of the limb are involved in chondrogenic differentiation, the central core tissue was surgically separated from the peripheral tissue. We have found that HA is by far the predominant GAG accumulated by cells comprising the distal subridge region (representing greater than 50% of the total GAG accumulated during a 90-min labelling period with [3H]glucosamine), and that there is a progressive decline in HA accumulation along the proximodistal axis. The relative and total amount of HA accumulated is highest in the distal subridge region (segment 1), intermediate i(i the proximal subridge region (segment 2) and lowest in the proximal central core regions Of the limb (segments 3 through 6). The striking decrease in HA accumulation in the central core of segment 3 is accompanied by a striking increase in the accumulation of chondroitin sulphate, one of the major constituents of cartilage matrix. In contrast to the central core regions of segments 3 through 6, the relative and total amount of HA accumulated by the peripheral non-chondrogenic regions of these segments remains relatively high, being similar to the accumulation observed in the proximal subridge region. These results indicate that there is a gradation of HA accumulation along the proximodistal axis of both stages-24 and -25 limb buds which correlates with distance of cells from the AER and the state of differentiation of the cells.