Identification of glycosaminoglycans in the chondrocranium of the chick embryo before and at the onset of chondrogenesis

It appears that hyaluronate is associated with cell migration and the chondroitin sulphates with differentiation during morphogenesis of the chick embryo. The aim of this study was to see if such a correlation could be made for chondrocranium morphogenesis. Specifically, the purpose of this study was (1) to determine the proportion of extracellular matrix (ECM) to cell area and total head mesenchymal area during chondrocranium morphogenesis; and (2) to identify the location, types, and relative amounts of glycosaminoglycans (GAG) being synthesized in the presumptive chondrocranium at the onset of chondrogenesis and prior to this time. Morphometric analyses were made on median and parasagittal sections of heads of stage-24 and -33 embryos in order to determine relative contributions of cells and ECM to the total area of head mesenchyme at these stages. Presumptive chondrocrania (heads minus eyes) of these stage embryos were also analysed histochemically and biochemically in order to identify the GAGs present in the ECM. Sections of whole heads were stained with alcian blue at low and high pH as well as digested prior to staining with hyaluronidase ( Streptomyces and testicular). Identification of GAGs was done by pulse labelling embryos with [ 3 H]glucosamine, digesting homogenates with hyaluronidase ( Streptomyces or testicular), precipitating the undigested GAGs with cetylpyridinium chloride and countingthe dissolved precipitates using scintillation spectrophotometry. The types and relative amounts of GAGs present in the presumptive chondrocranium were determined by comparing the amount of radioactivity in the precipitates of the non-digested GAG with the counts in the precipitates of the predigested GAGs. This study reports that chondrogenesis begins in the presumptive chondrocranium of the chick embryo at stage 33 and that the area of the head mesenchyme increases 60-fold between stages 24 and 33. Little change in cell density and individual cell area as well as in the relative proportion of total area allocated to cells and ECM occurs. GAGs are localized exclusively in the presumptive chondrocranium. These GAGs are restricted to the ventral half of the presumptive chondrocranium. Within this region, the GAGs are further localized to the presumptive facial area, perichordal region, ethmoid, sphenoid and periotic regions. The types of GAG being synthesized in the head mesenchyme of both stage-24 and -33 embryos are hyaluronate, the chondroitins and unidentified sulphated GAGs (dermatan, keratan, heparin and heparan sulphate). At stage 24, hyaluronate, chondroitin and the unidentified sulphated GAGs constitute about 33% each of the GAG being synthesized. At stage 33, the level of hyaluronate synthesis drops to 2%, the chondroitins to 24% and the unidentified sulphated GAGs increase to 74%. There is an 18·5-fold decrease in the percentage of hyaluronate, a 1·5-fold decrease in the amount of chondroitins and a 2·7-fold increase in the percentage of unidentified sulphated GAGs being synthesized as chondrocranium morphogenesis proceeds.