Abstract
Changes in glycosaminoglycan (GAG) content and distribution are vital for joint development. However, their precise character has not been established. We have used immunohistochemistry (IHC) and "critical electrolyte" Alcian blue staining to assess such changes in developing chick and rabbit joints. IHC showed chondroitin sulfate labeling in chick epiphyseal cartilage but not in interzones. In contrast, prominent labeling for keratan sulfate (KS) was restricted to chick cartilage-interzone interfaces. In rabbit knees, KS labeling was also prominent at presumptive cavity borders, but weak in interzone and cartilage. Selective pre-digestion produced appropriate loss of label and undersulfated KS was undetectable. Quantification of Alcian blue staining by scanning and integrating microdensitometry showed prominent hyaluronan-like (HA-like) interzone staining, with chondroitin sulfate and weaker KS staining restricted to epiphyseal cartilage. Hyaluronidase decreased HA-like staining in the interzone. Surprisingly, keratanases also reduced HA-like but not sulfated GAG (sGAG-like) staining in the interzone. Chondroitinase ABC had little effect on HA-like staining but decreased sGAG staining in all regions. Rabbit joints also showed HA-like but not KS staining in the interzone and strong chondroitin sulfate-like staining in epiphyseal cartilage. Our findings show restricted KS distribution in the region close to the presumptive joint cavity of developing chick and rabbit joints. Alcian blue staining does not detect this moiety. Therefore, it appears that although histochemistry allows relatively insensitive quantitative assessment of GAGs, IHC increases these detection limits. This is particularly evident for KS, which exhibits immunolabeling patterns in joints from different species that is consistent with a conserved functional role in chondrogenesis.
Highlights
SUMMARY Changes in glycosaminoglycan (GAG) content and distribution are vital for joint development
Rabbit joints showed HA-like but not keratan sulfate (KS) staining in the interzone and strong chondroitin sulfate-like staining in epiphyseal cartilage
Our findings show restricted KS distribution in the region close to the presumptive joint cavity of developing chick and rabbit joints
Summary
Embryonic White Leghorn chick hind limbs from Hamburger and Hamilton stages 39 and 41 (at least three chicks at each stage) were mounted on cork, covered with 10% PVA (grade GO4/140; Waker Chemicals, Surrey, UK) and immersed in n-hexane (grade low in aromatic hydrocarbons; BDH, Poole, UK) cooled to Ϫ70C. Serial longitudinal cryostat sections were cut at 10 m using a cabinet temperature of Ϫ30C and were stored at Ϫ70C until used. Hind limbs distal to the mid-femoral region were removed from New Zealand White rabbit fetuses aged 17, 20, and 25 days (at least two rabbits at each stage). Sections of both chick and rabbit joints were stained with 0.1% Toluidine blue in 0.1 M acetate buffer at pH 6.2 or with hematoxylin and eosin
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