Histochem Cell Biol (2006) 126:713–722 DOI 10.1007/s00418-006-0203-4 123 ORIGINAL PAPER Spatiotemporal distribution of heparan sulfate epitopes during murine cartilage growth plate development Ronald R. Gomes Jr · Toin H. Van Kuppevelt · Mary C. Farach-Carson · Daniel D. Carson Accepted: 16 May 2006 / Published online: 12 July 2006  Springer-Verlag 2006 Abstract Heparan sulfate proteoglycans (HSPGs) are abundant in the pericellular matrix of both devel- oping and mature cartilage. Increasing evidence sug- gests the action of numerous chondroregulatory molecules depends on HSPGs. In addition to speciWc functions attributed to their core protein, the complex- ity of heparan sulfate (HS) synthesis provides extraor- dinary structural and functional heterogeneity. Understanding the interactions of chondroregulatory molecules with HSPGs and their subsequent outcomes has been limited by the absence of a detailed analysis of HS species in cartilage. In this study, we characterize the distribution and variety of HS species in developing cartilage of normal mice. Cryo-sections of femur and tibia from normal mouse embryos were evaluated using immunostaining techniques. A panel of unique phage display antibodies speciWc to particular HS species were employed and visualized with secondary antibodies conjugated to Alexa-Xuor dyes. Confocal microscopy demonstrates that HS species are dynamic structures within developing growth plate cartilage and the perichondrium. GlcNS6S-IdoUA2S-GlcNS6S spe- cies are down regulated and localization of GlcNS6S- IdoUA-GlcNS6S species within the hypertrophic zone of the growth plate is lost during normal development. Regional diVerences in HS structures are present within developing growth plates, implying that interac- tions with and responses to HS-binding proteins also may display regional specialization. Keywords Glycosaminoglycan · Heparan sulfate proteoglycan · Chondrogenesis · Growth plate Introduction The chondrocyte is the resident cell of cartilage, a prominent tissue in the embryo that serves as a tem- plate for development of skeletal elements. At each stage along its diVerentiation continuum, the chondro- cyte expresses and secretes a distinct inventory of col- lagens, proteoglycans and other extracellular matrix (ECM) molecules (Karsenty and Wagner 2002). Thus, the cartilage cell resides in a vast and increasingly com- plex environment, which it depends upon for survival, proliferation, diVerentiation, and function. Proteoglycans are proteins that have a glycosamino- glycan (GAG) chain covalently attached to a core pro- tein. In the case of heparan sulfate proteoglycans (HSPGs), the GAG moiety consists of heparan sulfate (HS), an unbranched chain of 30–150 disaccharides. Each HS disaccharide unit, consisting of a glucosamine and an uronic acid residue, can be modiWed by N- deacetylation, N-sulfation, 2-O, 3-O, and/or 6-O sulf- ation, and C-5 epimerization. Not surprisingly, the totality of these modiWcations potentially lends HS to assume great structural and functional diversity. In recent years the importance of HS as both morphogen R. R. Gomes Jr · M. C. Farach-Carson · D. D. Carson (&) Department of Biological Sciences, University of Delaware Newark, Newark, DE 19716, USA e-mail: dcarson@udel.edu T. H. Van Kuppevelt Department of Biochemistry, University Medical Center, NCMLS, Nijmegen, The Netherlands Present address: R. R. Gomes Jr Department of Orthopaedics and Rehabilitation, Penn State College of Medicine Hershey, Hershey, PA 17033, USA