Anthony A. Capehart

Versican knock-down compromises chondrogenesis in the embryonic chick limb.

Mesenchymal cell aggregation is critical for cartilage formation in the vertebrate limb. The extracellular matrix (ECM) plays a critical role in governing cell behavior and cell phenotype in this tissue, and the hyalectin versican is highly expressed in the ECM of precartilage mesenchymal cells and developing synovial joints. Although several in vitro studies have been conducted in an attempt to address versicans role during limb mesenchymal condensation, factors such as differences in cell density in culture, variations between chondrogenic cell lines, and the inability to prolong the viability of limb explants have led to conflicting data, mandating an in vivo analysis. By using a morpholino directed strategy in ovo, we performed knock‐down of versican expression in the presumptive ulnar region of the developing chick wing at time points critical to skeletogenesis. These data indicate that in ovo misexpression of versican compromised mesenchymal condensation with resulting ulnar cartilages reduced in length distally by an average of 53% relative to contralateral control limbs. In select versican morphants the olecranon process was also reduced in size proximally and failed to cup the humerus, likely impairing joint morphogenesis. This study represents the first report assessing the role of versican in the developing chick limb in ovo, further demonstrating the importance of versican proteoglycan expression during chondrogenesis and extending previous findings to suggest a role for versican during synovial joint development. Anat Rec, 291:19–27, 2007.

Production of a Monoclonal Antibody by In Vitro Immunization That Recognizes a Native Chondroitin Sulfate Epitope in the Embryonic Chick Limb and Heart

We report the production of a monoclonal antibody (d1C4) by in vitro immunization that has immunoreactivity with a native chondroitin sulfate epitope in embryonic chick limb and heart. Murine lymphocytes were stimulated by direct exposure to unfixed, unsolubilized precartilage mesenchymal aggregates in high-density micromass culture derived from Stage 22–23 chick limb buds. Specificity of d1C4 reactivity was demonstrated by sensitivity of immunohistochemical staining to pretreatment with chondroitinase ABC or AC, preferential immunoreactivity with chondroitin-6-sulfate glycosaminoglycan (CS-C GAG) in ELISA, and competition of immunohistochemical staining with CS-C GAG. Immunohistochemical analysis of the expression of the d1C4 epitope revealed a striking localization of immunoreactivity in the extracellular matrix (ECM) of precartilage aggregates of chick limb mesenchyme in high-density micromass culture by 16 hr and the prechondrogenic limb core at Stage 23 in vivo. Immunoreactivity in both cultured limb mesenchyme and the embryonic limb continued through differentiation of prechondrogenic condensations into cartilage tissue. In the developing chick heart, d1C4 staining was found throughout the ECM of atrioventricular cushion tissue by Stage 25, but was localized to mesenchyme adjacent to the myocardium in the outflow tract cushions. There was an abrupt demarcation between d1C4-reactive intracardiac mesenchyme and unreactive extracardiac mesenchyme of the dorsal mesocardium in the Stage 22 embryo. This study demonstrates the efficacy of in vitro immunization of lymphocytes for the production of MAbs to native ECM constituents, such as CS-GAGs. Immunohistochemical data utilizing d1C4 suggest that CS-GAGs bearing this epitope may be important in early morphogenetic events leading to cartilage differentiation in the limb and valvuloseptal morphogenesis in the heart. (J Histochem Cytochem 45:1567–1581, 1997)

Proteolytic Cleavage of Versican During Limb Joint Development

Versican is highly expressed in developing joint interzones during limb morphogenesis. This study was undertaken to examine whether proteolytic cleavage of versican occurs that could potentially impact its function during the process of embryonic synovial joint formation. Using an antibody to the DPEAAE neoepitope generated by ADAMTS proteolysis, versican amino terminal cleavage fragments were detected in joint interzones at 12–16 days post coitum (dpc). ADAMTS‐1 localization overlapped that of DPEAAE‐reactive versican fragments suggesting it as one possible protease activity involved in processing of versican in the interzone. Results show that increased cleavage of versican in the interzone accompanies cavitation and suggests that proteolytic modification of versican may be important during the process of synovial joint maturation. Anat Rec, 293:208–214, 2010.

Inhibition of prostaglandin synthesis reduces cyclic AMP levels and inhibits chondrogenesis in cultured chick limb mesenchyme

The present study investigated effects of inhibiting the synthesis of prostaglandins (PGs) on cyclic AMP concentrations and chondrogenesis in cultured chick limb mesenchyme. Indomethacin produced concentration-dependent inhibition of both PGE(2) synthesis and chondrogenesis over a concentration range of 50--200 microM. Half maximal inhibition of PGE(2) was achieved with 50 microM concentrations of the drug which also produced visibly reduced amounts of cartilage matrix in cell cultures as evaluated by Alcian green staining on day 6 of culture. The inhibitory effects of indomethacin on chondrogenesis were largely reversed by addition of 1 mM dibutyryl cAMP, indicating that cells could still respond to cyclic AMP stimulation. Endogenous levels of cyclic AMP, which increased by 6 fold during the six days of culture in control cells, did not increase significantly from dissociated cells at the time of plating (day 0) in indomethacin- treated cultures. The results indicate that inhibition of the prechondrogenic rise in PGE(2) concentrations in limb mesenchyme prevents the increase in cyclic AMP levels which occur during this same period resulting in inhibition of chondrogenesis. The data provide further support for the hypothesis that PGE(2), through its effects on the adenylate cyclase-cAMP system, plays an important role in the differentiation of cartilage.

Versican G1 domain and V3 isoform overexpression results in increased chondrogenesis in the developing chick limb in ovo.

Previous work has shown that versican proteoglycan is highly expressed in the extracellular matrix of precartilage limb mesenchyme. Although much of versicans role in chondrogenesis has been attributed to its glycosaminoglycan complement, N‐ and C‐terminal G1 and G3 domains of versican have been shown to possess distinct functions when expressed ectopically. This study was undertaken to test the hypothesis that overexpression of the versican G1 domain and short V3 isoform, comprised of only G1 and G3, in the chick wing in ovo would result in increased chondrogenesis, suggesting function for discrete versican domains in limb skeletal development. Recombinant adenoviruses encoding G1 and V3 proteins were microinjected into proximal HH19–25 chick wing buds which resulted in significant enlargement of humeral primordia at HH35. Enhanced cartilage deposition appeared due to increased chondrogenic aggregation as a result of recombinant G1 or V3 overexpression, further implicating versican in early stages of limb development. Anat Rec 293:1669–1678, 2010.

Versican knockdown reduces interzone area during early stages of chick synovial joint development.

Much has been learned regarding factors that specify joint placement, but less is known regarding how these molecular instructions are translated into functional joint tissues. Previous studies have shown that the matrix chondroitin sulfate proteoglycan, versican, exhibits a similar pattern of expression in the embryonic joint rudiment of chick and mouse suggesting conserved function during joint development. In this study, versicans importance in developing joints was investigated by specific inhibition of its expression in the early joint interzone, tissue that gives rise to articular cartilages and joint cavity. In ovo microinjection of adenoviral shRNA constructs into the HH25 chick wing was employed to silence endogenous versican protein in developing appendicular joints. Results showed statistically significant (12–14%) reduction of nonchondrogenic elbow joint interzone area in whole‐mount specimens at HH36 in response to versican knockdown. Attenuated expression of key versican‐associated molecules including hyaluronan, tenascin, CD44, and link protein was also noted by histochemical and immunohistochemical analysis. Versican knockdown also lowered collagen II expression in presumptive articular chondrocytes indicating possible delay in chondrogenesis. Results suggest that versican functions interactively with other matrix/cell surface molecules to facilitate establishment or maintenance of early joint interzone structure. Anat Rec, 2012.

Selective and transient expression of a native chondroitin sulfate epitope in Deiters' cells, pillar cells, and the developing tectorial membrane

The tectorial membrane (TM) is an acellular connective tissue overlying the sensory hair cells of the organ of Corti. Association of the tectorial membrane with the stereocilia of the sensory hair cells is necessary for proper auditory function. During development, the mature tectorial membrane is thought to arise by fusion of a “major” and “minor” tectorial membrane (Lim, Hear Res 1986;22:117–146). Several proteins and glycoconjugates have been detected in the developing TM; however, the specific molecules which mediate fusion of the two components of the TM have not been identified.

Identification of γA‐like protocadherin expressed during chick development

The protocadherins are calcium‐dependent cell adhesion molecules of the cadherin superfamily that have been described in numerous species. Although less well characterized than classical cadherins, the protocadherins are also thought to facilitate critical cell–cell interactions during embryonic development. We have cloned a novel protocadherin from the embryonic chick utilizing a monoclonal antibody produced against a peanut agglutinin‐binding fraction of cultured chick limb tissue to screen a λZAP cDNA expression library from the stage 25 limb. A 2.8 kb cDNA clone was obtained that encoded multiple cadherin‐like ectodomains. Northern blotting revealed a single 4.6 kb RNA transcript that was highly enriched in the stage 43 chick brain. Utilization of 3′ Rapid amplification of cDNA ends (RACE) identified the entire 2.4 kb reading frame. The chick protocadherin contained five cadherin‐like extracellular repeats and a highly conserved cytoplasmic domain. Amino acid alignment of the extracellular domains revealed marked identity to the human γA protocadherin subfamily. In situ hybridization showed low levels of mRNA localization in several developing chick tissues, but stronger expression in the neural tube and dorsal root ganglia at stage 27. In the stage 43 chick brain, protocadherin mRNA was noted in discrete regions, particularly within the developing optic lobe. As for protocadherins described in other species, these results suggest that this novel γA‐like protocadherin may also play a role in chick neural development.

Heterogeneity of chondroitin sulfate glycosaminoglycan localization during early development of the striped bass (Morone saxatilis).

Recent studies have suggested important functions for proteoglycan‐associated chondroitin sulfate glycosaminoglycans (GAGs) during embryonic and larval development in numerous organisms, including the teleost. Little is known, however, about the specific distribution of different chondroitin sulfate GAGs during early development. The present study utilized immunohistochemistry to localize chondroitin sulfate GAG antigens during development of the striped bass (Morone saxatilis). Immunoreagents utilized were monoclonal antibodies (MAbs) TC2, d1C4, and CS‐56, which recognize, respectively, native epitopes on glycosaminoglycan chains enriched in chondroitin‐4‐, chondroitin‐6‐, and both chondroitin‐4‐ and ‐6‐sulfate. Little or no immunoreactivity was observed in gastrulating embryos at 18 hr postfertilization with any MAb tested. By 24 hr (8 somites), the CS‐56 epitope was localized around the notochord. At hatching (48 hr) and early larval (72 hr) stages, d1C4 and CS‐56 antigens codistributed in some sites (e.g., the notochord and myosepta), but a striking heterogeneity of chondroitin sulfate GAG localization was observed in other developing tissues, including the eye and specific subsets of basement membrane. At these latter time points, TC2 reacted primarily with the extracellular matrix of the developing heart, particularly the ventricular and conotruncal segments. Heterogeneous patterning of these chondroitin sulfate GAG epitopes suggests dynamic regulation of proteoglycan function during critical morphogenetic events in early development of the striped bass. Anat Rec 268:47–58, 2002.

Heterogeneous Expression of Chondroitin Sulfate Glycosaminoglycans and Versican Proteoglycan during Early Development of Rathke’s Pouch

While much is known regarding morphogenetic factors involved in specification and differentiation of Rathke’s pouch, less attention has been given to extracellular matrix (ECM) interactions involved in its formation. The present research investigated localization of two different chondroitin sulfate glycosaminoglycans (CS-GAGs), TC2 and d1C4, and versican CS-proteoglycan (PG) to identify additional ECM molecules involved in formation of the pituitary rudiment. Immunohistochemical evaluation of anterior pituitary primordia between HH15 and HH28 showed these ECM molecules prevalent in basement membrane and surrounding ECM underlying Rathke’s epithelia and to a lesser extent between pouch epithelial cells. TC2/d1C4 CS-GAGs and versican showed changing and heterogeneous localization during pouch development that suggested specific roles in cell-ECM interaction during pituitary morphogenesis. TC2 antigen colocalized with versican at early stages in an asymmetric pattern, with particularly strong staining between ventral diencephalon and roof of Rathke’s pouch while d1C4 CS-GAG encompassed the entire pouch by HH22 indicating association with a different CSPG. The heparan sulfate proteoglycan, perlecan, used to verify basement membrane structure, was a consistent component of Rathke’s pouch. Data show a dynamic and heterogeneous pattern of CS-GAG and versican expression during early chick Rathke’s pouch development that suggests new possibilities for ECM function in its establishment and growth.