Matrix remodeling associated 5 expression in trunk and limb during avian development.

Matrix remodeling associated 5 (MXRA5) is an extracellular protein that is upregulated in several cancers, but little is known regarding its spatial and temporal localization in the developing embryo. The present study was undertaken to investigate MXRA5 transcript expression in the trunk and limb of the embryonic chick to provide groundwork for future investigation of its developmental function. In situ hybridization utilizing digoxigenin-labeled sense control and experimental antisense probes was performed in paraffin sections of chick embryos from Hamburger and Hamilton (HH) stages 18-38. MXRA5 expression was initially low and restricted in extent at the earliest stages examined, but expression increased in strength and tissue distribution with developmental age. Transcripts were largely found in cells of mesodermal origin, including gut associated mesenchyme, tendon and ligament rudiments, intervertebral discs, dermal papillae of feather buds, heart valve precursors and leaflets, as well as in appendicular joint primordia. The present study has provided initial information on MXRA5 gene expression in the trunk and limb of early to mid-stage avian embryos. Results show that MXRA5 was expressed most strongly at sites undergoing change and remodeling of the extracellular matrix during transition of embryonic tissues into the functional adult morphology.

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, versican's 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.

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 versican's 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.