Distinct spatial and temporal distributions of aggrecan and versican in the embryonic chick heart

Abstract

Although chondroitin sulfate proteoglycans (CSPGs) are major components of the embryonic extracellular matrix, little attention has been paid to specific CSPGs in early heart development, in part because appropriate antibodies were not available. Therefore we prepared specific polyclonal antibodies against chicken aggrecan, versican, neurocan, and phosphacan. Western blotting and immunohistochemical studies revealed the presence of aggrecan and versican in stages 12-21 chicken embryo hearts in distinctive spatial and temporal patterns. Because this is the first demonstration of aggrecan in heart tissue, we further used RT-PCR to confirm that aggrecan is expressed in the heart and in situ hybridization to confirm the pattern of expression determined using antibodies. Versican is found in the myocardium and the myocardial basement membrane. In contrast, aggrecan is specifically colocalized with several groups of migrating cells including endocardial cushion tissue cells, epicardial cells, a mesenchymal cell population in the outflow tract that may be of neural crest origin, and a mesenchymal cell population in the inflow tract. The combined observations indicate that versican and aggrecan are expressed in unique patterns and suggest that they play very different roles in development.

Versican proteolysis mediates myocardial regression during outflow tract development

“…Cardiac neural crest cells are at least one cell type in the developing OFT known to express MMP-2 (Cai et al, 2000). In the chick, versican expression is similar to the mouse, including a heterogeneity that the authors attribute to ECM proteolysis and led to the hypothesis that versi- can proteolysis was occurring during OFT development and may be initiated by the migration of cardiac neural crest cells (Capehart et al, 1999;Zanin et al, 1999). The observed correlation between versican cleavage and loss of the distal myocardium in the truncus raised the possibility that proteolytic cleavage of versican may not only inactivate versican activity but also generate fragments with activities distinct from the intact versican molecule.…”

“…Versican is an extracellular matrix (ECM) chondroitin sulfate proteoglycan with abundant expression in several regions of the developing heart (Henderson and Copp, 1998;Mjaatvedt et al, 1998;Zanin et al, 1999). All four versican variants V 0 , V 1 , V 2 , and V 3 are present throughout cardiac development Zako et al, 1995;Kern et al, 2006).…”

Immunolocalization of chick periostin protein in the developing heart

“…The peptide was synthesized with an additional C-residue at the N-terminus to facilitate its attachment to Sulfolink Coupling Gel (Pierce, Rockford, IL). The injection of rabbits, the purification of total IgG, and the affinity purification of IgG using the peptide attached to Sulfolink Coupling Gel were all performed as previously described (Zanin et al, 1999).…”

“…Tissues to be analyzed were homogenized in at least five volumes of 2% SDS/25 mM Tris (pH 8.0) containing a cocktail of protease inhibitors (Zanin et al, 1999) and sonicated for 30 sec using a Fisher sonic dismembrator Model 300. The homogenates were then clarified by centrifugation at 14,000 g for 5 min and their protein concentration was determined using the Micro BCA Protein Assay (Pierce).…”

“…The gel was transblotted and the transfer incubated overnight with primary antibody (0.15 g/ml). Secondary antibodies and detection procedures were as previously described (Zanin et al, 1999), using enhanced chemiluminescence to detect HRP-conjugated secondary antibody. For lane 4 of Figure 1, the primary antibody was preincubated with 0.5 g/ml of the immunogenic peptide for 1 hr at room temperature before use on Western blots.…”