Role of heparan sulfate in dextral heart looping in chick

Heparan sulfate (HS) has been shown to be involved in left-right asymmetry formation, including the process of dextral heart looping during embryonic development. The structural features of HS required in this process, however, have not been explored. In this study, we examined the structure of HS from the heart-forming regions (or heart fields) of Hamburger and Hamilton stage 5-9 chick embryos. No significant differences were found in HS to chondroitin sulfate (CS) ratio, HS chain length, or [35S] sulfate incorporation at HS disaccharide level between the left and the right heart fields. Compared to other parts of the embryo, however, lower ratio of HS to CS, shorter HS chain length, and higher [35S] sulfate incorporation at 6-O position of the glucosamine residue in the HS chains were observed in the heart-forming regions. Moreover, HS from the left and the right heart fields exhibit differential cleavage by heparanase, an endo-beta-d- glucuronidase that cleaves specific sequences within the HS chain. In embryo culture, microinjection of the active human heparanase enzyme into the right but not the left pericardial cavity at stage 7-8+ resulted in reversed heart looping in a dose-dependent manner. Heart reversal following microinjection of heparin or heparin derivatives suggests the involvement of N- and 6-O-sulfation but not 2-O-sulfation in the heart looping process.