Proteoglycans in neurulation

Glycosaminoglycans are made up of repeating disaccharide subunits consisting of a hexosamine and a hexose or hexuronic acid. They occur freely or as carbohydrate side chains in proteoglycans. Although widely distributed, their importance in embryogenesis is incompletely understood. This thesis investigates their roles in neurulation and neural crest migration. Primary neurulation occurs between E8.5 and E10.5 in the mouse embryo. Neural tube closure in the spinal region is accomplished by bending at the median hinge point and at paired dorsolateral hinge points. Histochemical and immunohistochemical studies show that sulphated glycosaminoglycans are present in the basement membrane of the closing neural tube, as well as elsewhere in the embryo. Culturing E8.5 CD1 mouse embryos in the presence of chlorate, a competitive inhibitor of glycosaminoglycan sulphation, suppressed median hinge point formation. This was associated with increased bending at the dorsolateral hinge points, resulting in accelerated posterior neuropore closure. Suppression of median hinge point formation was prevented by addition of heparan sulphate, but not chondroitin sulphate, de-N- or de-O-sulphated heparan sulphate, to the culture medium. This may be due to the requirement for heparan sulphate in Sonic hedgehog induction of median hinge point formation. Chondroitin sulphate also influenced posterior neuropore closure. Chondroitinase treatment of CD1 mouse embryos in culture retarded closure of the neuropore, whereas exogenous chondroitin sulphate accelerated closure. However, the median and dorsolateral hinge points were unaffected. The mechanism of action of chondroitin sulphate is unknown and requires further investigation. Besides their role in neurulation, chondroitin sulphate proteoglycans are known to inhibit cell migration. Splotch mouse mutants, where mutations in Pax-3 result in deficiencies of neural crest derived structures, have been shown by in situ hybridisation to over-express versican mRNA. To determine whether the glycosaminoglycan component was increased, chondroitin sulphate was quantified in E9.5 splotch embryos photospectroscopically using a 1,9-dimethylmethylene blue binding assay. It was found that mutants contained larger amounts of chondroitin sulphate than wild type embryos. On the other hand, there was no difference in the net synthetic rate, as determined by 35S-labelling of chondroitin sulphate in cultured splotch embryos. This suggests that the defect may lie in the mutants ability to degrade chondroitin sulphate, resulting in its accumulation and inhibition of neural crest migration. These experiments underline the importance of sulphated glycosaminoglycans in mouse development and raise the possibility that these molecules may contribute to development of neural tube defects and neurocristopathies in humans