Toxicity of heparin in postimplantation whole-embryo culture

Cranial neural tube defects occur when heparin is added to the culture media of postimplantation rat embryos undergoing organogenesis in vitro. Timed-exposure studies were undertaken to determine whether the defects caused by heparin were the result of defective folding and fusion of the neural folds, or due to reopening of a closed neural tube. Experiments were also undertaken to elucidate whether the in vitro toxicity of heparin was due to an effect of heparin at the level of the culture medium, at the level of the visceral yolk sac, or at the level of the embryo proper. Heparin was found to cause defective folding and closure of the neural folds at between 9.5 and 10.5 days' gestation. Neural tube defects did not occur when embryos were cultured in media prepared using a culture medium depleted of heparin ligands by heparin-agarose affinity chromatography. However, studies with [G-(3)H]heparin demonstrated visceral yolk sac uptake and transfer of the radiolabel to the embryo proper. In addition, microinjection of heparin directly into the amniotic cavity of early head-fold embryo explants resulted in cranial neural tube defects similar to those caused by the addition of heparin to culture medium. These data indicate that heparin causes closure defects of cranial neurulation, primarily by an effect at the tissue level of the embryo proper.