Immunohistochemical demonstration of hyaluronan and its possible involvement in axolotl neural crest cell migration

Hyaluronan (HA), an extracellular matrix component, is involved mainly in t he control of cell proliferation, neural crest and tumor cell migration, an d wound repair. We investigated the effect of hyaluronan on neural crest (N C) cell migration and its ultrastructural localization in dark (wild-type) and white mutant embryos of the Mexican axolotl (Ambystoma mexicanum, Amphi bia). The axolotl system is an accepted model far studying mechanims of NC cell migration. Using a biotinylated hyaluronan binding protein (HABP), maj or extracellular matrix (ECM) spaces, including those of NC cell migration, reacted equally positive on cryosections through dark and white embryos. S ince neural crest-derived pigment cells migrate only in subepidermal spaces of dark embryos, HA does not seem to influence crest cell migration in viv o. However, when tested on different alternating substrates in vitro, migra ting NC cells in dark and white embryos prefer HA to fibronectin. In vivo, such an HA migration stimulating effect might exist as well, but be counter acted to differing degrees in dark and white embryos. The ultrastructural l ocalization of HA was studied by means of transmission electron microscopic immunohistochemistry using HABP and different protocols of standard chemic al fixation, cryofixation, embedding, and immunolabeling. The binding react ion of HA to HABP was strong and showed an equal distribution throughout EC M spaces after both standard chemical fixation/freeze substitution and cryo fixation. A preference for the somite or subepidermal side was not observed . Following standard fixation/freeze substitution HABP-labeled "honeycomb"- like networks reminiscent of fixation artifacts were more prominent than la beled fibrillar or irregular net-like structures. The latter predominated i n adequately frozen specimens following high-pressure freezing/freeze subst itution. For this reason fibrillar or irregular net-like structures very li kely represent hyaluronan in the complex subepidermal matrix of the axolotl embryo in its native arrangement. (C) 2000 Academic Press.