Pigment cell pattern formation in Taricha torosa: The role of the extracellular matrix in controlling pigment cell migration and differentiation

Abstract

The neural crest is a population of highly migratory mesenchymal cells that ultimately localize in specific sites and differentiate into a variety of cell types. This report describes studies on the factors governing the migratory pathways, differentiation, and ultimate localization of the neural crest-derived pigment cells (black melanophores and yellow xanthophores) in the California newt, Taricha torosa. Melanophores first appear scattered in the dorsal portion of the lateral neural crest migratory pathway (between the somites and the ectoderm). These cells are eventually found in two stripes: a dorsal stripe that runs along the apex of the somites, and a midbody stripe near the somite-lateral plate mesoderm border. Melanophores are not seen in the dorsal fin of prehatching embryos. Xanthophores can be identified with the light microscope using NH 4OH-induced autofluorescence of pteridines and in the transmission electron microscope (TEM) by the presence of pterinosomes. Xanthophores first appear scattered among the melanophores over the surface of the somites; these cells eventually are found between the two melanophore stripes and in the dorsal fin. We were interested in determining the roles of the extracellular matrix (ECM) in controlling the formation of pigment cell patterns in T. torosa. Immunocytochemistry, Alcian blue staining of paraffin sections and ruthenium red staining of thin sections (accompanied by Streptomyces hyaluronidase and chondroitinase ABC digestion) were used to identify the composition and distribution of the ECM surrounding the pigment cells at various stages during development. The adhesive glycoprotein fibronectin is found in the dorsal portion of the lateral neural crest migratory pathway as well as in the dorsal fin matrix. Glycosaminoglycans (GAG) are found primarily in the dorsal fin and in the ECM surrounding the notochord. The dorsal fin ECM contains hyaluronate (HA), which was identified in the TEM as Streptomyces hyaluronidase-sensitive 3–5 nm microfibrils, as well as sulfated proteoglycan aggregates. We then confronted T. torosa neural crest cells in vitro with known ECM molecules. When neural folds are explanted onto tissue culture plastic in half-strength L-15 medium containing 10% fetal calf serum (FCS), cells migrate from the explant and differentiate into melanophores after 6 to 9 days. Xanthophores appear in the cultures 2 to 4 days after the appearance of melanophores. When cultured on three-dimensional collagen gels, xanthophores migrate significantly farther ( P < 0.01) onto and into the collagen than melanophores (336 ± 183 vs 196 ± 160 μm from the edge of the explant). When 2.5 mg/ml chondroitin sulfate (CS) is present in the collagen gel, the distance that both pigment cell types migrate from the explant is reduced, with the result being that only xanthophores invade the GAG-rich matrix. When 1 mg/ml HA is present in the collagen gel, the differentiation of pigment cells is inhibited. Melanophores appear 48 hr later than in control gels without HA, and the number of melanophores in the explant after 10 days is significantly reduced ( P < 0.01; 26.6 vs 1.1 melanophores/explant). When 1 mg/ml of HA is added to the FCS-enriched medium over neural crest cells spreading on tissue culture plastic, there is a similar delay and inhibition of pigment cell differentiation. With 2 mg/ml of CS there is no effect on pigment cell differentiation in vitro. Melanophores eventually appear in the dorsal fin of T. torosa several weeks after hatching. When fragments of dorsal fin that contain no apparent melanophores are transferred onto tissue culture plastic, melanophores appear in the explants after a few days in culture. These results suggest the following model of ECM-cell interactions during pigment cell pattern formation in T. torosa: Pigment cells differentiate in regions of the embryo that contain relatively little GAG. Xanthophores are able to invade the GAG-rich dorsal fin, but melanophores can not. The melanophores that eventually appear in the dorsal fin are derived from the neural crest cells that invaded the fin during early development, and were delayed in differentiating by the presence of HA.