Structural analysis of extracellular matrix prior to the migration of cephalic neural crest cells.

Cephalic neural crest cells enter cell free areas containing abundant extracellular matrix (ECM). Previous histochemical studies have identified both sulfated and non-sulfated glycosaminoglycans within this matrix. In the present study, ultrastructural examination of the ECM demonstrated an anastomosing network of pleomorphic, cetyl pyridinium chloride-dependent strands within cell free spaces and in association with the basement membrane of the surface ectoderm. Thin section analysis revealed that the strands consisted of three components: (1) 3-5 nm filament meshwork; (2) electron dense amorphous material and (3) 30 nm granules. In contrast, the ECM associated with the basement membrane consisted principally of a continuum of electron dense, amorphous material. The molecular ordering of ECM within crest cell pathways was compared to the well-characterized, hyaluronate-rich, premigratory matrix of cardiac jelly.

A new technique for the rapid screening and selection of large pieces of tissue for ultrastructural evaluation.

A simple and rapid technique is described for the screening of Epon embedded organ slices for the location, isolation, and removal of small specific sites for ultrastructural study with the transmission electron microscope. This procedure consists of perfusion fixation followed by making 1 to 21/2 mm thick slices of relatively large pieces of the organs, control of the degree and evenness of the osmium staining by addition of 3% sodium iodate, and infiltration with a fluorescent dye prior to embedment in Epon. Tissue slices are embedded in wafer-shaped blocks, generally with several slices in one "wafer", and are examined in a controlled manner using a rapid form of serial surface polishing. Each level of the polished wafer is examined using an epi-illuminated fluorescence microscope, and selected sites are chosen at each level for ultrastructural study. Methods are also described for marking each selected site using a conventional slide marker, and for the removal of the selected site in the form of a small disc of Epon, after which the Epon wafer can be further serially polished and the examination continued. Areas to be thin-sectioned are removed using a core drill mounted on a model-maker's drill press. The technique is simple, does not require the destruction of remaining tissues to evaluate more critically a single small site, allows for the easy maintenance of tissue orientation, and the most time-consuming portions of the technique can be quickly taught to a person with no previous histological training.

A histochemical analysis of polyanoinic compounds found in the extracellular matrix encountered by migrating cephalic neural crest cells.

Neural crest cells destined to form craniofacial primordia initially are "seeded" into and subsequently migrate through the extracellular matrix (ECM) of a cell free space (CFS) between the surface ectoderm and the underlying mesoderm. Utilizing histochemical procedures for polyanionic compounds, we have demonstrated that both sulfated and nonsulfated glycosaminoglycans (GAG) are present in the CFS of the cephalic region of the chick embryo and that their distribution and structural organization vary with the passage of neural crest or mesodermally derived (MD) mesenchymal cells through it. In stages 7 and 8 embryos a predominance of fine filamentous strands composed primarily on nonsulfated, carboxyl-rich GAG is seen spanning intercellular spaces between adjacent tissues and MD mesenchymal cells. In older embryos (stages 9 and 10) much of the filamentous material is replaced by coarse fibrillar strands or amorphous material which coats the surfaces of MD mesenchymal and neural crest cells as they invade the CFS. Using enzymatic digestions (Streptomyces and testicular hyaluronidase) and the critical electrolyte concentration procedure, data suggest that the fine filamentous matrix onto which the neural crest cells migrate consists mainly of hyaluronate with lesser amounts of chondroitin and some sulfated GAG present. The coarse fibrillar matrix that appears after passage of either neural crest or MD mesenchymal cells through the original CFS contains strongly sulfated polyanionic material, predominantly chondroitin sulfates A, C. Since GAG is located ubiquitously within the ECM of embryos at various stages, the role of GAG, if any, in the transfer of developmental information may be of a general nature (ie. stimulus of motility) rather than of specific morphogenetic cues (for specific differentiation into craniofacial primordia).