Retention and structure of extracellular matrix in early chick embryos after quick-freezing and freeze-substitution

Early chick embryos, stages 11 to 14, were isolated, quick-frozen by immersion in isopentane/propane cryogen (-185 degrees C) and freeze-substituted for study by scanning electron microscopy. Emphasis was placed on the extracellular matrix (ECM) in the axial region of the segmental plate and developing somites. Ultrarapid freezing, followed by delicate freeze-substitution, immobilizes and retains much more ECM than chemical fixatives that include tannic acid (TA). The matrix on the dorsal surface of the neural tube is preserved as delicate filaments which are expressed bilaterally over the tube in a dorso-ventral orientation. These parallel primary ridges of ECM have a spacing of 1 to 3 micron, forming grooves on the wall of the neural tube. Interrupting this pattern are funnel-shaped ridges about 80 to 100 micron apart along the neural tube. The ridges become decorated with cross-bridges creating a dense lattice in the region of somite development, to the extent that a basal lamina composed of dense fibrillar network and amorphous mats of matrix accumulates on the lateral wall of the neural tube. Heavy strands and fenestrated lamellae of ECM interconnect the neural tube, notochord and somites, and attach the overlying epithelium to the upper surface of the somites. The pattern of ECM is complimentary to the migratory pathways of ventrally migrating neural crest cells and is the basis for suggesting that a physical substratum influencing the direction of neural crest cell migration is an idea that should be revived.