The development of proline-containing extracellular connective tissue fibrils by chick notochordal epithelium in vitro.

Notochords were isolated from Hamburger-Hamilton stages 13-15 chick embryos by trypsinization and microdissection. These were shown by electron microscopy to be completely devoid of extracellular materials or mesenchymal contaminants. Cultivation of notochordal isolates was carried out on a non-collagenous (Falcon Plastic) substratum for 0 to 48 hours. At 12 hours of in vitro incubation, a discontinuous basal lamina could be demonstrated on the surface of notochordal cells. This was followed by the appearance of microfibrils of various sizes and other components of the extracellular matrix. By 48 hours of in vitro incubation, the same extracellular materials which surround the notochord in vivo (notochord sheath) could be demonstrated in vitro. Autoradiographic studies show that tritiated proline is taken up by notochordal cells and secreted to the extracellular space where label is associated with basal lamina, microfibrils and ground substance. When cis-hydroxyproline, a known collagen-specific inhibitor is added to the system, tritiated proline label is located primarily intracellularly and fewer areas of active fibrillogenesis are noted. This suggests that ultrastructurally recognizable materials produced by notochordal cells in vitro may be at least partially collagenous. Significantly, these materials are produced in vivo at the same time (following stage 10) that notochordal tissues actively induce somite differentiation and cartilage formation. It seems reasonable that a biochemically or ultrastructurally identifiable component of the extracellular matrix may possibly mediate such induction.