Notochord of chick embryos secretes short‐form type IX collagen prior to the onset of vertebral chondrogenesis

Abstract

The notochord of embryonic chicks produces type IX collagen, as well as type II collagen, prior to the onset of vertebral chondrogenesis. To address the question of whether the notochord secretes the “long‐form” type IX collagen found in cartilage or the “short‐form” type IX found in the cornea and vitreous humor, we examined immunoreactivity of the notochordal type IX collagen using two different monoclonal antibodies. The antibody 2C2 recognizes an epitope close to the carboxyl‐terminus of the HMW fragment, which is present in both the long‐ and short‐form type IX collagens, whereas another antibody 4D6 recognizes an epitope in the NC4 domain of the long‐form type IX collagen, which is absent in the short‐form type IX collagen. Therefore, the long‐form is recognized by its reaction with both 2C2 and 4D6, while the short‐form by its reaction with only 2C2 and no reaction with 4D6. Immunostaining of vertebral sections with 2C2 shows an identical distribution of staining with that for type II collagen, although the staining with 2C2 is less intense. The 2C2‐reactive type IX collagen is found within the notochord at stage 14 and in the notochordal sheath at stage 20. Deposition of this collagen in the perinotochordal matrix increases with time and reaches a level comparable with that for type II at stage 31. In contrast, the 4D6‐reactive type IX collagen is not found within the notochord nor in the notochordal sheath. The collagen becomes detectable, however, in the perinotochordal matrix at stages 27 to 28 and is markedly increased at stage 29, reaching the levels for the 2C2‐reactive type IX at stage 31. A highly sensitive immuno‐dot blot assay has confirmed that the notochord from chick embryos at stages 19 to 20 produces type IX collagen that reacts with 2C2 but does not react with 4D6. The present study indicates (1) that the short‐form type IX collagen is secreted from the notochord and also from the sclerotome cells, and (2) that the switching in the production from the short‐form type IX collagen to the long‐form type IX occurs during stage 27 and stage 31 when sclerotome cells differentiate into chondrocytes in the developing vertebral body.