COLLAGEN TYPE-IX AND DEVELOPMENTALLY-REGULATED SWELLING OF THE AVIAN PRIMARY CORNEAL STROMA

A critical event in avian corneal development occurs when the acellula r primary stroma swells and becomes populated by mesenchymal cells tha t migrate from the periphery. These cells then deposit the mature stro mal matrix that exhibits the unique features necessary for corneal fun ction. Our previous work correlated the disappearance of collagen type IX immunoreactivity at stage 27 (51/2-6 days) with matrix swelling an d invasion, To investigate further the mechanism of this disappearance , we employed immunohistochemistry after tissue fixation with Histocho ice, a non-crosslinking fixative, immunoblot analysis of protein extra cts, and gel substrate chromatography (zymography) to detect endogenou s proteolytic activity. We found that corneas fixed in Histochoice ret ain immunoreactivity for type IX collagen for 1-2 days after corneal s welling. This immunoreactivity, however, becomes extractable from tiss ue sections of unfixed corneas at the time of initiation of stromal sw elling and mesenchymal cell invasion. Immunoblot analysis confirmed th at, following swelling, immunoreactivity for collagen IX decreased sub stantially in corneas, but not in the vitreous body, which served as a comparison. Analysis of ammonium sulfate (AS) fractions of such extra cts indicated that, at the time of swelling, much of the immunoreactiv ity for type IX collagen in cornea shifted from the AS precipitate (co ntaining high molecular weight molecules) to the AS supernatant (conta ining smaller fragments), In contrast, collagen IX immunoreactivity fr om the vitreous was precipitated by ammonium sulfate throughout the pe riod of study. Collagen type II, a major fibrillar collagen in both th e corneal stroma and vitreous, remained in the high molecular weight f raction at all times examined. Zymography detected the presence of the latent (proenzyme) form of gelatinase A (MMP-2) before corneal swelli ng and invasion (4 days), and both the latent and active forms of the enzyme after corneal swelling. This suggests tissue-specific, developm entally regulated proteolysis of collagen IX as a trigger for corneal matrix swelling. (C) 1998 Wiley-Liss, Inc.