ANALYSIS OF TRANSCRIPTIONAL ISOFORMS OF COLLAGEN TYPE-IX, TYPE-II, AND TYPE-I IN THE DEVELOPING AVIAN CORNEA BY COMPETITIVE POLYMERASE CHAIN-REACTION

The genes for the alpha 1(IX), alpha 1(II), and alpha 2(I) collagen ch ains can give rise to different isoforms of mRNA, generated by alterna tive promotor usage [for alpha 1(IX) and alpha 2(I)] or alternative sp licing [for alpha 1(II)]. In this study, we employed competitive rever se transcriptase PCR to quantitate the amounts of transcriptional isof orms for these genes in the embryonic avian cornea from its inception (about 3 1/2 days of development) to 11 days. In order to compare valu es at different time points, the results were normalized to those obta ined for the ''housekeeping'' enzyme, glycerol-3-phosphate dehydrogena se (G3PDH). These values were compared to those obtained from other ti ssues (anterior optic cup and cartilage) that synthesize different com binations of the collagen isoforms. We found that, in the cornea, tran scripts from the upstream promotor of alpha 1(IX) collagen (termed ''l ong IX'') were predominant at stage 18-20 (about 3 1/2 days), but then fell rapidly, and remained at a low level. By 5 days (just before str omal swelling) the major mRNA isoform of alpha 1(IX) was from the down stream promotor (termed ''short IX''). The relative amount of transcri pt for the short form of type IX collagen rose to a peak at about 6 da ys of development, and then declined. Throughout this period, the pred ominant transcriptional isoform of the collagen type II gene was IIA ( i.e., containing the alternatively spliced exon 2). This indicates tha t the molecules of type II collagen that are assembled into heterotypi c fibrils with type I collagen possess, at least transiently, an amino -terminal globular domain similar to that found in collagen types I, I II, and V. For type I, the ''bone/tendon'' mRNA isoform of the alpha 2 (I) collagen gene was predominant; transcripts from the downstream pro motor were at basal levels. In other tissues expressing collagen types IX and II, long IX was expressed predominantly with the IIA form in t he anterior optic cup at stage 22/23; in 14 1/2 day cartilage, long IX was expressed predominantly along with the IIB form of alpha 1(II). T he downstream transcript of the alpha 2(I) gene (I-cart) was found at high levels only in cartilage. (C) 1995 Wiley-Liss, Inc.