Differential co-expression of long and short form type IX collagen transcripts during avian limb chondrogenesis in ovo

Using RNA blot analysis of developmentally staged avian limb buds, we demonstrate that transcripts of several cartilage marker genes appear in limb tissue prior to overt chondrogenesis. Type II collagen mRNA, cartilage proteoglycan core protein mRNA, alpha 2(IX) collagen mRNA, and transcripts of the short form alpha 1(IX) collagen chain derived from the downstream promoter are co-expressed in limb tissue approximately 24–36 hours before the appearance of the respective polypeptides in differentiating cartilagenous tissue. Transcripts of the long form alpha 1(IX) collagen chain derived from the upstream promoter appear somewhat later in development; nearly coincident with the immunolocalization of type IX collagen in the cartilage elements of the limb. The spatial distribution of type II and type IX collagen transcripts was analyzed by in situ hybridization. Type II collagen and the long form alpha 1(IX) collagen transcripts co-localized in the chondrogenic elements of the developing forelimb. In contrast, short form alpha 1(IX) collagen transcripts which lack the 5′ region encoding the NC4 globular amino-terminal domain were distributed throughout the non-chondrogenic, non-myogenic mesenchymal regions of the limb and were not detectable above background levels in the limb chondrogenic elements. The precocious appearance of several cartilage marker gene transcripts prior to chondrogenesis suggests that multiple levels of gene regulation including alternative promoter use, alternative RNA splicing, alternative polyadenylation, and other post-transcriptional as well as translational mechanisms are active prior to, and during avian limb chondrogenesis.