Fig. 2

Temporal expression and splicing patterns of minor fibrillar collagens. RT-PCR was performed using RNA extracted from wild type embryos at 4, 6, 10, 16, 24, 36, 48, 72 hpf. (A) Multiple bands were observed when cDNA was amplified within the region between exons 5 and 9 in the case of Col11a1a on chr24, Col11a2 on chr19, and Col5a3 on chr3. Single amplification products were observed in the case of Col5a1 and Col11a1b on chr2. (B) Splicing patterns are depicted, reflecting the exon usage for those genes undergoing alternative splicing within the variable region. Boxes represent individual exons within the VR, whereas lines in between represent introns. Relative size of all elements depicted reflects relative actual size in basepairs. For Col11a1a on chr24, the predominant spliceform at the earliest stages examined in this study included exons 6A, 7 and 8 within the variable region. Other minor transcripts observed included exons 6A and 6B or 7 alone. For Col11a2, exons 6, 7 or 8 were observed to be either included or excluded in a complex pattern. An additional exon of approximately 1.2 kb was observed, replacing exons 6, 7 and 8 in the case of the largest alternatively spliced form. In the case of Col5a3, alternative splicing involved exon 7. (C). Each amplification product was excised and sequenced to confirm identity, intron–exon boundaries, and exon usage in comparison to genomic sequence currently available. Variable region exon sequences in C are color-coded to match the block diagram in (B).