Worm Breeder's Gazette 13(2): 82 (February 1, 1994)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
The alpha-2(IV) collagen gene let-2 is alternatively spliced, generating transcripts that contain either exon 9 or exon 10, but not both. These exons encode very similar products, differing primarily in the sequence of a 9 -10 amino acid Gly-X-Y interruption, viz: We have shown that the expression of these alternatively spliced transcripts is developmentally regulated (Sibley et al., 1993, J Cell Biol 123 255-264). In embryos over 90% of the alpha-2(IV) collagen mRNA contains exon 9, while larval and adult RNAs contain 80-90% exon 10. We are currently studying developmental changes in the expression of exons 9 and 10 at the protein level, using polyclonal anti-peptide antibodies raised in rabbits. For each exon, the peptides used for antibody production contain the interruption sequence and 1-2 Gly-X-Y repeats. Both anti-peptide antibodies react against a 200 kD band on Western blots, which is approximately the size of a single alpha-2(IV) collagen chain. We are using these antibodies in immunofluoresent staining of embryos and adults, and report here some preliminary results of these experiments.
Embryos show staining with the anti-exon 9 Ab beginning just before the lima stage of embryogenesis. Staining first appears intracellularly in presumptive body wall muscle cells, and this Ab continues to show staining in muscle cells throughout embryogenesis. Starting at the comma stage, staining with the anti-exon 9 Ab begins to show characteristics of a basement membrane, in that staining appears sheetlike around the pharynx and intestine. By the 2-fold stage, staining around the gonad also becomes evident. In adults, there is quite a different pattern of staining with the anti-exon 9 Ab, in that staining is seen only overlying body wall muscle cells; there is no staining of pharynx, intestine or gonad in adults. The adult exon 9 muscle staining appears to perfectly align with myo I staining, forming two stripes with no staining where myo A or the I band are localized.
In embryos, anti-exon 10 Ab first shows staining at the 1.5-fold stage. Anti-exon 10 Ab stains muscle cells intracellularly, in a pattern similar to the anti-exon 9 Ab at this stage, and continues to show muscle staining throughout the remainder of embryogenesis. The anti-exon 10 Ab does not stain pharynx, intestine or gonad in embryos. Results of staining adults with the anti-exon 10 Ab are as yet in progress.
At this point, it appears that muscle cells may be a major site of synthesis of both forms of alpha-2(IV) collagen. Collagen needed in the synthesis of basement membrane around the pharynx, intestine, hypodermis, and gonad may come from body wall muscle cells. The delayed initiation of anti-exon 10 Ab staining is consistent with our RNA results, in which the relative level of exon 10-containing transcripts does not significantly increase until early L1 (<1 hour after hatching). The alignment of exon 9-containing collagen and myo B is interesting, but its significance is as yet unclear.