Worm Breeder's Gazette 11(4): 59
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.
Coordinate expression. We have examined expression of the sqt-1 and rol-6 collagen genes by Northern and slot blot analyses. Both genes are expressed at the L1, L2, L3, and L4 molts, but no transcripts are detectable in mixed-stage embryo RNA or L2d-dauer RNA. By quantitative slot blot analysis, the relative levels of sqt-1:rol-6 mRNAs are approximately 2:1 at each of the molts at which they are expressed. These results are consistent with the proposal, based on genetic interactions and structural similarity, that sqt-1 and rol-6 physically interact and suggest that they could be present in a 2:1 ratio within a single heterotrimeric collagen molecule. The rol-6 mRNA is trans-spliced with SL1, while sqt-1 has a cis-spliced intron in its 5'UTR; another example in which one of a pair of related collagen genes has switched its mode of splicing (the other is col- 12/13). If SL1 affects translation, then the sqt-1:rol-6 polypeptide ratio may not be 2:1, but would still be constant at all stages. Expression at the L2 stage. sqt-1 and rol-6 transcripts are produced during the L1-L2 molt at levels similar to those seen in later molts, but mutant animals had been reported not to show phenotypes at the L2 stage (Cox et al. Genetics 95:317, 1980). We find that at the L2 stage e1350/+, e187 and sv1006 animals are strong RRol; some e1350 animals have slightly abnormal tails and move abnormally; sc13 animals are indistinguishable from N2. Thus, sqt-1 and rol-6 expression at the L1 molt can produce the RRol phenotype, but apparently not the LRol phenotype. Possibly some components of the cuticle required to produce the LRol phenotype are not expressed until the L2-L3 molt. The dilemma of dauers sqt-1 and rol-6 mutant animals display the Rol phenotype as dauers, but we could not detect transcripts from either gene in two different preparations of L2d-dauer RNA. To substantiate these results, we isolated RNA from daf-2(e1368) animals at multiple time points throughout the L1-L2d-dauer period and probed slot blots with sqt-1, -specific collagen) specific probes. sqt-1 and rol-6 transcripts were detected at and immediately following the L1-L2d molt (16-22 hours after plating L1s at 25 C), but were undetectable at all later time points (26-54 hrs). In contrast, col-2 transcripts were first detected when animals began the L2d-dauer molt (30 hrs), peaked at 38 hrs., and were barely detectable when animals had completed the molt (54 hrs). Given the level of sensitivity of these experiments, the levels of the sqt-1 and rol-6 transcripts must be at least 50-100 fold lower at the L2d-dauer molt than at other molts. We examined sqt-1 and rol-6 mutant animals at the L2d and dauer stages. Similar to L2 stage animals, e1350/+, e187 and su1006 animals are strong RRol as dauers. sc13 and e1350 dauer animals display variable LRol or RRol phenotypes, respectively. The term variable is used to indicate that some animals show strong Rol phenotype, some are weak Rol, and some are not observed to roll. The L2d phenotypes for these animals are the same as their dauer phenotypes. Note that the sqt-1 alleles display different phenotypes at the L2 and L2d stages, directly demonstrating a functional difference between these cuticles. Inheritance of pattern? How can the apparent lack of gene expression during formation of the dauer cuticle be reconciled with the appearance of mutant phenotypes at that stage (recall that the null phenotypes for both sqt-1 and rol-6 are WT)? It is possible that there is a very low level of sqt-1 and rol-6 expression and that it is sufficient to cause a phenotype. This seems an unlikely explanation for these structural proteins, given that the expression is at least 50-fold less than at an equivalent stage (L2-L3). It is possible that sqt-1 and rol-6 collagens could be extracted from the L2d cuticle and reutilized for assembly of the dauer cuticle. The strongest argument against this possibility is that the collagens in the cuticle are crosslinked with di- and trityrosine residues (D. Eyre and J. Kramer, unpublished results). There is no know mechanism to sever these crosslinks, short of hydrolyzing the proteins. Another possibility is that dauers are rollers because the preceding L2d stage animals were rollers, i.e., the pattern is maintained from one stage to the next in the absence of expression of the mutant collagen. We favor this explanation based on the arguments that follow. At each molt a new cuticle is assembled beneath the old cuticle, so connections of the hypodermis and muscles to the cuticle must be broken during the molt. It seems likely that some sort of scaffold must form at this time to maintain organization in the absence of these cuticle attachments. Jim Priess showed that elongation of the embryo is dependent on bundles of microfilaments and microtubules circumferentially oriented in the hypodermis and that these filaments also appear to be involved in formation of the L1 cuticle (Priess and Hirsh Dev. Biol. 117:156, 1986). When the L1 cuticle has formed the filaments disappear. He has found that at each molt the actin filaments reform, and could act as the postulated scaffold. When we stained sc13 and e187 animals with TRITC-phalloidin, the hypodermal syncitia and seam cells were seen to remain helically twisted throughout the entire L2d-dauer molting period. Bundles of circumferential actin filaments were seen in the hypodermal syncitia, while seam cells stained diffusely. The filaments are oriented perpendicular to the seam-syncitium junction and appear to attach to the belt desmosomes. This cytoskeletal arrangement presumably prevents the hypodermis from changing its organization, and locks in the helical structure present before the molt began. Since the dauer cuticle is synthesized by a helically twisted hypodermis it may not require mutant collagen to generate its twist. Thus, dauers inherit the pattern that existed in the L2d stage animal. This proposal predicts that the dauer phenotype must be the same as the L2d phenotype, and this is true for the sqt-1 and rol-6 mutants we have examined. It may also explain why, for certain morphological mutants, adults derived from dauers have a different phenotype than adults derived from L3s. The persistent dumpiness induced by levamisole treatment (Lewis et al. 95:905,1980) could also be explained as maintenance of the hypercontracted morphology by the hypodermal filament system. If this proposal is correct, the phenotypes of some morphological mutants could be a result of the combined effects of the morphology at previous stages and the composition of the current cuticle. [See Figure 1]