Worm Breeder's Gazette 15(2): 33 (February 1, 1998)
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.
Department of Cell and Molecular Biology, Northwestern University Medical School, Chicago, IL 60611.
The adult cuticle of Caenorhabditis elegans consists of cortical and basal layers connected through a medial layer by struts. The medial layer and struts are unique to the adult cuticle. The struts are periodically placed within the cuticle, lying roughly in rows along either side of the annulae, oriented perpendicular to the plane in which the rest of the cuticle is arranged. The bli-1 and bli-2 genes have been physically and genetically mapped. Transformation rescue has been used to show that the bli-1 gene corresponds to sequence CO9G5.6, which encodes an unusual cuticle collagen. The amino- and carboxyl-terminal domains of this protein are roughly 400 amino acids, approximately 10 times larger than the corresponding domains in most cuticle collagens. These terminal domains contain large proportions of proline and charged residues. Although no known motifs or domains implicated in protein interactions are evident in these terminal domains, the large size and number of charges found in these domains imply that BLI-1 may connect the layers flanking the medial layer by interacting with other cuticle components. The mutant phenotype of bli-2 is rescued by cosmid C32E11, which contains an open reading frame encoding a more standard cuticle collagen. The mutant phenotype of these genes is cuticles covered by fluid-filled swellings. We have made double mutant combinations of bli-1 and bli-2 with several heterochronic genes and observed that such combinations yield blisters only when an adult cuticle is present. Using a temperature-sensitive allele of bli-2, we showed that the function of its product is necessary in the late L4 stage, the time during which the adult cuticle is synthesized. In addition, mRNAs for the bli-1 and bli-2 genes are expressed highly only during the L4 stage of development (B. D. Ackley and J. M. K., unpub.). These data together imply a defect specific to the adult cuticle. Transmission electron microscopy of bli-1 and bli-2 mutants shows that the struts are absent from the medial layer, which is filled by granular electron dense material or morphologically abnormal struts, implying that these proteins are involved in strut construction. The hypodermis of these mutants can vary in width and can also contain large membrane bound structures. These may be due to secretory defects caused by incorrectly processed collagens, as have been noted previously in vertebrates and for collagen type IV in the worm. Some bli-1 and bli-2 mutant animals have abnormalities in the three-pronged adult alae, such as four-pronged alae or gaps and small discontinuities in the treads. Because tread assembly is controlled by the lateral seam, we have examined its integrity. Animals mutant in bli-1 and bli-2 have normal numbers of seam cells that appear to fuse correctly. Some mutant animals also exhibit irregularities in the spacing of the annulae. It is thought that the circumferential actin bundles that arise in the hypodermis prior to each molt may aid in spacing these indentations. We have examined these actin bundles in animals undergoing the L4-adult molt; there do not appear to be any abnormalitites in bundle orientation or distribution in mutant animals. Blistered animals can have greatly shortened lifespans (3-4 days) and reproductive capabilities due to the severity of the blistering. We have observed that blisters can "heal" in a small percentage of animals, allowing them to live essentially a normal lifespan. Although the severity of the blistering phenotype differs between alleles, the frequency of healing events observed is about 10% in every case. We are unsure how this healing occurs, but it may involve the fusion of the cortical and basal layers. Mutations in bli-1 and bli-2 exhibit allele-specific, partially penetrant intergenic non-complementation, suggesting that their gene products may interact. We have also found similar interactions with the other Bli genes. In addition, we have TEM evidence that the medial layer is the layer affected in animals mutant in all of the Bli genes. These data lead us to believe that these genes may constitute the proteins necessary for constructing struts. Therefore, this system could be a valuable one for understanding the assembly and maintenance of a discrete extracellular structure.