The adult cuticle of C. 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 morphology of the struts is defective in
bli-1 and
bli-2 mutants. The Bli phenotype is dependent upon the animal having an adult cuticle. Analysis of a temperature-sensitive allele of
bli-2 showed that its function is necessary in the late L4 stage, the time during which the adult cuticle is synthesized. In addition, mRNAs for these 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. Transformation rescue was used to show that
bli-1 and
bli-2 correspond to sequences C09G5.6 and F59E12.b. The
bli-2 gene encodes a standard cuticle collagen, while
bli-1 encodes an unusual cuticle collagen. The length of triple-helical domain in these collagens is sufficient to span the medial layer. The amino- and carboxy-terminal domains of BLI-1 are roughly 400 amino acids each, approximately 10 times larger than the corresponding domains in most cuticle collagens, and they contain large proportions of proline and charged residues. Although no known motifs implicated in protein interactions are evident in these terminal domains, the large size and number of charged residues suggest that BLI-1 may connect the layers flanking the medial layer by interacting with other cuticle components. The medial layer of
bli-1 and
bli-2 mutants is filled by granular electron dense material or contains morphologically abnormal struts. The secretory hypodermis of these mutants can vary in width and can also contain abnormal, large membrane bound structures. The other layers of
bli-1 and
bli-2 adult cuticles are thinner than in wild type animals, indicating that other cuticle components may not be secreted efficiently. Similar secretory defects caused by incorrectly processed collagens have been noted previously in vertebrates and for collagen type IV in C. elegans. Other evidence indicates that secretory defects may exist in mutant worms due to incorrectly processed collagens. These mutants can have subtle abnormalities in the alae and annular furrows. These are not the primary defects of mutations in
bli-1 and
bli-2, and they may simply reflect a need to simultaneously secrete interacting proteins to aid the assembly of a complex extracellular structure like the cuticle. Blistered animals can have 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. Healing may occur by the fusion of the external and basal cuticular layers. Mutations in
bli-1 and
bli-2 exhibit genetic interactions with the other four blister genes. In addition, we have TEM evidence that the medial layer is defective in animals mutant in all six blister genes. These data lead us to believe that these genes may constitute the proteins necessary for constructing struts, meaning that this system is a valuable one for understanding the assembly and maintenance of a discrete extracellular structure.