Worm Breeder's Gazette 9(3): 106
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.
During a sabbatical year at the MRC Laboratory in Cambridge, England, I isolated mutants that affect formation of the copulatory structures of the adult male tail (mab), and studied the process of tail morphogenesis. The most obvious feature of the adult male tail is the cuticular fan with its nine rays. This structure, consisting of a large fold in the outer layer of the cuticle, is formed when the cells that inhabit the posterior region of the L4 larva retract just before the L4/adult molt (Sulston et al., Dev. Biol. 78, 542-576 (1980)). As the cells withdraw anteriorly, the outer layer of the newly-formed adult cuticle is left behind and eventually folds in a precise way to form the fan. Two questions arise concerning this process: what is the mechanism of cell withdrawal, and how is the cuticular folding accomplished in such a precise way, with reproducible and sharp boundaries? It can be seen, by comparison of the volume of the animal before ar_ after withdrawal of the posterior cells, that retraction must be accompanied by extrusion of a large amount of fluid from the body. This fluid passes into the growing space between the retreating cells and the L4 cuticle, and is under pressure. In order to see whether the pressure affected the morphogenetic movements, a hole was made in the L4 cuticle with the laser microbeam during the morphogenetic process. When the cuticle was punctured, the fluid escaped in a rush, and morphogenesis stopped. The partially retracted cells bulged back against the L4 cuticle. This result could mean that retraction of the posterior cells is driven by pressure between the L4 and adult cuticles. Alternatively, it might simply mean that the newly-forming adult cuticle is not yet strong enough to resist internal body pressure. To test this idea worms were observed in hypertonic medium ( e.g., 6% glucose), where pressure is reduced in both the space in the tail between the L4 and adult cuticles, and in the body generally, because of loss of water through the cuticle. In hypertonic medium, morphogenesis of the tail is also arrested. This result makes it appear that liquid pressure in the space between the adult and L4 cuticles is necessary per se for the morphogenetic movement. This pressure would act along with retraction caused by muscles in the tail, and possibly other cellular mechanisms, to mold the final shape of the adult body. As retraction proceeds, folding of the fan occurs against the taut L4 cuticle, which may act as a sort of scaffold or mold for the process. This may help to explain the preciseness of the shape of the fan, but it cannot account for its boundaries. Retraction of the body occurs well beyond the boundaries of the fan. Beyond the fan the inner and outer layers of the adult cuticle no longer separate. Instead, the outer cuticular layer adheres to the inner layer and the surface of the body. The outer cuticular layer and its behavior can be observed under Nomarski optics during the morphogenetic process. A critical aspect of fan formation must involve synthesis of a special type of cuticle, in which inner and outer layers are not joined, over the surface where the fan will eventually form during retraction. Electron micrographs of serial sections of males in the late L4 stage were examined to determine what cells might be responsible for making this special cuticle. The electron micrographs were made by Sulston and coworkers in their earlier studies of the male. Much of the tale hypodermis in the late L4 larva is made up from the posterior daughters of the ray precursor cells, Rn (n=1-9), which come to lie roughly underneath the region where the outer cuticular layer will separate to form the fan. Further analysis of electron micrographs, along with laser ablation experiments, are necessary to determine whether these and possibly other hypodermal cells are indeed responsible for making the fan. In genetic studies, nine new mab mutations were isolated by screening males in clones of a mutagenized him strain for abnormalities. One mutation is a new allele of the previously- identified gene mab-11, and the others are probably in new mab genes. Abnormalities affect various structures, from the spicules to the fan and rays, and ranged from subtle to severe. One severe mutant was examined in more detail, and surprisingly was found to undergo a nearly normal morphogenetic process, up until the time that the L4 cuticle was breached in molting. At this point, the normally-shaped tail bulged back out and spoiled the nearly-normal fan, in somewhat the same manner as occurred when the L4 cuticle was prematurely punctured with the laser. This gene therefore appears to encode a product required to hold the definitive fan in place. The same mutant is ts for alae formation in the adults of both sexes, and has severe cuticle defects in the L1 larva, which is osmotically sensitive. Therefore the protein may also be used to maintain alae ( present exclusively in L1's, dauers, and adults - dauers were not examined in these experiments). Alae, like the fan, consist of a cuticular fold, and are formed by seam cells. (The ray precursor cells, Rn, are descended from seam cells.) We propose that this mutation affects a cuticle component required to maintain the structure of both alae and fan, and may identify a component of a morphogenetic 'subprogram' used in making these two structures.