248 Journal o[ Nematology, Volume 14, No. 2, April 1982 17. Kimble, J., and D. Hirsh. 1979. The post- 1980. The Caenorhabditis elegans male: postem- emhryonic cell lineages of the hermaphrodite and brynnic development of nongonadal structures. male gonads in Caenorhabditis elegans. Develop. Develop. Biol. 78:542-576. Biol. 70:396-417. 27. Sulston, J., and J. Hodgkin. 1979. A diet of 18. Kimble, J., and J. White. 1981. On the con- worms. Nature 279:758-759. lrol of germ cell developmenl in Caenorhabditis 28. Sulston, J., and R. Horvitz. 1977. Postem- elegans. Develop. Biol. 81:208-219. hryonic cell lineages of the nematode Caenorhabditis 19. Krieg, C., T. Cole, U. Deppe, E. Schieren- elegans. Develop. Biol. 56:110-156. berg, D. Schmin, B. Yodel and G. yon Ehrenstein. 29. Sulston, J., and R. Horvitz. 1981. Ahnormal 1978. The cellular anatomy of embryos of the nema- cell lineages in mutants of the nematode Caenorhah- tode Caenorhabditis elegans. Analysis and recon- ditis elegans. Develop. Biol. 82:41-55. struction of serial section electron micrographs. 30. Sulston, J., and J. White. 1980. Regulation Develop. Biol. 65:193-215. and cell atttonomy during postembryonic develop- 20. Luc, M. 1981. Observations on some Xi- ment of Caenorhabditis elegans. Develop. Biol. 78: phinema species with the female anterior branch 577-597. reduced or ahsent (Nematoda: tongidoridae). Revue 31. Triantaphyllou, A., and H. Hirschmann. Nematol. 4:157-167. 1980. Cytogenetics and morphology in relation to 21. Nigon, V. 1965. Development et reproduction evolution and speciation of plant-parasitic nema- des nematodes. In P. P. Grasse, ed. Traite de Zool- lodes. Annu. Rev. Phytopathol. 18:333-359. ogle. Tome IV. Massou et Cie Paris. 32. Ward, S., N. Thomson, J. White, and 22. Riddle, D. 1978. The genetics of development S. Brelmer. 1975. Electron microscopical reconstruc- and behavior in Caenorhabditis elegaus. J. Nema- lion t)f the anterior sensory anatomy of the nema- tology 10:1-16. tode Caelmrhabditis elegans. J. Comp. Neurol. 160: 23. Roman, J., and H. Hirschmann. 1969. Em- 313-338. bryogenesis attd postembryogenesis in species ot~ 33. Ware, R., C. Clark, K. Crossland, and Pratylenchus (Nematoda: Tylenchida). Proc. R. Russell. 1975. The nerve ring of the nematode Hehninthol. Soc. Wash. 36:164-174. Caenorhabditis elegans. J. Comp. Neurol. 162:71-110. 24. Sternberg, P., and R. Horvitz. 1981. Gonadal 34. White. J., and R. Horvitz. 1979. Laser micro- cell lineages of the nematode Panagrellus redivivus beam techniques in hiological research. Electro- and implications for evolution by the modification optical Systems Design, August, pp. 23-24. of cell lineage. Develop. Biol. 88:147-166. 35. White, J., E. Southgate, N. Thomson, and 25. Sternberg, P., and R. Horvitz. Postem- S. Brenner. 1976. The structure of the ventral nerve hryonic nongonadal cell lineages of the nematode cord of Caenorhabditis elegans. Phil. Trans. Roy. Panagrellus redivivus: description and comparison Soc. Lond. B 275:327-248. with those of Caenorhabditis elegans. Snbmitted for 36. Wood, W. B., J. s. Laufer, and S. Strome. publication. 1982. Developmental Determinants in Embryos of 26. Sulston, J., I). Alberlson, and N. Thomson. Caenorhahditis elegans. J. Nematology 14:267-273. The Cuticle of Caenorhabditis elegans ~ ROBERT S. EDGARa, GEORGE N. Cox :¢, ]~'IEREDITH KUSCH 2, AND JOAN C. POL1TZ'-' Journal of Nematology 14(2):248-258. 1982. The nematode cnticle i s among ttle most Studies so far largely have been of a de- complex extracelhdar structnres produced scriptive nature, involving attempts to char- by a living organism. For the last few years acterize the morphology and composition of the work in our laboratory has been de- the cuticle and to isolate and stndy mutants voted to the study of the cuticle of tile free altered in genes tltat control attd regulate living nematode, Caenorhabditis elegans. cuticle formation. Our long-term intercst is to tmderstand tile genetic control and regu- lSymposinm for presentation at Society of Nematologists lation of complex processes such as cnticle 20ttl Annual Meeting, Seattle. Washington. Augnst 1981. formation. This research was supported by Prants PCM 76-11481 and PCM 78-09439 from the Nati~mal Science Foundation and grants 5-SSO7RR07135 and 1 ROI GM28311-01AI from the ADULT MORPHOLOGY National Institutes of Health. -~l)epartment of Biology, Thimaml Laboratories, Univer- sity of California, Santa Cruz. CA 95064. The external and internal morpltology at)epartment of Molecular, Cellular, and Developmental o[ the adult cuticle of C. elegans differs little Biology; University of Colorado, Boulder, CO 80309. We thank H. Boedtker l)oty for tile chick collagen re- from that of the sibling species, C. briggsae, Fomhinant plasmid. examined by Zuckerman et al. (17). On the Caenorhabditis elegans: Edgar et at. 249 basis of our studies (3,5), the external Trausmission electron microscopy of the cuticle sur[ace revealed in SEM has more internal structure of the adult cuticle re- than 1,000 annulae 1.0 /~m wide separated veals two major cuticle lax, ers separated by by furrows framing circum[erentiaIly a space, presumably fluid filled. Tile outer around the animal. These annulae are inter- cortical layer is composed of an electron rupted at the lateral sides o[ the animal 1)y dense surface layer and an amorphous under three-pronged alae that extend from heat[ layer. The immr basal layer is composed of to tail (Fig. 1), two fiber layers running in different direc- DORSALHYPODERMAL CORD ANNULAE NUCLEI ~~\\,~ LATERALALAE / HYPODERMAL SEAM CELLS IN LATERAL HYPODERMAL HYPODERMALCORD SYHCYTIUM VENTRAL NYPODERMALCORO A ANNULAE ~ / f•,•//,"/'f~ f f f'K / f .1 (~ ~ . " - - CORTICALLAYER, . ~-"~ STRUTS MEDIANLAYER. ~ FIBERLAYERS BASALLAYER .-.+....','. Fig. 1. Diagrammatic sketch of the adult C. elegans cuticle. (a) Cross-section of an adult nematode showing the general organization of the cuticle and underlying hypodermis. (b) Magnilied view of internal anatomy of cuticle. Neither figure is drawn to scale. Reprinted from Cox et al. (4). 250 Journal of Nematology, Volume 14, No. 2, April 1982 tions, probably helically about the animal, pharynx, uterus, anus, and excretory pore. and an inner amorphous layer. Tile basal Enzymatic digestion of isolated cuticles has and cortical layers are joined by electron been followed by phase contrast microscopy. dense columnar structures we call struts; Pronase and collagenase digest first the these run in circumferential paired rows struts, to produce a double bag, and then under the annular furrows. Struts are also tile inner layer of the cuticle, but do not found irregularly between the paired rows. affect the outer layer. This outer layer of In the region of the alae, the basal and the cuticle is digested only by elastase which cortical layers appear fused, the struts are also digests the struts and inner layer. The not evident, and the fiber layers appear un- pharyngeal lining is not digested by col- changed. IVlany of these features of adult lagenase or elastase but is digested by cuticle structure are evident in Figures 2 pronase. These results suggest that the and 3 and are diagrammed in Figure 1. cortical layer, the struts and basal layer, and The cuticle is an extracellular secretion the pharyngeal lining all have different of the hypodernfis. The hypodermis in C. structural features that render them differ- elegans is, for the most part, a syncytium entially sensitive to these three enzymes. We (16). The nuclei are located in the lateral have found that treatment of the isolated ridges; none are present in the dorsal or cuticles with reducing agents snch as ventral ridges. These ridges are connected /3-mercaptoethanol (BME) gives results by a thin cytoplasmic bridge enclosing the comparable to enzymatic digestion with muscle bundles. A row of hypodermal seam collagenase: first solubilization of the struts, cells lies under each ala, 16 cells on each then the basal layer. The cortical layer, or a side; these cells fuse to form a seam cell portion of it, is resistant to solubilization by synctium after the adult molt. Laser abla- BME. tion of individual seam cells results in inter- Since cuticle integrity is little altered by ruptions of the alae, indicating that the hot detergents, cuticle proteins are held to- individual seam cells control the formation gether by covalent bonds. The solubiliza- of the ala above them (15). tion of the basal layer and the struts by BME suggests that the proteins composing these COMPOSITION OF ADULT structures are held together by disulphide To prepare cuticles for biochemical bonds. The insolubility in BME of a por- study, we sonicate a suspension of animals tion of the cortical layer suggests the ex- to rupture the cuticle (3). Extensive wash- istence of more extensive nonredncible ing of the cuticles in low ionic strength covalent cross-linking of proteins in tile buffer removes most of the cellular material, cortical layer. but some contaminating material still re- These observations are in accord with mains. This can be removed by treatment earlier studies on the cuticle of Ascaris with boiling detergent, such as SDS (sodium lumbricoides (7,10) which revealed an in- dodecyl sulfate), without major alteration ner layer composed of collagen-like proteins in cuticle morphology, as determined by cross-linked by disulphide bridges and all phase contrast light microscopy and trans- outer layer composed of covalently cross- mission electron microscopy of SDS-cleaned linked proteins that do not show the low cuticles. These cuticles show a slight bi- angle X-ray pattern characteristic of col- refringence. The direction of birefringence lagens and so are inferred to be a different is at right angles to the long axis of the class of fibrous proteins, termed cuticlins. animal and presumably derives from the The cuticle proteins solubilized by BME helically wound fiber layers that are oriented range in apparent molecular weight on SDS predominantly at right angles to the long polyacrylamide gels from 60,000 to 210,000 axis of the animal.