C. elegans cuticle collagen genes

TIG January 2000, volume 16, No. 10168-9525/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved. PII: S0168-9525(99)01857-0 21

Reviews

The exoskeleton of the free-living nematodeCaenorhabditis elegans consists predominantly of small

collagen-like proteins encoded by a multi-gene family ofapproximately 154 members1,2. These molecules are syn-thesized by specialized epithelial cells and after secretion,polymerize on the apical surface of the epithelium to forma complex multi-layered structure3,4. The entire process ofcuticle synthesis is repeated five times during development,once in the embryo before hatching and then towards theend of each of the four larval stages before moulting5.During each synthesis, different collagen genes areexpressed in discrete temporal periods, the reason forwhich could relate to the mechanism of control of collagenpolymerization and formation of different structural com-ponents of the cuticle6. The basic temporal pattern isrepeated during each synthesis. The developmental processcontrolling collagen gene expression and exoskeletal for-mation appears to be reset after embryonic hatching andthen after each of the larval moults, the cyclic repeats of theprocess being terminated by the larval to adult moult.

Collagens are structural proteins involved in the syn-thesis of a variety of extracellular matrices in animals7,8.Collagen occurs as a triple helix, the result of trimerizationof three monomeric collagen chains9,10. The amino acidsequence of the monomeric collagens is repetitive, withglycine at every third residue. This Gly-X-Y repeat occursin blocks, the length and organization of which dependson the type of collagen. The residues at the X and Ypositions within the repeats are frequently proline orhydroxyproline. In nematodes, as typified by the free-living species C. elegans, collagens are involved in the for-mation of two distinct structures, the basement membraneand the cuticle. It is the cuticular collagens, and the multi-gene family that encode this class of proteins that are thesubject of this review.

The nematode cuticleThe nematode cuticle is an exoskeleton that is synthesizedby the underlying epithelial tissue, called the hypoder-mis1,2,11. Cuticle functions include provision of a barrierbetween the animal and its environment, maintenance ofpost-embryonic body shape12–14 and movement via attach-

ments to muscle15,16. The cuticle is synthesized five timesduring development and is shed at each moult, first duringembryogenesis when the cuticle of the L1 larva is formed,and then again during each of the four larval stages beforeeach moult. Cuticular material is synthesized and secretedapically from epithelial cells and polymerizes on the exter-nal epithelial surface (Fig. 1). During synthesis, sub-membranous actin filaments form within the hypodermisand are organized circumferentially around the cylindricalbody of the worm. These filaments are coincident withfurrows that form on the epithelial cell membrane duringcuticle synthesis and subsequently with circumferentialfurrows that delineate the annulae (Fig. 1) on the surfaceof the polymerized cuticle17. This suggests that the shapeof the polymerized cuticle is influenced by the shape of thesurface on which it polymerizes. In addition to the an-nulae, longitudinal ridges, termed alae, exist on the lateralsurfaces of the cuticle at certain developmental stages,most notably the adult (Figs 1, 2)3. Although both of thesestructures can be visualized with a light microscope, thecuticle is transparent giving a deceptive view of its truestructural complexity. It consists of possibly six definablelayers that are distinct in their ultrastructure, as imaged byelectron microscopy4,18. The precise nature of the structureand layering varies at different developmental stages3; themechanisms by which proteins polymerize to form theseordered structures are currently unknown.

Mutant collagen genesThe major protein components of the C. elegans cuticleare small collagen-like polypeptides. These are encoded bya multi-gene family, consisting of approximately 154members. From random mutagenesis of C. elegans,approximately 45 loci have been identified that affectbody shape. Currently, 17 of these loci have been assignedto known gene sequences, and nine of these (bli-1, bli-2, dpy-2, dpy-7, dpy-10, dpy-13, rol-6, sqt-1 and sqt-3) arecuticular collagen genes. Among the remainder are genesencoding enzymes that are thought to be involved in collagen processing and genes involved in X-chromosomedosage compensation. Phenotypes associated withmutations in the collagen genes fall predominantly into

Iain L. Johnstonegbga14@udcf.gla.ac.uk

The Wellcome Centre forMolecular Parasitology,Anderson College,University of Glasgow,Glasgow, UK G11 6NU.

Collagen is a structural protein used in the generation of a wide variety of animal extracellular matrices. Theexoskeleton of the free-living nematode, Caenorhabditis elegans, is a complex collagen matrix that is tractableto genetic research. Mutations in individual cuticle collagen genes can cause exoskeletal defects that alter theshape of the animal. The complete sequence of the C. elegans genome indicates upwards of 150 distinctcollagen genes that probably contribute to this structure. During the synthesis of this matrix, individual collagengenes are expressed in distinct temporal periods, which might facilitate the formation of specific interactionsbetween distinct collagens.

Cuticle collagen genesexpression in Caenorhabditis elegans

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three basic types described as: (1) blister (bli), which is ablistering of cuticular material away from the surface of theanimal; (2) dumpy (dpy), which is a shortening in thelength of the animal as compared with wild type; and (3)roller (rol), which is a helical twisting of the animal’s body(see Fig. 2). The phenotypes are not necessarily exclusive.The blister phenotype (Fig. 2) is restricted to the adult stageand might identify a cuticular structure that is specific toadults. The dumpy phenotype (see dpy-10; Fig. 2) canaffect all post-embryonic developmental stages, althoughmutations in different dpy genes can cause greater or lessereffects at different stages. The defective exoskeleton in dpymutants significantly alters body shape. This is also true forthe roller phenotype. The alae in the cuticle (see Fig. 1) runparallel to the side of the animal in the wild type (Fig. 2dand e, left panels), but are helically twisted along the lengthof the animal in roller mutants, as indicated by their angledaspect relative to the side of the animal (Fig. 2d and e, rightpanels). The helical distortion in roller mutants is notrestricted to the exoskeleton itself. In the roller phenotype,the exoskeleton, the underlying ectodermal cells and mus-culature are all helically twisted along the length of the ani-mal with a similar turn to that seen for the alae19.

Mutations that cause the blister, dumpy and the com-bined dumpy and roller phenotypes are generally recessive;

mutations that cause the roller non-dumpy phenotype aremore frequently dominant. Among the recessive alleles aretrue genetic nulls, but most are glycine substitutionswithin the collagen domains1,11. Dominant alleles includemutations at the proposed ‘subtilisin-like’ cleavage site,which is believed to be required for processing of themature collagen20,21. Some of the mutations that cause thediscussed phenotypes are recessive and lead to loss offunction, indicating that at least some of the collagenshave specific roles in the formation of the exoskeleton1.

Cuticular collagen structureA typical polypeptide structure has been predicted for thecuticular collagen genes (Fig. 3). The nine genes discussedabove also fit this basic structure13,14,22–24. They are pre-dicted to encode two main blocks of Gly-X-Y collagen-like polypeptide that are flanked and separated by threeclusters of cysteine residues, which are termed Domains I,II and III in this review (Fig. 3). The smaller N-terminalGly-X-Y block typically contains 8–10 Gly-X-Y collagenrepeats, the larger C-terminal block has 40–42 Gly-X-Yrepeats. Generally, the C-terminal collagen block has oneor two very small disruptions within the Gly-X-Y repeats,the precise position and size of which vary between theproducts of different genes. In addition to these collagen-like regions, the molecules are predicted to have a non-collagen N-terminal region that varies considerably in size,but that contains conserved regions including a predictedsignal peptide and a proposed subtilisin-like cleavage site,which might be involved in the generation of a maturepolypeptide from a pro-collagen precursor21. Mutationsthat disrupt this proposed cleavage site have been shownfor some genes to be neomorphic2, generating a phenotypethat is distinct from both the wild-type and loss-of-functionalleles. This could be explained by its insertion into thecuticle of collagen with its pro-domain still attached.

In addition to the neomorphic alleles that affect the pro-posed subtilisin-like pro-collagen maturation site, variousother lesions have been characterized for the group of ninegenes discussed above13,14,20,23,24. The most common lesion issubstitution of a Gly within the Gly-X-Y collagen regions.For some of the genes, true genetic nulls have been identi-fied. Where data are available, the Gly substitutions seem tobehave as loss of function. DPY-7 protein containing a Glysubstitution appears to accumulate in the cytoplasm duringcuticle synthesis, but very little mutant protein gets incorpo-rated into the cuticle (L. McMahon, pers. commun.). Itseems likely that collagen that contains such Gly substitu-tions is recognized as being aberrant by cellular machineryand is not incorporated into the exoskeletal structure. Thisphenomenon of removal of Gly substitution mutant collagen has also been described for vertebrate collagens8.

Cuticular collagen gene familiesThe complete sequence of the C. elegans genome permitsan accurate indication of all possible collagen-like genes.In the absence of experimental data, the number of pre-dicted cuticular collagen genes must depend on the precisecriteria used to define this class. There are approximately154 predicted cuticular collagen genes (http://www.worms.gla.ac.uk/collagen/cecolgenes.htm); in addition,there are several gene predictions that contain some butnot all of these regions (shown in Fig. 3). Similarly sizedcollagen gene families have been described for other nematode species25.

FIGURE 1. The cuticle and hypodermis of Caenorhabditis elegans

(a) A section of the cylindrical body of the nematode. Annulae are present on the surface of the cuticle atall stages, alae are visible only on the L1, the dauer and the adult cuticle3. The bottom two drawingsrepresent cross sections through the cuticle and underlying hypodermal cells. (b) The presence of furrowsin the apical surface of the hypodermis and juxtaposed bundles of actin filaments that form during cuticlesecretion. These are coincident with the positioning of the annular furrows in the newly formed cuticle. (c)The organization of the hypodermis after cuticle secretion, after dissociation of the actin bundles.

Annular furrows

Annulus(a)

(b)

(c)

External cortical layer

Cell membrane

Actin filamentbundles

Cortical layersHypodermal cells

Ventral hypodermis

Dorsal hypodermis

Lateralseam cells

Striated layers

Alae

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Phylogenetic analysis of this gene family is complicatedby the high levels of similarity within collagen-encodingregions. The family can be divided into groups based onthe pattern of conserved cysteines of their predictedencoded collagens (Fig. 3). The evolutionary significanceof these groupings is supported by homologies within theN- and C- non-collagen regions, which place the genesinto groups that agree with the groupings that are basedon the cysteine patterns alone. Of the 154 genes, 68belong to Group 1, 12 to Group 1a, 38 to Group 2 and 31to Group 3. The dpy-7 and dpy-2 groups have three andtwo members, respectively. Of the genes defined bymutation, bli-1, sqt-1 and rol-6 belong to Group 1; bli-2belongs to Group 2; dpy-13 and sqt-3 belong to Group 3;dpy-7 belongs to the dpy-7 Group and dpy-2 and dpy-10constitute the dpy-2 Group. Group 1a is not representedamong the genes that are defined mutationally.

The nine mutationally defined genes have been identi-fied from random mutagenic screens and selected on thebasis that the mutation caused an easily detectable changein body shape. For these nine genes, multiple alleles ofeach have been obtained. There are only approximately 20shape-change mutants not yet assigned to a gene definedby sequence. It therefore seems likely that most of thecuticular collagen genes that are defined by sequencemight not be mutated readily to generate detectablephenotypes. Because of the high similarities between manymembers of this gene family, many of the gene productsmight have overlapping functions. No single, molecularlydefinable characteristic unites all of the nine genes so faridentified. Only the proposed collagen Group 1a is notrepresented among them, and this is a very highly

conserved group. Four of the nine might be consideredunusual as their encoded proteins, DPY-2, DPY-10, DPY-7 and BLI-1 all have significantly longer than aver-age C-terminal tails. In each case, loss-of-function allelescause significant phenotypes when they are homozygotic,implying a specific function for these proteins. Theremaining five have no obvious distinguishing feature;however, two of these, sqt-1 and rol-6, have very weak orundetectable phenotypes associated with loss-of-functionalleles2. All of the original alleles of these two genes aregain-of-function, loss-of-function only being obtained asintragenic suppressors of gain-of-function alleles.

These groups are not unique to C. elegans. Genes thatare closely related to the different proposed groups in C. elegans have been described for various distantlyrelated nematode species; examples for all of the mainproposed groups can be found in the sequence databases,indicating that the different groups must have evolvedbefore the divergence of many (if not all) modern daynematodes. This is supported by the observation thatsome individual members of groups within C. eleganshave closer relatives within other species than within theirgroup in C. elegans. As an example, a gene termed Mjcol-3from the plant parasitic nematode species Meloidogynejavanica26 is predicted to encode a collagen that has signifi-cantly greater similarity with that predicted for dpy-7 ofC. elegans than dpy-7 is predicted to have with the othertwo members of its group within C. elegans.

Regulated expression of cuticular collagen genesCuticular collagen genes are subject to strict spatial andtemporal modes of regulation. As stated earlier, the cuticle

FIGURE 2. Phenotypes associated with mutated collagen genes

The top three images are adult animals of (a) the wild-type strain N2, (b) a bli-2 (e768) mutant and (c) a dpy-10 (e128 ) mutant. Blistering of the cuticle in bli-2 isindicated by arrows. dpy-10 is significantly shorter and fatter than wild type. The bottom panels show high magnification images of (d) a wild-type N2 strain and (e)a mutant rol-6 (su1006 ). In each case, the animal’s body lies longitudinal from top to bottom of the image and the image represents approximately half the width ofthe nematode. Both left-hand panels show a deep optical section where part of the side of the animal is in focus. The orientation of the animal’s side is emphasizedby the black vertical line. Cuticular structures termed alae are indicated (black arrow) in a top optical section for both wild type and rol-6 mutant in the right-handpanels. As can be seen, the alae run parallel to the side of the animal in the wild type, but are at an angle in the rol-6 mutant.

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is synthesized by specialized epithelial cells termed thehypodermis5. Reporter gene fusions between the promotersplus regulatory sequences, for several cuticular collagengenes and either lac-Z or green fluorescent protein (GFP)give specific expression of the reporter gene within thehypodermis of transgenic animals6,27. (To date, we havetested eight cuticular collagen genes in this way.)Transgene expression of dpy-7/GFP has been shown withinhypodermal cells (Fig. 4). For the gene dpy-7, we have beenable to show that only a relatively short region of upstream

gene sequence, approximately 125 bp, is sufficient to driveapparently wild-type tissue-specific expression of thisgene27. We have recently generated a specific monoclonalantibody against the C-terminal tail of the DPY-7 protein.Preceding the secretion of the cuticle, DPY-7 protein isdetected within hypodermal cells; after secretion, it local-izes to the annular furrows of the cuticle (L. McMahon andI.L. Johnstone, unpublished). The cellular localization ofpre-secreted DPY-7 is similar to the predicted expressionpattern indicated by the reporter gene assays.

FIGURE 3. Organization of cuticular collagen

The predicted organization of a typical cuticular collagen. The positions of the two blocks of Gly-X-Y sequence are indicated, as are the three cysteine-containingdomains. Underneath this, the actual sequence for the three cysteine Domains I, II and III for representative examples of genes for all of the proposed classes. The genenames are those used by the Caenorhabditis elegans genome project that are GENEFINDER predictions39. The number of amino acid residues between conserved cysteinesis indicated above the sequence for each class. Three examples from within each proposed group have been selected for comparison avoiding close neighbours withingroups, except dpy-7 and dpy-2 groups that contain three and two members. Group 1 has 68 members, Group 1a has 12 members, Group 2 has 38 members and Group3 has 31 members. The molecules are predicted to have a carboxy non-collagen tail of varying lengths, but frequently very short. For many of the genes, the entirecarboxy tail is the region indicated as Domain III in the upper section. However, some genes are predicted to encode significant extensions beyond this domain. Thismulti-gene family is generally dispersed throughout the genome; however, there are several examples of local clusters. In those instances where genes are adjacent toone another in the genome, they are frequently closely related by sequence and are hence within the same group.

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AG..QCNCGAQSSGCPA VGIVSE.GGPCIKCPA AAYCPCPARSVAVQRSAGFEQCNCGPKSEGCPA VAITHDIPGGCIKCPP AGYCPCPSRAAYKARSNSQCSCGLPSQGCPA IPIPNDFPKECIKCPA AEYCPCPERKRRRV

GLPAWCQCEPTKPTCPP TYAPINCPQVSFDCIKCPA AAYCACPPRSAVFLSRHGLPAWCQCEPTKPTCPP TFAPLTCAPVSQDCVKCPE AAYCACPPRSAVFVSRHGLPAWCQCEPAKPVCPP TFAPITCPPKDPSCVKCPE AAYCPCPPRSAVFVNRFAH SGGGSCCSCGV NQPAGPDSF.CFDCPA GACDHCPPPRTAPGY GGGGGCCGCGV ATPAPNYDW.CFDCPP GGCEHCPPPRTAPGY EPAPQCCTCQQ APSDGLQSEPCMICPP GDCFHCPTPRTPPGY

VGNGQCEGCCLP PKEPCEPITPPPCKPCPE GVCPKYCALDGGVFFED ASTGGCDACCLP PQQPCDPITPPPCQPCPQ GICPKYCAIDGGVFFED QEHNSCDGCCLP PQSPCEPLTPPPCPACPP GICPKYCAIDGGVFFED

GVLHQCSQCTRLHCPQ VDLEPQEELPCSICPA TYCPSDCGVNTILSQFG SDNQQCTSCVQLRCPP VPLDPEPAFPCVICPA GHCPSSCGVQEIVAPSV MGGAFCKGCFLLSCPQ IQPESEPELPCVICPA SYCPSDCGVQPILTEMF

PLETECPGCCIP NQTCPLNQVREPPPCRPCPK GVCVCQNVDSILLIN PQFQECPACCIP NASCIPERVFEPPPCLPCPQ GTCVCQDTEVVMNDE

Group 1 c50b6.4zk1193.1f27c1.8

Group 1af54d1.3b0222.8f17c8.2 Group 2k02d7.3f46c8.2f14h12.1

Group 3c53b4.5f55c10.3t05a1.2

dpy-7f56d5.1f46c8.6c31h2.2

dpy-2t14b4.7t14b4.6

Domain I Domain IIIDomain II

41–42 aa ~133 aa

42 aa 133 aa

45 aa ~130 aa

43–44 aa ~132 aa

42 aa 134 aa

47 aa 131 aa

Gly-X-Yc c c c cc c

N CGly-X-Y

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In addition to the tissue-specific regulated expression,cuticular collagen genes are under tight temporal control.During the life cycle, a cuticle is synthesized five times, once inthe embryo before hatching, and then at the end of each ofthe four larval stages before moulting (Fig. 5). As might beexpected, cuticular collagen genes are expressed in a temporalpattern that reflects this repetitious set of cuticle-synthesisevents, once per synthesis. However, genes are not allexpressed at the same time during synthesis (Fig. 5). Cleartemporal differences can be detected6, for example, dpy-7mRNA shows a peak of abundance approximately 4 h beforeeach moult. By contrast, col-12 mRNA abundance peaks 4 hlater, at each moult. We have analyzed approximately 20cuticular collagen genes in this way, the data for six of whichhave been published6. In addition to the early and late expres-sion times represented here by the genes dpy-7 and col-12, wefind genes such as dpy-13 and sqt-1 that are expressed inbetween these two temporal extremes. For those cuticularcollagen genes that show such oscillating peaks of mRNAabundance relative to the larval life cycle, time of expressionrelative to the moult is effectively constant. For example, agene that is expressed 4 h before the L1 moult is expressedapproximately 4 h before each moult. In addition to this repetitious larval expression pattern, we have detected genesthat are expressed at a significant level only in the adult afterthe moulting cycle is complete. Also, for the genes that areexpressed in an oscillating pattern during larval development,some are expressed in the adult after the L4 to adult moultand some are not – col-12 is, dpy-7 is not (Fig. 5). The adultanimal grows significantly without moulting. It is possiblethat a limited set of collagen genes is expressed in the adult aspart of an adult cuticle growth mechanism.

Assembly of collagensVery little is known about the control of cuticular collagenassembly into the multi-protein cuticle structure. If themolecules are true collagens, the primary polymerizationmust be the formation of a trimer with the collagen triplehelix, followed by subsequent higher order interactionsbetween trimers that ultimately form the cuticle structure.Mutant alleles predicted to cause glycine substitutionswithin the collagen-like Gly-X-Y repeats for some of themutationally defined genes discussed above have beenshown to interfere with function, supporting the involve-ment of these peptide sequences in trimer for-mation1,11,13,14. Similar lesions can destabilize the collagentriple helix in vertebrate collagens8,28. The cuticle is a com-plex, multi-layered structure. The temporal series ofcuticular collagen gene expressions described above mightexist to promote the formation of distinct substructures(possibly layers) within the cuticle. If this is the case, dis-tinct cuticle collagens would localize to specific regions ofthe cuticle, and collagens expressed at the same time mightlocalize to the same sub-cuticular structure or region, or atleast to a structure created at the same relative time.

The proposed primary interaction of cuticular collagentrimer formation is only likely to happen between mol-ecules that are expressed at the same time. For reasonsthat relate to the behaviour of glycine-substitutionmutants, trimerization of cuticular collagens is likely to berestricted either to homo-trimerization or trimerizationbetween very closely related members of cuticle collagenfamilies1. For some vertebrate collagens, the C-terminalnon-collagen domain has been shown to be involved in theprimary act of trimer formation29 forming a nucleus for

the ‘zipper’-like mechanism that propagates in a C- to N-terminal direction to form the collagen triple helix30. Anobvious feature of the C-terminal non-collagen domain ofall predicted cuticular collagens is the two cysteineresidues in Domain III (Fig. 2) that are positioned immedi-ately C-terminal to the last Gly-X-Y repeats. Extraction ofcollagens from the C. elegans cuticle requires the presenceof reducing agents18; clearly disulphide bonds could beinvolved in interactions between the three monomers of atrimer or with higher order interactions between trimers.

Given the very small size of most of the cuticular col-lagen C-terminal tails (typically 15–20 residues) and the

FIGURE 4. Transgenic dpy-7/GFP reporter genefusion

An animal transgenic for a dpy-7/GFP reporter gene fusion. (a) Differentialinterference contrast (DIC) image and (b) fluorescence for green fluorescentprotein (GFP). The GFP is localized to the nucleus by a nuclear localization signal.This reporter transgene is expressed in most hypodermal cells. The strain wasgenerated by Eric Stewart. Scalebar represents 0.1 mm.

FIGURE 5. Temporal changes in mRNA abundance

The graphs show life-cycle temporal changes in mRNA abundance (as measured relative to a controltranscript, detailed in Ref. 6) for the two cuticular collagen genes dpy-7 and col-12. Time is indicated onthe x-axis as hours after release from an L1 larval developmental arrest that is induced by hatchingembryos in the absence of food. Timing of the four moults and the life cycle stages are indicated below.

12

10

8

6

4

2

01 10

L1 L2 L3 L4 Adultmoultmoultmoultmoult

20 30 40Rel

ativ

e m

RN

A a

bund

ance

Hours post L1 developmental arrest

dpy-7col-12

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close proximity of the conserved cysteine residues withinthe tail to the Gly-X-Y repeats, it is worth considering thepossible involvement of these cysteine residues in inter-chain disulphide bond formation as part of the generationof a nucleus for triple helix formation or in providing cor-rect register between monomers. The absolute conser-vation of these residues in all predicted cuticular collagensindicates their functional importance, but does not distin-guish between importance for assembly of the trimer andsubsequent interactions or function. Ethyl methane-sulphonate (EMS)-induced mutations in the sqt-1 gene thatcause a substitution of one of these cysteines with eithertyrosine or serine are able to generate a recessive left-rollerphenotype that is distinct from the almost wild-type pheno-type of null mutations in this gene20,21. That the phenotypeis distinct from the complete loss of the SQT-1 proteinmight indicate the presence of the mutant protein withinthe cuticle, and suggests that the single-cysteine-substitu-tion SQT-1 protein is assembled to some degree. Thephenotype of these mutants might therefore be the result ofthe presence of aberrant protein within the cuticle asopposed to the loss of wild-type protein. However, therecessive nature of these alleles over wild type suggests thateither in the presence of wild-type protein, mutant proteinfails to produce a phenotype, or wild-type protein is moreefficiently assembled into the cuticle than the mutant. Invitro substitution of either C-terminal cysteine in SQT-1 orROL-6 (a closely related collagen to SQT-1) can generate asimilar left-roller phenotype when transgenically expressedin the respective null mutant background20,21.

Existing data is not conclusive as to the precise functionof these conserved cysteines, especially whether they formbonds between the three members of a trimer or betweentrimers. Indeed, these two distinct possibilities need not beexclusive. It is at least conceivable that disulphide bondsformed within a trimer during its assembly could be brokenand reformed between trimers at a later stage in collagenbiosynthesis. The class of enzyme that could perform suchan exchange of disulphide bonds is protein disulphide isom-erase (PDI). In vertebrates, PDIs have been shown to beinvolved, along with other chaperones, in the trimerizationof some collagens31. C. elegans has two PDI genes, pdi-1and pdi-2. The gene pdi-1 exists as part of an operon witha gene termed cyp-9. Reporter-gene assays suggest that thisoperon is transcribed specifically within the same hypoder-mal cells that synthesize cuticular collagen32. Interestingly,cyp-9 encodes a putative peptidyl prolyl cis–trans isom-erase, another enzyme implicated in promoting collagentriple helix formation in vertebrates33. There is, therefore, apossible functional relationship of cuticle collagen biosyn-thesis for these two co-transcribed C. elegans genes. ThePDI, at least in theory, provides the enzymatic function fordisulphide bond partner exchanges.

The developmental decision to synthesize acuticleMoulting in insects is hormonally controlled, and a de-rivative of cholesterol, 20-hydroxyecdysone, plays a central

role in moulting in Drosophila melanogaster, its activitybeing mediated through its interaction with nuclear hor-mone receptor (NHR) class transcription factors. Evidencein Drosophila suggests a cascade of gene expression inresponse to the ecdysone hormone, with the transcriptionof genes, including NHR genes, being induced early inresponse to the hormone34. The early response genes arethought to be involved in the subsequent transcriptionalcontrol of late response genes. C. elegans has a very largefamily of nuclear-hormone-receptor-like transcription fac-tor genes; approximately 270 are predicted by the C. ele-gans genome project35. One of these, nhr-23, is expressedin the hypodermis and its activity has been implicated as arequirement for normal moulting36.

Recently, analysis of a C. elegans gene, lrp-1, predictedto encode a member of the low density lipoprotein recep-tor family of proteins, was shown to be required for nor-mal execution of moulting in C. elegans37. C. elegans has anutritional requirement for sterol. Sterol starvation canmimic the phenotype, including moulting defects, of lossof function of lrp-1 (Ref. 37). LRP-1 is synthesized withinand secreted apically from the major hypodermal cells –those cells also responsible for most of the cuticle synthe-sis. Yochem et al. speculate that the role of LRP-1 mightbe to endocytose sterols from extracellular fluids37. Thatlrp-1 mutants and sterol starvation can both generatemoulting defects is interesting; however, as yet, it isunclear why this occurs. One possibility is a need forsterols for the synthesis of a signalling molecule likeecdysone used in controlling the moulting cycle. However,other possibilities, including more structural roles relatingto the mechanics of moulting or hypodermal membranebehaviour, must also exist.

Finally, a phylogenetic analysis of 18S ribosomal DNAsequences suggests a close relationship between arthropods,nematodes and all other moulting phyla38. Although theinvolvement of a cholesterol derivative such as ecdysone hasnot as yet been demonstrated in nematodes, there is a bodyof evidence that might point towards a common evolution-ary origin of moulting for these animal groups.

AcknowledgementsThe C. elegans Genome Project is responsible for thegeneration of most of the gene sequence data discussedhere and the contribution of all those who have worked onthis project is acknowledged. The developers and curatorsof ACeDB are also thanked for the essential provision ofinformation. Thanks to J. Kramer for discussion and dataon bli-1 and bli-2 before publication. I would also like tothank members of my lab, J. Muriel and L. McMahon forunpublished data and to Eric Stewart for the transgenicdpy-7/GFP C. elegans strain used for the images in Fig. 4.Tragically and very unexpectedly, Eric died earlier lastyear. His presence in the lab is sorely missed. Thanks alsoto T. Page for frequent discussions on worm cuticles andcollagen processing enzymes. I.L.J. is the recipient of a Medical Research Council Senior Fellowship inBiomedical Research.

References1 Johnstone, I.L. (1994) The cuticle of the nematode

Caenorhabditis elegans: a complex collagen structure.BioEssays 16, 171–178

2 Kramer, J.M. (1997) Extracellular Matrix. In C. elegans (Vol. II)(Riddle, D.L. et al., eds), pp. 471–500, Cold Spring Harbor

Laboratory Press3 Cox, G.N. et al. (1981) The cuticle of Caenorhabditis elegans. II.

Stage-specific changes in ultrastructure and protein compositionduring postembryonic development. Dev. Biol. 86, 456–470

4 Peixoto, C.A. and Desouza, W. (1995) Freeze-fracture anddeep-etched view of the cuticle of Caenorhabditis elegans.

Tissue Cell 27, 561–5685 Singh, R.N. and Sulston, J.E. (1978) Some observations on

moulting in Caenorhabditis elegans. Nematologica 24, 63–716 Johnstone, I.L. and Barry, J.D. (1996) Temporal reiteration of a

precise gene expression pattern during nematodedevelopment. EMBO J. 15, 3633–3639

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Reviews

In recent years, rapid progress has been made in under-standing how transforming growth factor-b (TGFb)

and related ligands signal, in part because of a wealth ofgenetic and developmental information previously avail-able on the pathways in which these ligands function.Model genetic systems show us how TGFb-related path-ways signal, how they are regulated and what cellularprocesses they control. As the Caenorhabditis elegansgenome is completely sequenced and the tools to analysethese basic cellular processes is expanding, C. eleganswill continue to play a major role in elucidating thesefunctions and networks. In this review, we discuss thegenetics and developmental biology of C. elegans TGFbsignaling.

The TGFb superfamily plays critical roles in severalimportant processes, such as cell proliferation, embryonicpatterning and cell-type specification1–6. Biochemical iden-

tification of serine-threonine kinase receptors as mediatorsof TGFb signaling was an important advance in the field,as was the identification of cytoplasmic and nuclear effec-tors belonging to the Smad family (a fusion of sma andMad gene names). In Drosophila, Mothers against dpp(Mad) was genetically identified as part of theDecapentaplegic (dpp) pathway, and its cDNA sequenceindicated it is a cytoplasmic protein, which is consistentwith a role as a mediator of receptor signaling7. Work inC. elegans revealed three Smads that function in the sameTGFb signaling pathway, suggesting that multiple Smadsmight be required in other pathways8. Cloning of mam-malian homologs demonstrated that these genes are con-served across diverse metazoan phyla8,9. Furthermore,developmental studies in Xenopus led to the identificationof Smad2, a potent mesoderm inducer10. These discoveriesspurred a flurry of Smad cloning and database harvesting.

C. elegans cuticle collagen genes

Garth I. Pattersonpatterson@waksman.rutgers.edu

Richard W. Padgett*padgett@waksman.rutgers.edu

Department of MolecularBiology andBiochemistry, 604 AllisonRoad, Rutgers University,Piscataway, NJ 08854,USA.*Waksman Institute, 190 Frelinghuysen Road,Rutgers University,Piscataway, NJ 08854,USA.

Genetic and molecular analysis in Caenorhabditis elegans has produced new insights into how TGFb-relatedpathways transduce signals and the developmental processes in which they function. These pathways areessential regulators of dauer formation, body-size determination, male copulatory structures and axonalguidance. Here, we review the insights that have come from standard molecular genetic experiments anddiscuss how the recently completed genome sequence has contributed to our understanding of these pathways.

TGFb-related pathwaysroles in Caenorhabditis elegans development

7 Prockop, D.J. (1998) What holds us together? Why do some of usfall apart? What can we do about it? Matrix Biol. 16, 519–528

8 Prockop, D.J. and Kivirikko, K.I. (1995) Collagens – molecularbiology, diseases, and potentials for therapy. Annu. Rev.Biochem. 64, 403–434

9 Ramachandran, G.N. (1967) Structure of collagen at themolecular level. In Treatise on Collagen (Ramachandran, G.N.,ed.), pp. 103–183, Academic Press.

10 Ramachandran, G.N. and Ramakrishnan, C. (1976) Molecularstructure. In Biochemistry of Collagen (Ramachandran, G.N.and Reddi, A.H., eds), pp. 45–81, Plenum Press

11 Kramer, J.M. (1994) Genetic analysis of extracellular matrix inC. elegans. Annu. Rev. Genet. 28, 95–116

12 Kramer, J.M. et al. (1988) The sqt-1 gene of C. elegansencodes a collagen critical for organismal morphogenesis.Cell 55, 555–565

13 Levy, A.D. et al. (1993) Molecular and genetic analyses of theCaenorhabditis elegans dpy- 2 and dpy-10 collagen genes – avariety of molecular alterations affect organismal morphology.Mol. Biol. Cell 4, 803–817

14 Johnstone, I.L. et al. (1992) Molecular analysis of mutations inthe Caenorhabditis elegans collagen gene dpy-7. EMBO J. 11,3857–3863

15 Francis, R. and Waterston, R.H. (1991) Muscle-cell attachmentin Caenorhabditis elegans. J. Cell Biol. 114, 465–479

16 Hresko, M. et al. (1999) Myotactin, a novel hypodermal proteininvolved in muscle-cell adhesion in Caenorhabditis elegans. J. Cell Biol. 146, 659–672

17 Costa, M. et al. (1997) The role of actin filaments in patterningthe Caenorhabditis elegans cuticle. Dev. Biol. 184, 373–384

18 Cox, G.N. et al. (1981) Cuticle of Caenorhabditis elegans: itsisolation and partial characterisation. J. Cell Biol. 90, 7–17

19 Higgins, B.J. and Hirsh, D. (1977) Roller mutants of the

nematode Caenorhabditis elegans. Mol. Gen. Genet. 150, 63–7220 Kramer, J.M. and Johnson, J.J. (1993) Analysis of mutations in

the sqt-1 and rol-6 collagen genes of Caenorhabditis elegans.Genetics 135, 1035–1045

21 Yang, J. and Kramer, J.M. (1994) In vitro mutagenesis ofCaenorhabditis elegans cuticle collagens identifies a potentialsubtilisin-like protease cleavage site and demonstrates thatcarboxyl domain disulfide bonding is required for normalfunction but not assembly. Mol. Cell. Biol. 14, 2722–2730

22 Kramer, J.M. et al. (1990) The Caenorhabditis elegans rol-6gene, which interacts with the sqt-1 collagen gene todetermine organismal morphology, encodes a collagen. Mol.Cell. Biol. 10, 2081–2089

23 van der Keyl, H. et al. (1994) Caenorhabditis elegans sqt-3mutants have mutations in the col-1 collagen gene. Dev. Dyn.201, 86–94

24 von Mende, N. et al. (1988) dpy-13: a nematode collagen genethat affects body shape. Cell 55, 567–576

25 Johnstone, I.L. et al. (1996) Cuticular collagen genes from theparasitic nematode Ostertagia circumcincta. Mol. Biochem.Parasitol. 80, 103–112

26 Koltai, H. et al. (1997) The first isolated collagen gene of theroot-knot nematode Meloidogyne javanica is developmentallyregulated. Gene 196, 191–199

27 Gilleard, J.S. et al. (1997) cis regulatory requirements forhypodermal cell-specific expression of the Caenorhabditiselegans cuticle collagen gene dpy-7. Mol. Cell. Biol. 17,2301–2311

28 Byers, P.H. (1990) Brittle bones – fragile molecules: disorders ofcollagen gene structure and expression. Trends Genet. 6, 293–300

29 Lim, A.L. et al. (1998) Role of the pro-alpha 2(I) COOH-terminal region in assembly of type I collagen: Truncation ofthe last 10 amino acid residues of pro-alpha 2(I) chain

prevents assembly of type I collagen heterotrimer. J. Cell.Biochem. 71, 216–232

30 Engel, J. and Prockop, D.J. (1991) The zipper-like folding ofcollagen triple helices and the effects of mutations thatdisrupt the zipper. Annu. Rev. Biophys. Chem. 20, 137–152

31 Wilson, R. et al. (1998) Protein disulfide isomerase acts as amolecular chaperone during the assembly of procollagen. J. Biol. Chem. 273, 9637–9643

32 Page, A.P. (1997) Cyclophilin and protein disulfide isomerasegenes are co-transcribed in a functionally related manner inCaenorhabditis elegans. DNA Cell Biol. 16, 1335–1343

33 Steinmann, B. et al. (1991) Cyclosporine-a slows collagentriple-helix formation in vivo – indirect evidence for aphysiological-role of peptidyl-prolyl cis- trans-isomerase. J. Biol. Chem. 266, 1299–1303

34 Russell, S.H. (1996) Nuclear hormone receptors and theDrosophila ecdysone response. Biochem. Soc. Symp. 62, 111–121

35 Ruvkun, G. and Hobert, O. (1998) The taxonomy ofdevelopmental control in Caenorhabditis elegans. Science282, 2033–2041

36 Kostrouchova, M. et al. (1998) CHR3: a Caenorhabditiselegans orphan nuclear hormone receptor required for properepidermal development and molting. Development 125,1617–1626

37 Yochem, J. et al. (1999) A gp330/megalin-related protein isrequired in the major epidermis of Caenorhabditis elegans forcompletion of molting. Development 126, 597–606

38 Aguinaldo, A.A. et al. (1997) Evidence for a clade ofnematodes, arthropods and other moulting animals. Nature387, 489–493

39 C. elegans sequencing consortium (1998) Genome sequence of the nematode C. elegans: A platform for investigating biology. Science 282, 2012–2018