Mutations in the Caenorhabditis elegans orthologs of human genes required for mitochondrial tRNA modification cause similar electron transport chain defects but different nuclear responses.

Several oxidative phosphorylation (OXPHOS) diseases are caused by defects in the post-transcriptional modification of mitochondrial tRNAs (mt-tRNAs). Mutations in MTO1 or GTPBP3 impair the modification of the wobble uridine at position 5 of the pyrimidine ring and cause heart failure. Mutations in TRMU affect modification at position 2 and cause liver disease. Presently, the molecular basis of the diseases and why mutations in the different genes lead to such different clinical symptoms is poorly understood. Here we use Caenorhabditis elegans as a model organism to investigate how defects in the TRMU, GTPBP3 and MTO1 orthologues (designated as mttu-1, mtcu-1, and mtcu-2, respectively) exert their effects. We found that whereas the inactivation of each C. elegans gene is associated with a mild OXPHOS dysfunction, mutations in mtcu-1 or mtcu-2 cause changes in the expression of metabolic and mitochondrial stress response genes that are quite different from those caused by mttu-1 mutations. Our data suggest that retrograde signaling promotes defect-specific metabolic reprogramming, which is able to rescue the OXPHOS dysfunction in the single mutants by stimulating the oxidative tricarboxylic acid cycle flux through complex II. This adaptive response, however, appears to be associated with a biological cost since the single mutant worms exhibit thermosensitivity and decreased fertility and, in the case of mttu-1, longer reproductive cycle. Notably, mttu-1 worms also exhibit increased lifespan. We further show that mtcu-1; mttu-1 and mtcu-2; mttu-1 double mutants display severe growth defects and sterility. The animal models presented here support the idea that the pathological states in humans may initially develop not as a direct consequence of a bioenergetic defect, but from the cell's maladaptive response to the hypomodification status of mt-tRNAs. Our work highlights the important association of the defect-specific metabolic rewiring with the pathological phenotype, which must be taken into consideration in exploring specific therapeutic interventions.

S6K links cell fate, cell cycle and nutrient response in C. elegans germline stem/progenitor cells.

Coupling of stem/progenitor cell proliferation and differentiation to organismal physiological demands ensures the proper growth and homeostasis of tissues. However, in vivo mechanisms underlying this control are poorly characterized. We investigated the role of ribosomal protein S6 kinase (S6K) at the intersection of nutrition and the establishment of a stem/progenitor cell population using the C. elegans germ line as a model. We find that rsks-1 (which encodes the worm homolog of mammalian p70S6K) is required germline-autonomously for proper establishment of the germline progenitor pool. In the germ line, rsks-1 promotes cell cycle progression and inhibits larval progenitor differentiation, promotes growth of adult tumors and requires a conserved TOR phosphorylation site. Loss of rsks-1 and ife-1 (eIF4E) together reduces the germline progenitor pool more severely than either single mutant and similarly to reducing the activity of let-363 (TOR) or daf-15 (RAPTOR). Moreover, rsks-1 acts in parallel with the glp-1 (Notch) and daf-2 (insulin-IGF receptor) pathways, and does not share the same genetic dependencies with its role in lifespan control. We show that overall dietary restriction and amino acid deprivation cause germline defects similar to a subset of rsks-1 mutant phenotypes. Consistent with a link between diet and germline proliferation via rsks-1, loss of rsks-1 renders the germ line largely insensitive to the effects of dietary restriction. Our studies establish the C. elegans germ line as an in vivo model to understand TOR-S6K signaling in proliferation and differentiation and suggest that this pathway is a key nutrient-responsive regulator of germline progenitors.

The control of cell growth and body size in Caenorhabditis elegans.

One of the most important ways in which animal species vary is in their size. Individuals of the largest animal ever thought to have lived, the blue whale (Balaenoptera musculus), can reach a weight of 190 t and a length of over 30 m. At the other extreme, among the smallest multicellular animals are males of the parasitic wasp, Dicopomorpha echmepterygis, which even as adults are just 140 µm in length. In terms of volume, these species differ by more than 14 orders of magnitude. Since size has such profound effects on an organism's ecology, anatomy and physiology, an important task for evolutionary biology and ecology is to account for why organisms grow to their characteristic sizes. Equally, a full description of an organism's development must include an explanation of how its growth and body size are regulated. Here I review research on how these processes are controlled in the nematode, Caenorhabditis elegans. Analyses of small and long mutants have revealed that in the worm, DBL-1, a ligand in the TGFß superfamily family, promotes growth in a dose-dependent manner. DBL-1 signaling affects body size by stimulating the growth of syncytial hypodermal cells rather than controlling cell division. Signals from chemosensory neurons and from the gonad also modulate body size, in part, independently of DBL-1-mediated signaling. Organismal size and morphology is heavily influenced by the cuticle, which acts as the exoskeleton. Finally, I summarize research on several genes that appear to regulate body size by cell autonomously regulating cell growth throughout the worm.

Selenoprotein TRXR-1 and GSR-1 are essential for removal of old cuticle during molting in Caenorhabditis elegans.

Selenoproteins, in particular thioredoxin reductase, have been implicated in countering oxidative damage occurring during aging but the molecular functions of these proteins have not been extensively investigated in different animal models. Here we demonstrate that TRXR-1 thioredoxin reductase, the sole selenoprotein in Caenorhabditis elegans, does not protect against acute oxidative stress but functions instead together with GSR-1 glutathione reductase to promote the removal of old cuticle during molting. We show that the oxidation state of disulfide groups in the cuticle is tightly regulated during the molting cycle, and that when trxr-1 and gsr-1 function is reduced, disulfide groups in the cuticle remain oxidized. A selenocysteine-to-cysteine TRXR-1 mutant fails to rescue molting defects. Furthermore, worms lacking SELB-1, the C. elegans homolog of Escherichia coli SelB or mammalian EFsec, a translation elongation factor known to be specific for selenocysteine in E. coli, fail to incorporate selenocysteine, and display the same phenotype as those lacking trxr-1. Thus, TRXR-1 function in the reduction of old cuticle is strictly selenocysteine dependent in the nematode. Exogenously supplied reduced glutathione reduces disulfide groups in the cuticle and induces apolysis, the separation of old and new cuticle, strongly suggesting that molting involves the regulated reduction of cuticle components driven by TRXR-1 and GSR-1. Using dauer larvae, we demonstrate that aged worms have a decreased capacity to molt, and decreased expression of GSR-1. Together, our results establish a function for the selenoprotein TRXR-1 and GSR-1 in the removal of old cuticle from the surface of epidermal cells.

Caenorhabditis elegans numb inhibits endocytic recycling by binding TAT-1 aminophospholipid translocase.

Numb regulates endocytosis in many metazoans, but the mechanism by which it functions is not completely understood. Here we report that the Caenorhabditis elegans Numb ortholog, NUM-1A, a regulator of endocytic recycling, binds the C isoform of transbilayer amphipath transporter-1 (TAT-1), a P4 family adenosine triphosphatase and putative aminophospholipid translocase that is required for proper endocytic trafficking. We demonstrate that TAT-1 is differentially spliced during development and that TAT-1C-specific splicing occurs in the intestine where NUM-1A is known to function. NUM-1A and TAT-1C colocalize in vivo. We have mapped the binding site to an NXXF motif in TAT-1C. This motif is not required for TAT-1C function but is required for NUM-1A's ability to inhibit recycling. We demonstrate that num-1A and tat-1 defects are both suppressed by the loss of the activity of PSSY-1, a phosphatidylserine (PS) synthase. PS is mislocalized in intestinal cells with defects in tat-1 or num-1A function. We propose that NUM-1A inhibits recycling by inhibiting TAT-1C's ability to translocate PS across the membranes of recycling endosomes.

Tissue- and paralogue-specific functions of acyl-CoA-binding proteins in lipid metabolism in Caenorhabditis elegans.

ACBP (acyl-CoA-binding protein) is a small primarily cytosolic protein that binds acyl-CoA esters with high specificity and affinity. ACBP has been identified in all eukaryotic species, indicating that it performs a basal cellular function. However, differential tissue expression and the existence of several ACBP paralogues in many eukaryotic species indicate that these proteins serve distinct functions. The nematode Caenorhabditis elegans expresses seven ACBPs: four basal forms and three ACBP domain proteins. We find that each of these paralogues is capable of complementing the growth of ACBP-deficient yeast cells, and that they exhibit distinct temporal and tissue expression patterns in C. elegans. We have obtained loss-of-function mutants for six of these forms. All single mutants display relatively subtle phenotypes; however, we find that functional loss of ACBP-1 leads to reduced triacylglycerol (triglyceride) levels and aberrant lipid droplet morphology and number in the intestine. We also show that worms lacking ACBP-2 show a severe decrease in the ß-oxidation of unsaturated fatty acids. A quadruple mutant, lacking all basal ACBPs, is slightly developmentally delayed, displays abnormal intestinal lipid storage, and increased ß-oxidation. Collectively, the present results suggest that each of the ACBP paralogues serves a distinct function in C. elegans.

Defects in tRNA modification associated with neurological and developmental dysfunctions in Caenorhabditis elegans elongator mutants.

Elongator is a six subunit protein complex, conserved from yeast to humans. Mutations in the human Elongator homologue, hELP1, are associated with the neurological disease familial dysautonomia. However, how Elongator functions in metazoans, and how the human mutations affect neural functions is incompletely understood. Here we show that in Caenorhabditis elegans, ELPC-1 and ELPC-3, components of the Elongator complex, are required for the formation of the 5-carbamoylmethyl and 5-methylcarboxymethyl side chains of wobble uridines in tRNA. The lack of these modifications leads to defects in translation in C. elegans. ELPC-1::GFP and ELPC-3::GFP reporters are strongly expressed in a subset of chemosensory neurons required for salt chemotaxis learning. elpc-1 or elpc-3 gene inactivation causes a defect in this process, associated with a posttranscriptional reduction of neuropeptide and a decreased accumulation of acetylcholine in the synaptic cleft. elpc-1 and elpc-3 mutations are synthetic lethal together with those in tuc-1, which is required for thiolation of tRNAs having the 5'methylcarboxymethyl side chain. elpc-1; tuc-1 and elpc-3; tuc-1 double mutants display developmental defects. Our results suggest that, by its effect on tRNA modification, Elongator promotes both neural function and development.

The C. elegans P4-ATPase TAT-1 regulates lysosome biogenesis and endocytosis.

P-type adenosine triphosphatases (ATPases) of the Drs2p family (P4-ATPases) are multipass transmembrane proteins required to generate and maintain phospholipid asymmetry in membrane bilayers. In Saccharomyces cerevisiae, several members of this family control distinct transport events within the endosomal and secretory pathways. Comparatively, little is known about the functions of P4-ATPases in multicellular organisms. In this study, we analyzed the role of the Caenorhabditis elegans Drs2p homologue transbilayer amphipath transporter (TAT)-1 in intracellular trafficking. tat-1 is expressed in many tissues including the intestine, the epidermis and the nervous system. In intestinal cells, tat-1 loss-of-function mutants accumulate large vacuoles of mixed endolysosomal identity positive for the lysosomal protein LMP-1. In addition, they lack the same class of storage granules as lmp-1 mutants, suggesting that part of the tat-1 phenotype might result from LMP-1 sequestration in an aberrant compartment. Epidermal cells mutant for tat-1 contain acidified giant hybrid multivesicular bodies probably corresponding to endolysosomal intermediate compartments or deficient lysosomes. Finally, TAT-1 is required for yolk uptake in oocytes and an early step of fluid-phase endocytosis in the intestine. Hence, TAT-1 is required at multiple steps of the endolysosomal pathway, at least in part by ensuring proper trafficking of cell-specific effector proteins.

EGL-4 promotes turning behavior of C. elegans males during mating.

During mating, C. elegans males whose tails have reached the head or tail of their intended mates are able to switch to scanning the other side by performing a turn during which the male's tail curls ventrally all the while keeping in contact with the hermaphrodite. The ability to execute turns efficiently is dependent upon serotonergic neurons in the posterior ventral nerve cord that stimulate diagonal muscles by activating a serotonin receptor, SER-1. Here we show that turning behavior is abnormal in males lacking a cGMP-dependent protein kinase, EGL-4. egl-4 mutant males are also resistant to ventral tail curling induced by exogenous serotonin.

C. elegans TAT-6, a putative aminophospholipid translocase, is expressed in sujc cells in the hermaphrodite gonad.

In healthy eukaryotic cells, the two leaflets that make up plasma membranes are highly asymmetric with respect to the lipids they contain. In both unicellular eukaryotes and metazoans, the asymmetry in the distribution of aminophospholipids is maintained by P4-family transmembrane ATPases, which catalyze the movement of selected phospholipids from the outer leaflet to the inner. C. elegans has six P4-family ATPases, TAT-1 - TAT-6. TAT-1 - TAT-5 are expressed in many tissues and cells. Here we report that, in contrast, TAT-6 is much less broadly expressed and that, within the somatic gonad, expression of TAT-6 reporters is restricted to the spermathecal-uterine core cell (sujc) cells.

LRIG proteins regulate lipid metabolism via BMP signaling and affect the risk of type 2 diabetes.

Leucine-rich repeats and immunoglobulin-like domains (LRIG) proteins have been implicated as regulators of growth factor signaling; however, the possible redundancy among mammalian LRIG1, LRIG2, and LRIG3 has hindered detailed elucidation of their physiological functions. Here, we show that Lrig-null mouse embryonic fibroblasts (MEFs) are deficient in adipogenesis and bone morphogenetic protein (BMP) signaling. In contrast, transforming growth factor-beta (TGF-ß) and receptor tyrosine kinase (RTK) signaling appeared unaltered in Lrig-null cells. The BMP signaling defect was rescued by ectopic expression of LRIG1 or LRIG3 but not by expression of LRIG2. Caenorhabditis elegans with mutant LRIG/sma-10 variants also exhibited a lipid storage defect. Human LRIG1 variants were strongly associated with increased body mass index (BMI) yet protected against type 2 diabetes; these effects were likely mediated by altered adipocyte morphology. These results demonstrate that LRIG proteins function as evolutionarily conserved regulators of lipid metabolism and BMP signaling and have implications for human disease.

Caenorhabditis elegans num-1 negatively regulates endocytic recycling.

Much of the material taken into cells by endocytosis is rapidly returned to the plasma membrane by the endocytic recycling pathway. Although recycling is vital for the correct localization of cell membrane receptors and lipids, the molecular mechanisms that regulate recycling are only partially understood. Here we show that in Caenorhabditis elegans endocytic recycling is inhibited by NUM-1A, the nematode Numb homolog. NUM-1AGFP fusion protein is localized to the baso-lateral surfaces of many polarized epithelial cells, including the hypodermis and the intestine. We show that increased NUM-1A levels cause morphological defects in these cells similar to those caused by loss-of-function mutations in rme-1, a positive regulator of recycling in both C. elegans and mammals. We describe the isolation of worms lacking num-1A activity and show that, consistent with a model in which NUM-1A negatively regulates recycling in the intestine, loss of num-1A function bypasses the requirement for RME-1. Genetic epistasis analysis with rab-10, which is required at an early part of the recycling pathway, suggests that loss of num-1A function does not affect the uptake of material by endocytosis but rather inhibits baso-lateral recycling downstream of rab-10.

MAA-1, a novel acyl-CoA-binding protein involved in endosomal vesicle transport in Caenorhabditis elegans.

The budding and fission of vesicles during membrane trafficking requires many proteins, including those that coat the vesicles, adaptor proteins that recruit components of the coat, and small GTPases that initiate vesicle formation. In addition, vesicle formation in vitro is promoted by the hydrolysis of acyl-CoA lipid esters. The mechanisms by which these lipid esters are directed to the appropriate membranes in vivo, and their precise roles in vesicle biogenesis, are not yet understood. Here, we present the first report on membrane associated ACBP domain-containing protein-1 (MAA-1), a novel membrane-associated member of the acyl-CoA-binding protein family. We show that in Caenorhabditis elegans, MAA-1 localizes to intracellular membrane organelles in the secretory and endocytic pathway and that mutations in maa-1 reduce the rate of endosomal recycling. A lack of maa-1 activity causes a change in endosomal morphology. Although in wild type, many endosomal organelles have long tubular protrusions, loss of MAA-1 activity results in loss of the tubular domains, suggesting the maa-1 is required for the generation or maintenance of these domains. Furthermore, we demonstrate that MAA-1 binds fatty acyl-CoA in vitro and that this ligand-binding ability is important for its function in vivo. Our results are consistent with a role for MAA-1 in an acyl-CoA-dependent process during vesicle formation.

Treatment of Caenorhabditis elegans with Small Selenium Species Enhances Antioxidant Defense Systems.

SCOPE: Small selenium (Se) species play a key role in Se metabolism and act as dietary sources of the essential trace element. However, they are redox-active and trigger pro- and antioxidant responses. As health outcomes are strongly species-dependent, species-specific characteristics of Se compounds are tested in vivo. METHODS AND RESULTS: In the model organism Caenorhabditis elegans (C. elegans), immediate and sustained effects of selenite, selenomethionine (SeMet), and Se-methylselenocysteine (MeSeCys) are studied regarding their bioavailability, incorporation into proteins, as well as modulation of the cellular redox status. While all tested Se compounds are bioavailable, only SeMet persistently accumulates and is non-specifically incorporated into proteins. However, the protection toward chemically-induced formation of reactive species is independent of the applied Se compound. Increased thioredoxin reductase (TXNRD) activity and changes in mRNA expression levels of antioxidant proteins indicate the activation of cellular defense mechanisms. However, in txnrd-1 deletion mutants, no protective effects of the Se species are observed anymore, which is also reflected by differential gene expression data. CONCLUSION: Se species protect against chemically-induced reactive species formation. The identified immediate and sustained systemic effects of Se species give rise to speculations on possible benefits facing subsequent periods of inadequate Se intake.

Selenium species-dependent toxicity, bioavailability and metabolic transformations in Caenorhabditis elegans.

The essential micronutrient selenium (Se) is required for various systemic functions, but its beneficial range is narrow and overexposure may result in adverse health effects. Additionally, the chemical form of the ingested selenium contributes crucially to its health effects. While small Se species play a major role in Se metabolism, their toxicological effects, bioavailability and metabolic transformations following elevated uptake are poorly understood. Utilizing the tractable invertebrate Caenorhabditis elegans allowed for an alternative approach to study species-specific characteristics of organic and inorganic Se forms in vivo, revealing remarkable species-dependent differences in the toxicity and bioavailability of selenite, selenomethionine (SeMet) and Se-methylselenocysteine (MeSeCys). An inverse relationship was found between toxicity and bioavailability of the Se species, with the organic species displaying a higher bioavailability than the inorganic form, yet being less toxic. Quantitative Se speciation analysis with HPLC/mass spectrometry revealed a partial metabolism of SeMet and MeSeCys. In SeMet exposed worms, identified metabolites were Se-adenosylselenomethionine (AdoSeMet) and Se-adenosylselenohomocysteine (AdoSeHcy), while worms exposed to MeSeCys produced Se-methylselenoglutathione (MeSeGSH) and γ-glutamyl-MeSeCys (γ-Glu-MeSeCys). Moreover, the possible role of the sole selenoprotein in the nematode, thioredoxin reductase-1 (TrxR-1), was studied comparing wildtype and trxr-1 deletion mutants. Although a lower basal Se level was detected in trxr-1 mutants, Se toxicity and bioavailability following acute exposure was indistinguishable from wildtype worms. Altogether, the current study demonstrates the suitability of C. elegans as a model for Se species dependent toxicity and metabolism, while further research is needed to elucidate TrxR-1 function in the nematode.

Flagella-mediated secretion of a novel Vibrio cholerae cytotoxin affecting both vertebrate and invertebrate hosts.

Using Caenorhabditis elegans as an infection host model for Vibrio cholerae predator interactions, we discovered a bacterial cytotoxin, MakA, whose function as a virulence factor relies on secretion via the flagellum channel in a proton motive force-dependent manner. The MakA protein is expressed from the polycistronic makDCBA (motility-associated killing factor) operon. Bacteria expressing makDCBA induced dramatic changes in intestinal morphology leading to a defecation defect, starvation and death in C. elegans. The Mak proteins also promoted V. cholerae colonization of the zebrafish gut causing lethal infection. A structural model of purified MakA at 1.9 Å resolution indicated similarities to members of a superfamily of bacterial toxins with unknown biological roles. Our findings reveal an unrecognized role for V. cholerae flagella in cytotoxin export that may contribute both to environmental spread of the bacteria by promoting survival and proliferation in encounters with predators, and to pathophysiological effects during infections.

Diet-dependent depletion of queuosine in tRNAs in Caenorhabditis elegans does not lead to a developmental block.

Queuosine (Q), a hypermodified nucleoside,occurs at the wobble position of transfer RNAs (tRNAs)with GUN anticodons. In eubacteria, absence of Q affects messenger RNA (mRNA) translation and reduces the virulence of certain pathogenic strains. In animal cells,changes in the abundance of Q have been shown to correlate with diverse phenomena including stress tolerance, cell proliferation and tumour growth but the function of Q in animals is poorly understood. Animals are thought to obtain Q (or its analogues) as a micronutrient from dietary sources such as gut micro flora. However,the difficulty of maintaining animals under bacteria-free conditions on Q-deficient diets has severely hampered the study of Q metabolism and function in animals. In this study,we show that as in higher animals, tRNAs in the nematode Caenorhabditis elegans are modified by Q and its sugar derivatives. When the worms were fed on Q-deficient Escherichia coli, Q modification was absent from the worm tRNAs suggesting that C.elegans lacks a de novo pathway of Q biosynthesis. The inherent advantages of C.elegans as a model organism, and the simplicity of conferring a Q-deficient phenotype on it make it an ideal system to investigate the function of Q modification in tRNA.

Fourier transform infrared microspectroscopy for the analysis of the biochemical composition of C. elegans worms.

Changes in intermediary metabolism have profound effects on many aspects of C. elegans biology including growth, development and behavior. However, many traditional biochemical techniques for analyzing chemical composition require relatively large amounts of starting material precluding the analysis of mutants that cannot be grown in large amounts as homozygotes. Here we describe a technique for detecting changes in the chemical compositions of C. elegans worms by Fourier transform infrared microspectroscopy. We demonstrate that the technique can be used to detect changes in the relative levels of carbohydrates, proteins and lipids in one and the same worm. We suggest that Fourier transform infrared microspectroscopy represents a useful addition to the arsenal of techniques for metabolic studies of C. elegans worms.

A Caenorhabditis elegans mutant lacking functional nicotinamide nucleotide transhydrogenase displays increased sensitivity to oxidative stress.

Proton-translocating mitochondrial nicotinamide nucleotide transhydrogenase (NNT) was investigated regarding its physiological role in Caenorhabditis elegans. NNT catalyzes the reduction of NADP(+) by NADH driven by the electrochemical proton gradient, Deltap, and is thus a potentially important source of mitochondrial NADPH. Mitochondrial detoxification of reactive oxygen species (ROS) by glutathione-dependent peroxidases depends on NADPH for regeneration of reduced glutathione. Transhydrogenase may therefore be directly involved in the defense against oxidative stress. nnt-1 deletion mutants of C. elegans, nnt-1(sv34), were isolated and shown to grow essentially as wild type under normal laboratory conditions, but with a strongly lowered GSH/GSSG ratio. Under conditions of oxidative stress, caused by the superoxide-generating agent methyl viologen, growth of worms lacking nnt-1 activity was severely impaired. A similar result was obtained by using RNAi. Reintroducing nnt-1 in the nnt-1(sv34) knockout mutant led to a partial rescue of growth under oxidative stress conditions. These results provide evidence for the first time that nnt-1 is important in the defense against mitochondrial oxidative stress.

Increased or decreased levels of Caenorhabditis elegans lon-3, a gene encoding a collagen, cause reciprocal changes in body length.

Body length in C. elegans is regulated by a member of the TGFbeta family, DBL-1. Loss-of-function mutations in dbl-1, or in genes encoding components of the signaling pathway it activates, cause worms to be shorter than wild type and slightly thinner (Sma). Overexpression of dbl-1 confers the Lon phenotype characterized by an increase in body length. We show here that loss-of-function mutations in dbl-1 and lon-1, respectively, cause a decrease or increase in the ploidy of nuclei in the hypodermal syncytial cell, hyp7. To learn more about the regulation of body length in C. elegans we carried out a genetic screen for new mutations causing a Lon phenotype. We report here the cloning and characterization of lon-3. lon-3 is shown to encode a putative cuticle collagen that is expressed in hypodermal cells. We show that, whereas putative null mutations in lon-3 (or reduction of lon-3 activity by RNAi) causes a Lon phenotype, increasing lon-3 gene copy number causes a marked reduction in body length. Morphometric analyses indicate that the lon-3 loss-of-function phenotype resembles that caused by overexpression of dbl-1. Furthermore, phenotypes caused by defects in dbl-1 or lon-3 expression are in both cases suppressed by a null mutation in sqt-1, a second cuticle collagen gene. However, whereas loss of dbl-1 activity causes a reduction in hypodermal endoreduplication, the reduction in body length associated with overexpression of lon-3 occurs in the absence of defects in hypodermal ploidy.