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.     

Glypican LON-2 is a conserved negative regulator of BMP-like signaling in Caenorhabditis elegans

Bone morphogenetic protein (BMP) pathways are required for a wide variety of developmental and homeostatic decisions, and mutations in signaling components are associated with several diseases. An important aspect of BMP control is the extracellular regulation of these pathways. We show that LON-2 negatively regulates a BMP-like signaling pathway that controls body length in C. elegans. lon-2 acts genetically upstream of the BMP-like gene dbl-1, and loss of lon-2 function results in animals that are longer than normal. LON-2 is a conserved member of the glypican family of heparan sulfate proteoglycans, a family with several members known to regulate growth-factor signaling in many organisms. LON-2 is functionally conserved because the Drosophila glypican gene dally rescues the lon-2(lf) body-size defect. We show that the LON-2 protein binds BMP2 in vitro, and a mutant variation of LON-2 found in lon-2(e2140) animals diminishes this interaction. We propose that LON-2 binding to DBL-1 negatively regulates this pathway in C. elegans by attenuating ligand-receptor interactions. This is the first report of a glypican directly interacting with a growth-factor pathway in C. elegans and provides a mechanistic model for glypican regulation of growth-factor pathways.     

RNT-1, the C. elegans homologue of mammalian RUNX transcription factors, regulates body size and male tail development

The rnt-1 gene is the only Caenorhabditis elegans homologue of the mammalian RUNX genes. Several lines of molecular biological evidence have demonstrated that the RUNX proteins interact and cooperate with Smads, which are transforming growth factor-beta (TGF-beta) signal mediators. However, the involvement of RUNX in TGF-beta signaling has not yet been supported by any genetic evidence. The Sma/Mab TGF-beta signaling pathway in C. elegans is known to regulate body length and male tail development. The rnt-1(ok351) mutants show the characteristic phenotypes observed in mutants of the Sma/Mab pathway, namely, they have a small body size and ray defects. Moreover, RNT-1 can physically interact with SMA-4 which is one of the Smads in C. elegans, and double mutant animals containing both the rnt-1(ok351) mutation and a mutation in a known Sma/Mab pathway gene displayed synergism in the aberrant phenotypes. In addition, lon-1(e185) mutants was epistatic to rnt-1(ok351) mutants in terms of long phenotype, suggesting that lon-1 is indeed downstream target of rnt-1. Our data reveal that RNT-1 functionally cooperates with the SMA-4 proteins to regulate body size and male tail development in C. elegans.     

Hypodermal expression of Caenorhabditis elegans TGF-beta type I receptor SMA-6 is essential for the growth and maintenance of body length.

There are several transforming growth factor-beta (TGF-beta) pathways in the nematode Caenorhabditis elegans. One of these pathways regulates body length and is composed of the ligand DBL-1, serine/threonine protein kinase receptors SMA-6 and DAF-4, and cytoplasmic signaling components SMA-2, SMA-3, and SMA-4. To further examine the molecular mechanisms of body-length regulation in the nematode by the TGF-beta pathway, we examined the regional requirement for the type-I receptor SMA-6. Using a SMA-6::GFP (green fluorescent protein) reporter gene, sma-6 was highly expressed in the hypodermis, unlike the type-II receptor DAF-4, which is reported to be ubiquitously expressed. We then examined the ability of SMA-6 expression in different regions of the C. elegans body to rescue the sma-6 phenotype (small) and found that hypodermal expression of SMA-6 is necessary and sufficient for the growth and maintenance of body length. We also demonstrate that GATA sequences in the sma-6 promoter contribute to the hypodermal expression of sma-6.     

Molecular cloning of lin-29, a heterochronic gene required for the differentiation of hypodermal cells and the cessation of molting in C.elegans.

The lin-29 gene product of C.elegans activates a temporal developmental switch for hypodermal cells. Loss-of-function lin-29 mutations result in worms that fail to execute a stage-specific pattern of hypodermal differentiation that includes exist from the cell cycle, repression of larval cuticle genes, activation of adult cuticle genes, and the cessation of molting. Combined genetic and physical mapping of restriction fragment length polymorphisms (RFLPs) was used to identify the lin-29 locus. A probe from the insertion site of a Tc1 (maP1), closely linked and to the left of lin-29 on the genetic map, was used to identify a large set of overlapping cosmid, lambda and yeast artificial chromosome (YAC) clones assembled as part of the C.elegans physical mapping project. Radiolabeled DNA from one YAC clone identified two distinct allele-specific alterations that cosegregated with the lin-29 mutant phenotype in lin-29 intragenic recombinants. lin-29 sequences were severely under-represented in all cosmid and lambda libraries tested, but were readily cloned in a YAC vector, suggesting that the lin-29 region contains sequences incompatible with standard prokaryotic cloning techniques.     

Regulation of body length and male tail ray pattern formation of Caenorhabditis elegans by a member of TGF-beta family

We have identified a new member of the TGF-beta superfamily, CET-1, from Caenorhabditis elegans, which is expressed in the ventral nerve cord and other neurons. cet-1 null mutants have shortened bodies and male tail abnormal phenotype resembling sma mutants, suggesting cet-1, sma-2, sma-3 and sma-4 share a common pathway. Overexpression experiments demonstrated that cet-1 function requires wild-type sma genes. Interestingly, CET-1 appears to affect body length in a dose-dependent manner. Heterozygotes for cet-1 displayed body lengths ranging between null mutant and wild type, and overexpression of CET-1 in wild-type worms elongated body length close to lon mutants. In male sensory ray patterning, lack of cet-1 function results in ray fusions. Epistasis analysis revealed that mab-21 lies downstream and is negatively regulated by the cet-1/sma pathway in the male tail. Our results show that cet-1 controls diverse biological processes during C. elegans development probably through different target genes.     

Caenorhabditis elegans PMR1, a P-type calcium ATPase, is important for calcium/manganese homeostasis and oxidative stress response

The Caenorhabditis elegans PMR1, a P-type Ca2+/Mn2+ ATPase, is expressed in hypodermal seam cells, intestinal cells and spermatheca; localized in Golgi complex. Knock down of pmr-1 as well as overexpression of truncated Caenorhabditis elegans PMR1, which mimics dominant mutations observed in human Hailey-Hailey disease, renders the worm highly sensitive to EGTA and Mn2+. Interestingly, pmr-1 knock down not only causes animals to become resistant to oxidative stress but also suppresses high reactive oxygen species sensitivity of smf-3 RNA-mediated interference and daf-16 worms. These findings suggest that C. elegans PMR1 has important roles in Ca2+ and Mn2+ homeostasis and oxidative stress response.     

FGF signaling functions in the hypodermis to regulate fluid balance in C. elegans

Signaling by the Caenorhabditis elegans fibroblast growth factor receptor EGL-15 is activated by LET-756, a fibroblast growth factor, and attenuated by CLR-1, a receptor tyrosine phosphatase. Hyperactive EGL-15 signaling results in a dramatic Clr phenotype characterized by the accumulation of clear fluid within the pseudocoelomic space, suggesting that regulated EGL-15 signaling is essential for fluid homeostasis in C. elegans. To determine the cellular focus of EGL-15 signaling, we identified an enhancer element (e15) within the egl-15 promoter, which is both necessary for the promoter activity and sufficient when duplicated to drive either egl-15 or clr-1 rescue activity. This enhancer drives GFP expression in hypodermal cells. Consistent with this finding, immunofluorescence studies of EGL-15 indicate that EGL-15 is expressed in hypodermal cells, and hypodermal promoters can drive full clr-1 and egl-15 rescue activity. Moreover, a mosaic analysis of mpk-1, which acts downstream of egl-15, suggests that its suppression of Clr (Soc) function is required in the hypodermis. These results suggest that EGL-15 and CLR-1 act in the hypodermis to regulate fluid homeostasis in worms.     

M142.2 (cut-6), a novel Caenorhabditis elegans matrix gene important for dauer body shape

The cuticle of the nematode Caenorhabditis elegans is a collagenous extracellular matrix which forms the exoskeleton and defines the shape of the worm. We have characterized the C. elegans gene M142.2, and we show that this is a developmentally regulated gene important for cuticle structure. Transgenic worms expressing M142.2 promoter fused to green fluorescent protein showed that M142.2 is expressed in late embryos and L2d predauers, in the hypodermal cells which synthesize the cuticle. The same temporal pattern was seen by RT-PCR using RNA purified from specific developmental stages. A recombinant fragment of M142.2 was expressed in Escherichia coli and used to raise an antiserum. Immunohistochemistry using the antiserum localized M142.2 to the periphery of the alae of L1 and dauers, forming two longitudinal ribbons over the hypodermal cells. Loss-of-function of M142.2 by RNAi resulted in a novel phenotype: dumpy dauers which lacked alae. M142.2 therefore plays a major role in the assembly of the alae and the morphology of the dauer cuticle; because of its similarity to the other cut genes of the cuticle, we have named the gene cut-6.     

Caenorhabditis elegans teneurin, ten-1, is required for gonadal and pharyngeal basement membrane integrity and acts redundantly with integrin ina-1 and dystroglycan dgn-1

The Caenorhabditis elegans teneurin ortholog, ten-1, plays an important role in gonad and pharynx development. We found that lack of TEN-1 does not affect germline proliferation but leads to local basement membrane deficiency and early gonad disruption. Teneurin is expressed in the somatic precursor cells of the gonad that appear to be crucial for gonad epithelialization and basement membrane integrity. Ten-1 null mutants also arrest as L1 larvae with malformed pharynges and disorganized pharyngeal basement membranes. The pleiotropic phenotype of ten-1 mutant worms is similar to defects found in basement membrane receptor mutants ina-1 and dgn-1 as well as in the mutants of the extracellular matrix component laminin, epi-1. We show that the ten-1 mutation is synthetic lethal with mutations of genes encoding basement membrane components and receptors due to pharyngeal or hypodermal defects. This indicates that TEN-1 could act redundantly with integrin INA-1, dystroglycan DGN-1, and laminin EPI-1 in C. elegans development. Moreover, ten-1 deletion sensitizes worms to loss of nidogen nid-1 causing a pharynx unattached phenotype in ten-1;nid-1 double mutants. We conclude that TEN-1 is important for basement membrane maintenance and/or adhesion in particular organs and affects the function of somatic gonad precursor cells.     

Single-minded and the evolution of the ventral midline in arthropods

Glypicans are multifunctional proteoglycans with regulatory roles in several intercellular signaling pathways. Here, we examine the functional requirements for glypican regulation of bone morphogenetic protein (BMP)-mediated body length in C. elegans. We provide evidence that two parts of C. elegans glypican LON-2 can independently inhibit BMP signaling in vivo: the N-terminal furin protease product and the C-terminal region containing heparan sulfate attachment sequences. While the C-terminal protease product is dispensable for LON-2 minimal core protein activity, it does affect the localization of LON-2. Cleavage of LON-2 into two parts at the conserved furin protease site is not required for LON-2 to inhibit BMP-like signaling. The glycosyl-phosphatidylinositol (GPI) membrane anchor is also not absolutely required for LON-2 activity. Finally, we show that an RGD protein-protein interaction motif in the LON-2 N-terminal domain is necessary for LON-2 core protein activity, suggesting that LON-2 inhibits BMP signaling by acting as a scaffold for BMP and an RGD-binding protein.     

Present and not reporting for duty: dsRNAi in mammalian cells

Double‐stranded RNA interference (dsRNAi) represents a primary means of anti‐viral defense in plants, worms, and insects, yet appears mostly supplanted by the protein‐based interferon (IFN) response in vertebrates such as mammals. The degree to which dsRNAi is anti‐viral in mammals has been contentious. Maillard et al ( 2016 ) find that dsRNAi retains sequence‐specific silencing in mammalian cells incapable of triggering an IFN response, suggesting that dsRNAi is inhibited by the action of interferon‐stimulated genes. Importantly, they observe that while dsRNA can “vaccinate” against the incoming cognate virus though dsRNAi silencing, no dsRNAi is observed with viral infection alone, suggesting that this evolutionarily conserved anti‐viral pathway is present but functionally elusive in the cell types studied thus far.     

Heme utilization in the Caenorhabditis elegans hypodermal cells is facilitated by heme-responsive gene-2

The roundworm Caenorhabditis elegans is a heme auxotroph that requires the coordinated actions of HRG-1 heme permeases to transport environmental heme into the intestine and HRG-3, a secreted protein, to deliver intestinal heme to other tissues including the embryo. Here we show that heme homeostasis in the extraintestinal hypodermal tissue was facilitated by the transmembrane protein HRG-2. Systemic heme deficiency up-regulated hrg-2 mRNA expression over 200-fold in the main body hypodermal syncytium, hyp 7. HRG-2 is a type I membrane protein that binds heme and localizes to the endoplasmic reticulum and apical plasma membrane. Cytochrome heme profiles are aberrant in HRG-2-deficient worms, a phenotype that was partially suppressed by heme supplementation. A heme-deficient yeast strain, ectopically expressing worm HRG-2, revealed significantly improved growth at submicromolar concentrations of exogenous heme. Taken together, our results implicate HRG-2 as a facilitator of heme utilization in the Caenorhabditis elegans hypodermis and provide a mechanism for the regulation of heme homeostasis in an extraintestinal tissue.     

Cloning and developmental expression analysis of ltd-1, the Caenorhabditis elegans homologue of the mouse kyphoscoliosis (ky) gene

We have characterized the developmental expression pattern of the Caenorhabditis elegans homologue of the mouse ky gene. The Ky protein has a putative key function in muscle development and has homologues in invertebrates, fungi and a cyanobacterium. The C. elegans Ky homologue gene has been named ltd-1 for LIM and transglutaminase domains gene. The LTD-1::GFP construct is expressed in developing hypodermal cells from the twofold stage embryo through adulthood. These data define the ltd-1 gene as a novel marker for C. elegans epithelial cell development.     

mag-1, a homolog of Drosophila mago nashi, regulates hermaphrodite germ-line sex determination in Caenorhabditis elegans

The Caenorhabditis elegans gene mag-1 can substitute functionally for its homolog mago nashi in Drosophila and is predicted to encode a protein that exhibits 80% identity and 88% similarity to Mago nashi (P. A. Newmark et al., 1997, Development 120, 3197-3207). We have used RNA-mediated interference (RNAi) to analyze the phenotypic consequences of impairing mag-1 function in C. elegans. We show here that mag-1(RNAi) causes masculinization of the germ line (Mog phenotype) in RNA-injected hermaphrodites, suggesting that mag-1 is involved in hermaphrodite germ-line sex determination. Epistasis analysis shows that ectopic sperm production caused by mag-1(RNAi) is prevented by loss-of-function (lf) mutations in fog-2, gld-1, fem-1, fem-2, fem-3, and fog-1, all of which cause germ-line feminization in XX hermaphrodites, but not by a her-1(lf) mutation which causes germ-line feminization only in XO males. These results suggest that mag-1 interacts with the fog, fem, and gld genes and acts independently of her-1. We propose that mag-1 normally allows oogenesis by inhibiting function of one or more of these masculinizing genes, which act during the fourth larval stage to promote transient sperm production in the hermaphrodite germ line. When the Mog phenotype is suppressed by a fog-2(lf) mutation, mag-1(RNAi) also causes lethality in the progeny embryos of RNA-injected, mated hermaphrodites, suggesting an essential role for mag-1 during embryogenesis. The defective embryos arrest during morphogenesis with an apparent elongation defect. The distribution pattern of a JAM-1::GFP reporter, which is localized to boundaries of hypodermal cells, shows that hypodermis is disorganized in these embryos. The temporal expression pattern of the mag-1 gene prior to and during morphogenesis appears to be consistent with an essential role of mag-1 in embryonic hypodermal organization and elongation.     

The Caenorhabditis elegans pumilio homolog, puf-9, is required for the 3'UTR-mediated repression of the let-7 microRNA target gene, hbl-1

The Puf family of RNA-binding proteins directs cell fates by regulating gene expression at the level of translation and RNA stability. Here, we report that the Caenorhabditis elegans pumilio homolog, puf-9, controls the differentiation of epidermal stem cells at the larval-to-adult transition. Genetic analysis reveals that loss-of-function mutations in puf-9 enhance the lethality and heterochronic phenotypes caused by mutations in the let-7 microRNA (miRNA), while suppressing the heterochronic phenotypes of lin-41, a let-7 target and homolog of Drosophila Brat. puf-9 interacts with another known temporal regulator hbl-1, the Caenorhabditis elegans ortholog of hunchback. We present evidence demonstrating that puf-9 is required for the 3'UTR-mediated regulation of hbl-1, in both the hypodermis and the ventral nerve cord. Finally, we show that this regulation is dependent on a region of the hbl-1 3'UTR that contains putative Puf family binding sites as well as binding sites for the let-7 miRNA family, suggesting that puf-9 and let-7 may mediate hypodermal seam cell differentiation by regulating common targets.     

Metalloproteases with EGF, CUB, and thrombospondin-1 domains function in molting of Caenorhabditis elegans

Functional analysis using RNAi was performed on eleven genes for metalloproteases of the M12A family in Caenorhabditis elegans and the interference of the C17G1.6 gene (nas-37) was found to cause incomplete molting. The RNAi of the C26C6.3 gene (nas-36) also caused a similar molting defect but not so severely as that of the nas-37 gene. Both the genes encode an astacin-like metalloprotease with an epidermal growth factor (EGF)-like domain, a CUB domain, and a thrombospondin-1 domain, in this order. The promoter-driven green fluorescent protein (GFP) expression analysis suggested that they are expressed in hypodermal cells throughout the larval stages and in the vulva of adult animals. In the genetic background of rde-1(ne219), where RNAi does not work, the molting defect caused by the nas-37 interference was observed when the transgenic wild-type rde-1 gene was expressed under the control of the dpy-7 promoter, known to be active in the hypodermal cells, but not under the control of the myo-3 promoter, active in the muscular cells. Therefore these proteases are thought to be secreted by the hypodermal cells and to participate in shedding of old cuticles.     

CeHMT-1, a putative phytochelatin transporter, is required for cadmium tolerance in Caenorhabditis elegans

Phytochelatins (PCs), (gamma-Glu-Cys)n Gly polymers that were formerly considered to be restricted to plants and some fungal systems, are now known to play a critical role in heavy metal (notably Cd2+) detoxification in Caenorhabditis elegans. In view of the functional equivalence of the gene encoding C. elegans PC synthase 1, ce-pcs-1, to its homologs from plant and fungal sources, we have gone on to explore processes downstream of PC fabrication in this organism. Here we describe the identification of a half-molecule ATP-binding cassette transporter, CeHMT-1, from C. elegans with an equivalent topology to that of the putative PC transporter SpHMT-1 from Schizosaccharomyces pombe. At one level, CeHMT-1 satisfies the requirements of a Cd2+ tolerance factor involved in the sequestration and/or elimination of Cd x PC complexes. Heterologous expression of cehmt-1 in S. pombe alleviates the Cd2+-hypersensitivity of hmt- mutants concomitant with the localization of CeHMT-1 to the vacuolar membrane. Suppression of the expression of ce-hmt-1 in intact worms by RNA interference (RNAi) confers a Cd2+-hypersensitive phenotype similar to but more pronounced than that exhibited by ce-pcs-1 RNAi worms. At another level, it is evident from comparisons of the cell morphology of ce-hmt-1 and cepcs-1 single and double RNAi mutants that CeHMT-1 also contributes to Cd2+ tolerance in other ways. Whereas the intestinal epithelial cells of ce-pcs-1 RNAi worms undergo necrosis upon exposure to toxic levels of Cd2+, the corresponding cells of ce-hmt-1 RNAi worms instead elaborate punctate refractive inclusions within the vicinity of the nucleus. Moreover, a deficiency in CeHMT-1 does not interfere with the phenotype associated with CePCS-1 deficiency and vice versa. Double ce-hmt-1; ce-pcs-1 RNAi mutants exhibit both cell morphologies when exposed to Cd2+. These results and those from our previous investigations of the requirement for PC synthase for heavy metal tolerance in C. elegans demonstrate PC-dependent, HMT-1-mediated heavy metal detoxification not only in S. pombe but also in some invertebrates while at the same time indicating that the action of CeHMT-1 does not depend exclusively on PC synthesis.