dpy-18 encodes an alpha-subunit of prolyl-4-hydroxylase in caenorhabditis elegans

Collagen is an extracellular matrix (ECM) component encoded by a large multigene family in multicellular animals. Procollagen is post-translationally modified by prolyl-4-hydroxylase (EC 1.14.11.2) before secretion and participation in ECM formation. Therefore, collagen processing and regulation can be studied by examining this required interaction of prolyl-4-hydroxylase with procollagen. High-resolution polymorphism mapping was used to place the Caenorhabditis elegans dpy-18 gene on the physical map, and we show that it encodes a prolyl-4-hydroxylase alpha catalytic subunit. The Dpy phenotype of dpy-18(e364) amber mutants is more severe when this mutation is in trans to the noncomplementing deficiency tDf7, while the dpy-18(e499) deletion mutant exhibits the same phenotype as dpy-18(e499)/tDf7. Furthermore, dpy-18 RNA interference (RNAi) in wild-type worms results in Dpy progeny, while dpy-18 (RNAi) in dpy-18(e499) mutants does not alter the Dpy phenotype of their progeny. These observations suggest that the dpy-18 null phenotype is Dpy. A dpy-18::gfp promoter fusion construct is expressed throughout the hypodermis within the cells that abundantly produce the cuticle collagens, as well as in certain head and posterior neurons. While prolyl-4-hydroxylase has been studied extensively by biochemical techniques, this is the first report of a mutationally defined prolyl-4-hydroxylase in any animal.     

The C terminus of collagen SQT-3 has complex and essential functions in nematode collagen assembly

The nematode exoskeleton is a multilayered structure secreted by the underlying hypodermal cells and mainly composed of small collagens, which are encoded by a large gene family. In previous work, we reported analysis of the C. elegans dpy-31 locus, encoding a hypodermally expressed zinc-metalloprotease of the BMP-1/TOLLOID family essential for viability and cuticle deposition. We have generated a large set of extragenic suppressors of dpy-31 lethality, most of which we show here to be allelic to the cuticle collagen genes sqt-3 and dpy-17. We analyzed the interaction among dpy-31, sqt-3, and dpy-17 using a SQT-3-specific antiserum, which was employed in immunofluorescence experiments. Our results support a role for DPY-31 in SQT-3 extracellular processing and suggest that the SQT-3 C-terminal nontrimeric region serves multiple roles during SQT-3 assembly. Different missense mutations of this region have diverse phenotypic consequences, including cold-sensitive lethality. Furthermore, the biochemical and genetic data indicate that the extracellular assemblies of DPY-17 and SQT-3 are interdependent, most likely because the collagens are incorporated into the same cuticular substructure. We find that absence of DPY-17 causes extensive intracellular retention of SQT-3, indicating that formation of the SQT-3-DPY-17 polymer could begin in the intracellular environment before secretion.     

Molecular analysis of mutations in the Caenorhabditis elegans collagen gene dpy-7.

Collagens are a family of proteins contributing to the body structure of eukaryotes. They are encoded by a large and diverse gene family in the nematode Caenorhabditis elegans but by only a few genes in vertebrates. We have studied mutant alleles of the C. elegans dpy-7 gene, one of a large group of genes whose mutant phenotype is altered body form and several of which have previously been shown to encode cuticular collagens. We made use of the C. elegans physical map to screen specifically for collagen genes in the region of the X chromosome to which dpy-7 maps. This yielded a wild-type collagen gene clone which we showed, by micro-injection, could repair the dpy-7 mutant phenotype in transgenic animals. We cloned the homologous sequence from four dpy-7 mutant strains and by sequence analysis identified a single mutation in each case. All four mutations result in the substitution of a glycine with a larger residue in the conserved Gly-X-Y collagen domains. Similar substitutions in vertebrate collagens cause the heritable brittle bone disorder osteogenesis imperfecta. Whereas the human mutations are dominant, the dpy-7 mutations are recessive, and this may reflect different levels of complexity of collagenous macromolecular structures in the two organisms.     

Molecular and genetic analyses of the Caenorhabditis elegans dpy-2 and dpy-10 collagen genes: a variety of molecular alterations affect organismal morphology.

We have identified and cloned the Caenorhabditis elegans dpy-2 and dpy-10 genes and determined that they encode collagens. Genetic data suggested that these genes are important in morphogenesis and possibly other developmental events. These data include the morphologic phenotypes exhibited by mutants, unusual genetic interactions with the sqt-1 collagen gene, and suppression of mutations in the glp-1 and mup-1 genes. The proximity of the dpy-2 and dpy-10 genes (3.5 kilobase) and the structural similarity of their encoded proteins (41% amino acid identity) indicate that dpy-2 and dpy-10 are the result of a gene duplication event. The genes do not, however, appear to be functionally redundant, because a dpy-10 null mutant is not rescued by the dpy-2 gene. In addition, full complementation between dpy-2 and dpy-10 can be demonstrated with all recessive alleles tested in trans. Sequence analysis of several mutant alleles of each gene was performed to determine the nature of the molecular defects that can cause the morphologic phenotypes. Glycine substitutions within the Gly-X-Y portion of the collagens can result in dumpy (Dpy), dumpy, left roller (DLRol), or temperature-sensitive DLRol phenotypes. dpy-10(cn64), a dominant temperature-sensitive DLRol allele, creates an Arg-to-Cys substitution in the amino non-Gly-X-Y portion of the protein. Three dpy-10 alleles contain Tc1 insertions in the coding region of the gene. dpy-10(cg36) (DRLol) creates a nonsense codon near the end of the Gly-X-Y region. The nature of this mutation, combined with genetic data, indicates that DLRol is the null phenotype of dpy-10. The Dpy phenotype results from reduced function of the dpy-10 collagen gene. Our results indicate that a variety of molecular defects in these collagens can result in severe morphologic changes in C. elegans.     

Gene interactions in Caenorhabditis elegans define DPY-31 as a candidate procollagen C-proteinase and SQT-3/ROL-4 as its predicted major target

Zinc metalloproteases of the BMP-1/TOLLOID family (also known as astacins) are extracellular enzymes involved in important developmental processes in metazoans. We report the characterization of the Caenorhabditis elegans gene dpy-31, which encodes the first essential astacin metalloprotease identified in this organism. Loss-of-function mutations in dpy-31 result in cuticle defects, abnormal morphology, and embryonic lethality, indicating that dpy-31 is required for formation of the collagenous exoskeleton. DPY-31 is widely expressed in the hypodermal cells, which are responsible for cuticle secretion. We have investigated the dpy-31 function through reversion analysis. While complete reversion can be obtained only by intragenic suppressors, reversion of the Dpy-31 lethal phenotype also can be caused by dominant extragenic suppressors. Nine extragenic suppressors carry mutations in the uniquely essential collagen gene sqt-3, which we show is the same gene as rol-4. Most mutations exhibit the unusual property of exclusively dominant suppression and all affect the sequence of the SQT-3 collagen C terminus. This suggests that DPY-31 is responsible for C-terminal proteolytic processing of collagen trimers and is therefore a structural and functional homolog of vertebrate BMP-1. The results also demonstrate the critical importance of the collagen C-terminal sequence, which is highly conserved among all 49 members of the SQT-3 subfamily.     

A mutation in a cuticle collagen causes hypersensitivity to the endocrine disrupting chemical, bisphenol A, in Caenorhabditis elegans

A novel mutant gene, bis-1 (bisphenol A sensitive) has been isolated in the nematode, Caenorhabditis elegans, that affects the response to endocrine disrupting chemicals (EDC). The bis-1(nx3) allele is hypersensitive to bisphenol A (BPA), is allelic to a collagen gene (col-121), and is expressed in hypodermal cells. Among the collagen mutants so far studied, bis-1(nx3), dpy-2(e8), dpy-7(e88) and dpy-10(e128) showed BPA sensitivity. The isolated mutant may work as a useful tool for the assay of EDC toxicity since the physiological effect of the collagen mutation (glycine substitution) indicates an increased sensitivity to BPA.     

Identification of a novel gene family involved in osmotic stress response in Caenorhabditis elegans

Organisms exposed to the damaging effects of high osmolarity accumulate solutes to increase cytoplasmic osmolarity. Yeast accumulates glycerol in response to osmotic stress, activated primarily by MAP kinase Hog1 signaling. A pathway regulated by protein kinase C (PKC1) also responds to changes in osmolarity and cell wall integrity. C. elegans accumulates glycerol when exposed to high osmolarity, but the molecular pathways responsible for this are not well understood. We report the identification of two genes, osm-7 and osm-11, which are related members of a novel gene family. Mutations in either gene lead to high internal levels of glycerol and cause an osmotic resistance phenotype (Osr). These mutants also have an altered defecation rhythm (Dec). Mutations in cuticle collagen genes dpy-2, dpy-7, and dpy-10 cause a similar Osr Dec phenotype. osm-7 is expressed in the hypodermis and may be secreted. We hypothesize that osm-7 and osm-11 interact with the cuticle, and disruption of the cuticle causes activation of signaling pathways that increase glycerol production. The phenotypes of osm-7 are not suppressed by mutations in MAP kinase or PKC pathways, suggesting that C. elegans uses signaling pathways different from yeast to mount a response to osmotic stress.     

Properties of a Class of Genes Required for Ray Morphogenesis in Caenorhabditis Elegans

We have identified eight mutations that define at least five terminal differentiation genes (ram genes) whose products are required during the extension of the male-specific ray sensilla in Caenorhabditis elegans. ram gene mutations result in morphological abnormalities in the sensory rays but do not appear to interfere with ray functions. A similar ray morphology phenotype was observed in males harboring mutations in three previously defined genes, dpy-11, dpy-18 and sqt-1, that also affect body shape. One of these genes, sqt-1, is known to encode a collagen. Mutations in different ram genes failed to complement, from which we infer that their gene products functionally interact. For one ram gene, failure to complement was shown to result from haploinsufficiency. Intergenic noncomplementation did not extend to the body morphology genes. The temperature-sensitive periods of both ram and body morphology mutations corresponded to the period of development in which ray extension occurs. We propose that ram gene products act together in a critical interaction between the rays and the cuticle required for wild-type ray morphology.     

A novel zinc-carboxypeptidase SURO-1 regulates cuticle formation and body morphogenesis in Caenorhabditis elegans

Cuticle formation and molting are critical for the development of Caenorhabditis elegans. To understand cuticle formation more clearly, we screened for suppressors in transgenic worms that expressed dominant ROL-6 collagen proteins. The suro-1 mutant, which is mild dumpy, exhibited a different ROL-6::GFP localization pattern compared to other Dpy mutants. We identified mutations in three suro-1 mutants, and found that suro-1 (ORF R11A5.7) encodes a putative zinc-carboxypeptidase homologue. The expression of this enzyme in the hypodermis and the genetic interactions between this enzyme and other collagen-modifying enzyme mutants suggest a regulatory role in collagen processing and cuticle organization for this novel carboxypeptidase. These findings aid our understanding of cuticle formation during worm development.     

Molecular cloning and characterization of the dpy-20 gene of Caenorhabditis elegans

We describe the molecular analysis of the dpy20 gene in Caenorhabditis elegans. Isolation of genomic sequences was facilitated by the availability of a mutation that resulted from insertion of a Tc1 transposable element into the dpy-20 gene. The Tc1 insertion site in the m474:: Tc1 allele was identified and was found to lie within the coding region of dpy-20. Three revertants (two wild-type and one partial revertant) resulted from the excision of this Tc1 element. Genomic dpy-20 clones were isolated from a library of wild-type DNA and were found to lie just to the left of the unc-22 locus on the physical map, compatible with the position of dpy-20 on the genetic map. Cosmid DNA containing the dpy-20 gene was successfully used to rescue the mutant phenotype of animals homozygous for another dpy-20 allele, e1282ts. Sequence analysis of the putative dpy-20 homologue in Caenorhabditis briggsae was performed to confirm identification of the coding regions of the C. elegans gene and to identify conserved regulatory regions. Sequence analysis of dpy-20 revealed that it was not similar to other genes encoding known cuticle components such as collagen or cuticulin. The dpy-20 gene product, therefore, identifies a previously unknown type of protein that may be directly or indirectly involved in cuticle function. Northern blot analysis showed that dpy-20 is expressed predominantly in the second larval stage and that the mRNA is not at all abundant. Data from temperature shift studies using the temperature-sensitive allele e1282ts showed that the sensitive period also occurs at approximately the second larval stage. Therefore, expression of dpy-20 mRNA and function of the DPY-20 protein are closely linked temporally.