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.     

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.     

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.     

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.     

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.     

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.     

Prolyl 4-hydroxylase is an essential procollagen-modifying enzyme required for exoskeleton formation and the maintenance of body shape in the nematode Caenorhabditis elegans

The multienzyme complex prolyl 4-hydroxylase catalyzes the hydroxylation of proline residues and acts as a chaperone during collagen synthesis in multicellular organisms. The beta subunit of this complex is identical to protein disulfide isomerase (PDI). The free-living nematode Caenorhabditis elegans is encased in a collagenous exoskeleton and represents an excellent model for the study of collagen biosynthesis and extracellular matrix formation. In this study, we examined prolyl 4-hydroxylase alpha-subunit (PHY; EC 1.14.11.2)- and beta-subunit (PDI; EC 5.3.4.1)-encoding genes with respect to their role in collagen modification and formation of the C. elegans exoskeleton. We identified genes encoding two PHYs and a single associated PDI and showed that all three are expressed in collagen-synthesizing ectodermal cells at times of maximal collagen synthesis. Disruption of the pdi gene via RNA interference resulted in embryonic lethality. Similarly, the combined phy genes are required for embryonic development. Interference with phy-1 resulted in a morphologically dumpy phenotype, which we determined to be identical to the uncharacterized dpy-18 locus. Two dpy-18 mutant strains were shown to have null alleles for phy-1 and to have a reduced hydroxyproline content in their exoskeleton collagens. This study demonstrates in vivo that this enzyme complex plays a central role in extracellular matrix formation and is essential for normal metazoan development.     

Analysis of Mutations in the Sqt-1 and Rol-6 Collagen Genes of Caenorhabditis Elegans

Different mutations in the sqt-1 and rol-6 collagen genes of Caenorhabditis elegans can cause diverse changes in body morphology and display different genetic attributes. We have determined the nucleotide alterations in 15 mutant alleles of these genes. Three mutations in sqt-1 and one in rol-6 that cause dominant right-handed helical twisting (RRol) of animals are arginine to cysteine replacements. These mutations are all within a short conserved sequence, on the amino terminal side of the Gly-X-Y repeats, that is found in all C. elegans cuticle collagens. A recessive RRol mutation of rol-6 is a replacement of one of the same conserved arginines by histidine. In contrast, three sqt-1 mutations that cause recessive left-handed helical twisting (LRol) are replacements of a conserved carboxyterminal cysteine residue with either tyrosine or serine. These results suggest that disulfide bonding is important in collagen organization and that a deficit or surplus of disulfides may cause cuticle alterations of opposite handedness. In contrast to other collagens, glycine replacement mutations in the Gly-X-Y repeats of sqt-1 cause very mild phenotypes. Nonsense mutations of both sqt-1 and rol-6 cause nearly, but not totally, wild-type phenotypes. A nonsense mutation in sqt-1 suppresses the phenotype of rol-6 RRol mutations, suggesting that rol-6 collagen function is dependent on the presence of sqt-1 collagen. Mutations of sqt-1 are not suppressed by a rol-6 nonsense mutation, however, indicating that sqt-1 collagen can function independently of rol-6.     

The Dpy-30 Gene Encodes an Essential Component of the Caenorhabditis Elegans Dosage Compensation Machinery

The need to regulate X chromosome expression in Caenorhabditis elegans arises as a consequence of the primary sex-determining signal, the X/A ratio (the ratio of X chromosomes to sets of autosomes), which directs 1X/2A animals to develop as males and 2X/2A animals to develop as hermaphrodites. C. elegans possesses a dosage compensation mechanism that equalizes X chromosome expression between the two sexes despite their disparity in X chromosome dosage. Previous genetic analysis led to the identification of four autosomal genes, dpy-21, dpy-26, dpy-27 and dpy-28, whose products are essential in XX animals for proper dosage compensation, but not for sex determination. We report the identification and characterization of dpy-30, an essential component of the dosage compensation machinery. Putative null mutations in dpy-30 disrupt dosage compensation and cause a severe maternal-effect, XX-specific lethality. Rare survivors of the dpy-30 lethality are dumpy and express their X-linked genes at higher than wild-type levels. These dpy-30 mutant phenotypes superficially resemble those caused by mutations in dpy-26, dpy-27 and dpy-28; however, detailed phenotypic analysis reveals important differences that distinguish dpy-30 from these genes. In contrast to the XX-specific lethality caused by mutations in the other dpy genes, the XX-specific lethality caused by dpy-30 mutations is completely penetrant and temperature sensitive. In addition, unlike the other genes, dpy-30 is required for the normal development of XO animals. Although dpy-30 mutations do not significantly affect the viability of XO animals, they do cause them to be developmentally delayed and to possess numerous morphological and behavioral abnormalities. Finally, dpy-30 mutations can dramatically influence the choice of sexual fate in animals with an ambiguous sexual identity, despite having no apparent effect on the sexual phenotype of otherwise wild-type animals. Paradoxically, depending on the genetic background, dpy-30 mutations cause either masculinization or feminization, thus revealing the complex regulatory relationship between the sex determination and dosage compensation processes. The novel phenotypes caused by dpy-30 mutations suggest that in addition to acting in the dosage compensation process, dpy-30 may play a more general role in the development of both XX and XO animals.     

Genetic Studies of Unusual Loci That Affect Body Shape of the Nematode CAENORHABDITIS ELEGANS and May Code for Cuticle Structural Proteins

In Caenorhabditis elegans, four loci (sqt-1, sqt-2, sqt-3 and rol-8) in which mutations affect body shape and cuticle morphology have unusual genetic properties. Mutant alleles of sqt-1 can interact to produce animals with a variety of mutant phenotypes: left roller, right roller, dumpy and long. At least three mutant phenotypes are specified by mutations in the sqt-3 locus. Most alleles at these loci are either dominant or cryptic dominant (i.e., are dominant only in certain genetic backgrounds). Most alleles of these loci exhibit codominance. Two putative null alleles of the sqt-1 locus produce a wild-type phenotype. Many alleles of these genes demonstrate unusual intergenic interactions that are not the result of simple epistasis: animals doubly heterozygous for mutations at two loci often display unexpected and unpredictable phenotypes. We suggest that these genetic properties might be expected of genes, such as the collagen genes, the products of which interact to form the animal's cuticle, and which are member genes of a gene family.     

Collagens, modifying enzymes and their mutations in humans, flies and worms

Collagens and proteins with collagen-like domains form large superfamilies in various species, and the numbers of known family members are increasing constantly. Vertebrates have at least 27 collagen types with 42 distinct polypeptide chains, >20 additional proteins with collagen-like domains and approximately 20 isoenzymes of various collagen-modifying enzymes. Caenorhabditis elegans has approximately 175 cuticle collagen polypeptides and two basement membrane collagens. Drosophila melanogaster has far fewer collagens than many other species but has approximately 20 polypeptides similar to the catalytic subunits of prolyl 4-hydroxylase, the key enzyme of collagen synthesis. More than 1300 mutations have so far been characterized in 23 of the 42 human collagen genes in various diseases, and many mouse models and C. elegans mutants are also available to analyse the collagen gene family and their modifying enzymes.     

Genetic Mapping of CAENORHABDITIS ELEGANS Collagen Genes Using DNA Polymorphisms as Phenotypic Markers

In Caenorhabditis elegans collagens comprise a dispersed family of 40-150 genes, the majority of which probably code for collagen proteins found in the animal's cuticle. The conserved (Gly-X-Y)n triple helix coding sequence of collagen genes has facilitated the isolation of a large number of C. elegans collagen genes by recombinant DNA methods. We have begun a study of the chromosomal organization of these genes by screening laboratory strains of C. elegans for DNA polymorphisms in the regions surrounding collagen genes. Polymorphisms near seven genes have been identified and have been used as phenotypic markers in genetic crosses to assign the genes to linkage groups II, III, IV, and X. Four genes are shown by multifactor crosses to map to a 2-3 map unit interval between unc-24 and unc-22 on chromosome IV.     

The Caenorhabditis elegans rol-6 gene, which interacts with the sqt-1 collagen gene to determine organismal morphology, encodes a collagen.

The rol-6 gene is one of the more than 40 loci in Caenorhabditis elegans that primarily affect organismal morphology. Certain mutations in the rol-6 gene produce animals that have the right roller phenotype, i.e., they are twisted into a right-handed helix. The rol-6 gene interacts with another gene that affects morphology, sqt-1; a left roller allele of sqt-1 acts as a dominant suppressor of a right roller allele of rol-6. The sqt-1 gene has previously been shown to encode a collagen. We isolated and sequenced the rol-6 gene and found that it also encodes a collagen. The rol-6 gene was identified by physical mapping of overlapping chromosomal deficiencies that cover the gene and by identification of an allele-specific restriction site alteration. The amino acid sequence of the collagen encoded by rol-6 is more similar to that of the sqt-1 collagen than to any of the other ten C. elegans cuticle collagen sequences compared. The locations of cysteine residues flanking the Gly-X-Y repeat regions of rol-6 and sqt-1 are identical, but differ from those in the other collagens. The sequence similarities between rol-6 and sqt-1 indicate that they represent a new collagen subfamily in C. elegans. These findings suggest that these two collagens physically interact, possibly explaining the genetic interaction seen between the rol-6 and sqt-1 genes.     

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.     

Genes That Implement the Hermaphrodite Mode of Dosage Compensation in Caenorhabditis Elegans

We report a genetic characterization of several essential components of the dosage compensation process in Caenorhabditis elegans. Mutations in the genes dpy-26, dpy-27, dpy-28, and the newly identified gene dpy-29 disrupt dosage compensation, resulting in elevated X-linked gene expression in XX animals and an incompletely penetrant maternal-effect XX-specific lethality. These dpy mutations appear to cause XX animals to express each set of X-linked genes at a level appropriate for XO animals. XO dpy animals are essentially wild type. Both the viability and the level of X-linked gene expression in XX animals carrying mutations in two or more dpy genes are the same as in animals carrying only a single mutation, consistent with the view that these genes act together in a single process (dosage compensation). To define a potential time of action for the gene dpd-28 we performed reciprocal temperature-shift experiments with a heat sensitive allele. The temperature-sensitive period for lethality begins 5 hr after fertilization at the 300-cell stage and extends to about 9 hr, a point well beyond the end of cell proliferation. This temperature-sensitive period suggests that dosage compensation is functioning in XX animals by mid-embryogenesis, when many zygotically transcribed genes are active. While mutations in the dpy genes have no effect on the sexual phenotype of otherwise wild-type XX or XO animals, they do have a slight feminizing effect on animals whose sex-determination process is already genetically perturbed. The opposite directions of the feminizing effects on sex determination and the masculinizing effects on dosage compensation caused by the dpy mutations are inconsistent with the wild-type dpy genes acting to coordinately control both processes. Instead, the feminizing effects are most likely an indirect consequence of disruptions in dosage compensation caused by the dpy mutations. Based on the cumulative evidence, the likely mechanism of dosage compensation in C. elegans involves reducing X-linked gene expression in XX animals to equal that in XO animals via the action of the dpy genes.