Two sets of interacting collagens form functionally distinct substructures within a Caenorhabditis elegans extracellular matrix

A ubiquitous feature of collagens is protein interaction, the trimerization of monomers to form a triple helix followed by higher order interactions during the formation of the mature extracellular matrix. The Caenorhabditis elegans cuticle is a complex extracellular matrix consisting predominantly of cuticle collagens, which are encoded by a family of approximately 154 genes. We identify two discrete interacting sets of collagens and show that they form functionally distinct matrix substructures. We show that mutation in or RNA-mediated interference of a gene encoding a collagen belonging to one interacting set affects the assembly of other members of that set, but not those belonging to the other set. During cuticle synthesis, the collagen genes are expressed in a distinct temporal series, which we hypothesize exists to facilitate partner finding and the formation of appropriate interactions between encoded collagens. Consistent with this hypothesis, we find for the two identified interacting sets that the individual members of each set are temporally coexpressed, whereas the two sets are expressed approximately 2 h apart during matrix synthesis.     

Number and organization of collagen genes in Caenorhabditis elegans.

We analyzed the number and organization of collagen genes in the nematode Caenorhabditis elegans. Genomic Southern blot hybridization experiments and recombinant phage library screenings indicated that C. elegans has between 40 and 150 distinct collagen genes. A large number of recombinant phages containing collagen genes were isolated from C. elegans DNA libraries. Physical mapping studies indicated that most phage contained a single small collagen gene less than 3 kilobases in size. A few phage contained multiple collagen hybridizing regions and may contain a larger collagen gene or several tightly linked small collagen genes. No overlaps were observed between phages containing different collagen genes, implying that the genes are dispersed in the C. elegans genome. Consistent with the small size of most collagen genes, we found that the predominant class of collagen mRNA in C. elegans is 1.2 to 1.4 kilobases in length. Genomic Southern blot experiments under stringent hybridization conditions revealed considerable sequence diversity among collagen genes. Our data suggest that most collagen genes are unique or are present in only a few copies.     

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.     

Overlapping stage-specific sets of numerous small collagenous polypeptides are translated in vitro from Caenorhabditis elegans RNA

Caenorhabditis elegans synthesizes four morphologically distinct types of collagenous cuticles during its lifetime. We show that in RNA populations isolated early or late during the L4-to-adult molt, chick and nematode collagen DNAs hybridize strongly to RNAs of about 1.2 kb. Different but overlapping classes of correspondingly small collagenous polypeptides (310-460 residues in length) are translated in vitro from these two populations and from RNA isolated at the L2-to-dauer molt. Over 60 different collagenous translation products are identified. These collagenous polypeptides are smaller than mature cuticle collagens and smaller than most vertebrate collagens. They probably represent cuticle collagen precursors and the primary products of the cuticle collagen genes of C. elegans.     

Temporal reiteration of a precise gene expression pattern during nematode development.

The nematode Caenorhabditis elegans is contained within a multifunctional exoskeleton, the cuticle, that contains a large number of distinct collagens. As the nematode proceeds from the egg through four larval stages to the adult, transition between larval stages is marked by synthesis of a new cuticle and subsequent moulting of the old one. This is a cyclically repeated developmental event, frequently described as the moulting cycle. We have examined the temporal expression of a group of six genes encoding distinct cuticular collagens. As expected, mRNA abundance for each of the six genes tested is found to oscillate, peaking once during each larval stage. Unexpectedly, the periods of abundance for each gene do not coincide, different genes being expressed at different times relative to one another within the moulting cycle. We detect a programme of temporally distinct waves of collagen gene expression, the precise pattern of which is repeated during each of the four larval stages. This multiphasic pattern of oscillating cuticular collagen gene expression indicates an unexpected complexity of temporal control during the nematode moulting cycle and has implications for collagen trimerization and cuticle synthesis.     

Stage-specific patterns of collagen gene expression during development of Caenorhabditis elegans.

Collagens are the major protein components of the Caenorhabditis elegans cuticle and are encoded by a large family of 40 to 150 closely related but nonidentical genes. We have determined temporal patterns of mRNA accumulation for a large number of collagen genes by screening recombinant phages and plasmids containing cloned collagen genes under high stringency conditions with 32P-labeled cDNA preparations specific for eggs or three postembryonic molts. We find that collagen mRNA levels are regulated both temporally and quantitatively during C. elegans development. Most genes studied exhibit one of four patterns of mRNA accumulation which correlate with changes in cuticle morphology and collagen protein composition during development. Our results suggest that, in general, there is a progressive activation of new collagen genes during normal development.     

Otx/otd homeobox genes specify distinct sensory neuron identities in C. elegans

The mechanisms by which the diverse functional identities of neurons are generated are poorly understood. C. elegans responds to thermal and chemical stimuli using 12 types of sensory neurons. The Otx/otd homolog ttx-1 specifies the identities of the AFD thermosensory neurons. We show here that ceh-36 and ceh-37, the remaining two Otx-like genes in the C. elegans genome, specify the identities of AWC, ASE, and AWB chemosensory neurons, defining a role for this gene family in sensory neuron specification. All C. elegans Otx genes and rat Otx1 can substitute for ceh-37 and ceh-36, but only ceh-37 functionally substitutes for ttx-1. Functional substitution in the AWB neurons is mediated by activation of the same downstream target lim-4 by different Otx genes. Misexpression experiments indicate that although the specific identity adopted upon expression of an Otx gene may be constrained by the cellular context, individual Otx genes preferentially promote distinct neuronal identities.     

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.     

Visualization of Caenorhabditis elegans cuticular structures using the lipophilic vital dye DiI

The cuticle of C. elegans is a highly resistant structure that surrounds the exterior of the animal(1-4). The cuticle not only protects the animal from the environment, but also determines body shape and plays a role in motility(4-6). Several layers secreted by epidermal cells comprise the cuticle, including an outermost lipid layer(7). Circumferential ridges in the cuticle called annuli pattern the length of the animal and are present during all stages of development(8). Alae are longitudinal ridges that are present during specific stages of development, including L1, dauer, and adult stages(2,9). Mutations in genes that affect cuticular collagen organization can alter cuticular structure and animal body morphology(5,6,10,11). While cuticular imaging using compound microscopy with DIC optics is possible, current methods that highlight cuticular structures include fluorescent transgene expression(12), antibody staining(13), and electron microscopy(1). Labeled wheat germ agglutinin (WGA) has also been used to visualize cuticular glycoproteins, but is limited in resolving finer cuticular structures(14). Staining of cuticular surface using fluorescent dye has been observed, but never characterized in detail(15). We present a method to visualize cuticle in live C. elegans using the red fluorescent lipophilic dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), which is commonly used in C. elegans to visualize environmentally exposed neurons. This optimized protocol for DiI staining is a simple, robust method for high resolution fluorescent visualization of annuli, alae, vulva, male tail, and hermaphrodite tail spike in C. elegans.     

Comparisons of the complete sequences of two collagen genes from Caenorhabditis elegans

Several collagen genes have been isolated from the nematode Caenorhabditis elegans. The complete nucleotide sequences of two of these genes, col-1 and col-2, have been determined. These collagen genes differ from vertebrate collagen genes in that they contain only one or two introns, their triple-helical regions are interrupted by nonhelical amino acid sequences and they are smaller. A high degree of nucleotide and amino acid homology exists between col-1 and col-2. In particular, the regions around cysteines and lysines are most highly conserved. The C. elegans genome contains 50 or more collagen genes, the majority of which probably encode cuticle collagens; col-1 and col-2 apparently are members of this large family of cuticle collagen genes.     

Longevity pathways converge on autophagy genes to regulate life span in Caenorhabditis elegans

Aging is a multifactorial process with many mechanisms contributing to the decline. Mutations decreasing insulin/IGF-1 (insulin-like growth factor-1) or TOR (target of rapamycin) kinase-mediated signaling, mitochondrial activity and food intake each extend life span in divergent animal phyla. Understanding how these genetically distinct mechanisms interact to control longevity is a fundamental and fascinating problem in biology. Here we show that mutational inactivation of autophagy genes, which are involved in the degradation of aberrant, damaged cytoplasmic constituents accumulating in all aging cells, accelerates the rate at which the tissues age in the nematode Caenorhabditis elegans. According to our results Drosophila flies deficient in autophagy are also short-lived. We further demonstrate that reduced activity of autophagy genes suppresses life span extension in mutant nematodes with inherent dietary restriction, aberrant insulin/IGF-1 or TOR signaling, and lowered mitochondrial respiration. These findings suggest that the autophagy gene cascade functions downstream of and is inhibited by different longevity pathways in C. elegans, therefore, their effects converge on autophagy genes to slow down aging and lengthen life span. Thus, autophagy may act as a central regulatory mechanism of animal aging.     

Specificity and complexity of the Caenorhabditis elegans innate immune response

In response to infection, Caenorhabditis elegans produces an array of antimicrobial proteins. To understand the C. elegans immune response, we have investigated the regulation of a large, representative sample of candidate antimicrobial genes. We found that all these putative antimicrobial genes are expressed in tissues exposed to the environment, a position from which they can ward off infection. Using RNA interference to inhibit the function of immune signaling pathways in C. elegans, we found that different immune response pathways regulate expression of distinct but overlapping sets of antimicrobial genes. We also show that different bacterial pathogens regulate distinct but overlapping sets of antimicrobial genes. The patterns of genes induced by pathogens do not coincide with any single immune signaling pathway. Thus, even in this simple model system for innate immunity, striking specificity and complexity exist in the immune response. The unique patterns of antimicrobial gene expression observed when C. elegans is exposed to different pathogens or when different immune signaling pathways are perturbed suggest that a large set of yet to be identified pathogen recognition receptors (PRRs) exist in the nematode. These PRRs must interact in a complicated fashion to induce a unique set of antimicrobial genes. We also propose the existence of an "antimicrobial fingerprint," which will aid in assigning newly identified C. elegans innate immunity genes to known immune signaling pathways.     

Microarray analysis of gene expression with age in individual nematodes

We compare the aging of wild-type and long-lived C. elegans by gene expression profiling of individual nematodes. Using a custom cDNA array, we have characterized the gene expression of 4-5 individuals at 4 distinct ages throughout the adult lifespan of wild-type N2 nematodes, and at the same ages for individuals of the long-lived strain daf-2(e1370). Using statistical tools developed for microarray data analysis, we identify genes that differentiate aging N2 from aging daf-2, as well as classes of genes that change with age in a similar way in both genotypes. Our novel approach of studying individual nematodes provides practical advantages, since it obviates the use of mutants or drugs to block reproduction, as well as the use of stressful mass-culturing procedures, that have been required for previous microarray studies of C. elegans. In addition, this approach has the potential to uncover the molecular variability between individuals of a population, variation that is missed when studying pools of thousands of individuals.     

Gene expression profile of rabbit cartilage by expressed sequence tag analysis

Although the rabbit is commonly used as an animal model for the in vivo study of cartilage formation or regeneration, genetic approaches to the rabbit cartilage are rare. We constructed an expressed sequence tag (EST) library from rabbit cartilage tissue for the first time to establish the foundations for genetic study on rabbit cartilage. From our results, we identified 2387 unique genes among 4885 clones, corresponding to 1839 matched to characterized genes including 1618 genes with known function and 548 uncharacterized and novel genes. Gene expression profiles based on EST frequency show that type II collagen (COL2A1) and type X collagen (COL10A1) among collagen clones, proteoglycan 4 (PRG4) and decorin (DCN) among proteoglycan clones, and cartilage oligomeric matrix protein (COMP) and matrix Gla protein (MGP) among other extracellular matrix clones, are highly expressed in rabbit cartilage. In addition, gene expression analysis based on real-time PCR of these major extracellular matrix constituents showed that expression of col2a1 and col10a1 remains constant whereas the expression of prg4, dcn, and comp reveals substantial change with rabbit age. This EST library will provide a valuable resource with which to identify genes involved in the biochemical and physiological functions of rabbit cartilage, and will contribute to establishing the rabbit as an animal model for cartilage research.     

Three-dimensional collagen regulates collagen gene expression by a mechanism that requires serine/threonine kinases and is independent of mechanical contraction

Integrin alpha1beta1, one of the cellular collagen receptors, can participate in the regulation of collagen accumulation by acting as a negative feedback regulator. The molecular mechanism behind this phenomenon has been unknown. We have plated cells inside three-dimensional collagen and analyzed a set of chemical inhibitors for various signal transduction pathways. Only two wide-spectrum serine/threonine kinase inhibitors, H-7 and iso-H-7 could prevent the down-regulation of alpha1(I) collagen mRNA levels in cells exposed to three-dimensional collagen. In monolayer iso-H-7 slightly down-regulated collagen gene expression, indicating that inside collagen it affected integrin signaling rather than having a direct stimulatory effect on collagen mRNA levels. The effect of iso-H-7 was not dependent on its ability to inhibit protein kinases A, C, or G. H-7 and iso-H-7 could also inhibit collagen gel contraction, but this mechanism was independent of collagen gene regulation. Three-dimensional collagen could also up-regulate the mRNA levels of several matrix metalloproteinases (MMPs) but H-7 and iso-H-7 had no effect on the regulation of MMP genes. Our data indicate that three-dimensional collagenous matrix regulates distinct cellular signaling pathways and that collagen gene regulation is independent of the other effects of the matrix.     

Extracellular matrix density regulates the formation of tumour spheroids through cell migration

In this work, we show how the mechanical properties of the cellular microenvironment modulate the growth of tumour spheroids. Based on the composition of the extracellular matrix, its stiffness and architecture can significantly vary, subsequently influencing cell movement and tumour growth. However, it is still unclear exactly how both of these processes are regulated by the matrix composition. Here, we present a centre-based computational model that describes how collagen density, which modulates the steric hindrance properties of the matrix, governs individual cell migration and, consequently, leads to the formation of multicellular clusters of varying size. The model was calibrated using previously published experimental data, replicating a set of experiments in which cells were seeded in collagen matrices of different collagen densities, hence producing distinct mechanical properties. At an initial stage, we tracked individual cell trajectories and speeds. Subsequently, the formation of multicellular clusters was also analysed by quantifying their size. Overall, the results showed that our model could accurately replicate what was previously seen experimentally. Specifically, we showed that cells seeded in matrices with low collagen density tended to migrate more. Accordingly, cells strayed away from their original cluster and thus promoted the formation of small structures. In contrast, we also showed that high collagen densities hindered cell migration and produced multicellular clusters with increased volume. In conclusion, this model not only establishes a relation between matrix density and individual cell migration but also showcases how migration, or its inhibition, modulates tumour growth.